阅读说明：本技术 猪流行性腹泻病毒Nsp10蛋白、含该Nsp10蛋白的融合蛋白及其制备方法和应用 (Swine epidemic diarrhea virus Nsp10 protein, fusion protein containing Nsp10 protein, and preparation method and application thereof ) 是由 王婷 孔令保 朱丽婷 刘仕国 于 2021-08-04 设计创作，主要内容包括：本发明属于生物技术领域,具体涉及猪流行性腹泻病毒nsp10蛋白、含该nsp10蛋白的融合蛋白及其制备方法和应用。本发明将猪流行性腹泻病毒的nsp10蛋白进行了原核表达,其蛋白产量相当高,另外,本发明首次证明重组nsp10的原核表达可在小鼠体内诱导活跃的体液和细胞免疫,nsp10蛋白免疫鼠产生的nsp10抗体滴度达1:13000,MBP-nsp10蛋白免疫鼠产生的nsp10抗体滴度高达1:42000。同时证明了MBP可增强nsp10的免疫诱导能力,上述结果表明,PEDV的nsp10蛋白可用于制备PEDV诊断试剂和疫苗。(The invention belongs to the technical field of biology, and particularly relates to a nsp10 protein of porcine epidemic diarrhea virus, a fusion protein containing the nsp10 protein, and a preparation method and application thereof. The invention carries out prokaryotic expression on the nsp10 protein of the porcine epidemic diarrhea virus, the protein yield is quite high, in addition, the invention firstly proves that the prokaryotic expression of the recombinant nsp10 can induce active humoral and cellular immunity in mice, the nsp10 antibody titer generated by nsp10 protein immunized mice reaches 1:13000, and the nsp10 antibody titer generated by MBP-nsp10 protein immunized mice reaches 1: 42000. And the result shows that MBP can enhance the immunity induction capability of nsp10, and the nsp10 protein of PEDV can be used for preparing PEDV diagnostic reagents and vaccines.)

1. The fusion protein containing the porcine epidemic diarrhea virus nsp10 protein is characterized by consisting of MBP protein and porcine epidemic diarrhea virus nsp10 protein, wherein the amino acid sequence of the porcine epidemic diarrhea virus nsp10 protein is shown as SEQ ID NO. 1, and the amino acid sequence of the MBP protein is shown as SEQ ID NO. 2.

2. A recombinant expression vector comprising a nucleotide sequence for expressing the fusion protein of claim 1.

3. A host cell comprising the recombinant expression vector of claim 2.

4. Use of the nsp10 protein of porcine epidemic diarrhea virus and the fusion protein of claim 1 for the preparation of a vaccine for porcine epidemic diarrhea virus.

5. Use of the nsp10 protein of porcine epidemic diarrhea virus and the fusion protein of claim 1 for preparing diagnostic reagents for porcine epidemic diarrhea virus.

6. The method of producing the fusion protein of claim 1, comprising the steps of:

step 1: extracting porcine epidemic diarrhea virus RNA, and performing reverse transcription to obtain nsp10 cDNA;

step 2: designing and synthesizing primers of the nsp10 gene of the porcine epidemic diarrhea virus, and amplifying the nsp10 gene of the porcine epidemic diarrhea virus;

and step 3: connecting the nsp10 gene of the porcine epidemic diarrhea virus with a vector containing an MBP gene, and transforming competent cells after identification;

and 4, step 4: and (3) expressing, purifying and identifying the recombinant protein MBP-nsp 10.

7. The method for preparing a fusion protein according to claim 6, wherein the MBP-nsp10 protein has an optimal expression temperature of 16 ℃.

8. The method of claim 6, wherein the MBP-nsp10 protein is expressed for 24 hours optimally.

9. The method of claim 6, wherein the MBP-nsp10 protein is expressed at an optimal IPTG concentration of 1.4 mM.

10. The use according to claim 4, wherein the optimum expression temperature of the nsp10 protein of porcine epidemic diarrhea virus is 40 ℃, the optimum expression time is 8h, and the optimum IPTG concentration is 1.4mM when expressing.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a porcine epidemic diarrhea virus Nsp10 protein, a fusion protein containing the Nsp10 protein, and a preparation method and application thereof.

Background

Porcine Epidemic Diarrheal Virus (PEDV) is a strong infectious enterovirus that is responsible for the vomiting, Diarrhea and dehydration of pigs. PEDV was first released in 1971 in the uk, after which it was reported in several areas. PEDV epidemic was first reported in china in the eighties of the nineteenth century and PEDV virus strains were isolated. In 2010, PEDV mutant strains appeared in several countries in Asia, causing large-scale death of piglets and severe economic loss. The original vaccine has no good protection effect on PEDV mutant strains. In addition, the clinical symptoms of PEDV are highly similar to the pathological characteristics of infectious gastroenteritis and porcine rotavirus, and the existing detection means has cross reaction. Therefore, for researching PEDV mutant strains, the method is of great importance for the prevention, control and detection of PEDV in order to develop a specific detection method. PEDV nsp10 is an accessory protein of virus nucleoside-2-O '-methyltransferase, activates the activity of nucleoside-2-O' -methyltransferase, is a zinc binding protein, and a single nsp10 protein forms a 12-polymer structure and has no enzyme activity. nsp10 plays an important role in virus replication and can interact with the nonstructural protein nsp16 to antagonize interferon beta. Thus, the nsp10 protein of PEDV is a key regulatory subunit, an important regulator of viral RNA synthesis, and is essential for viral replication.

However, studies on the immunogenicity of nsp10 of PEDV have not been reported.

Disclosure of Invention

According to the invention, the Nsp10 protein of PEDV is subjected to prokaryotic expression, the protein yield is quite high, in addition, the invention firstly proves that the prokaryotic expression of recombinant Nsp10 can induce active humoral and cellular immunity in a mouse body, and simultaneously proves that MBP can enhance the immunity induction capability of Nsp10, and the results show that the Nsp10 protein of PEDV can be used for preparing PEDV diagnostic reagents and vaccines.

The technical scheme of the invention is as follows:

in order to obtain a large amount of NSP10 recombinant protein of PEDV, the target protein nsp10 is cloned into pMAL-c2x-MBP and pET-28a vectors. Maltose Binding Protein (MBP) is generally used for improving solubility, reducing protein degradation, increasing protein expression amount and stability, and subunit vaccines expressed by fusing MBP are found to have enhanced protection. The invention transforms the recombinant vector into Escherichia coli BL21 strain, and optimizes the protein expression condition: temperature, time and IPTG concentration, and Ni-NTA medium affinity chromatography technology is utilized, and a large amount of recombinant protein is obtained for subsequent research by purifying target protein through high-concentration competitive eluent. Purified MBP-nsp10, nsp10 and MBP-_TFFImmunizing Kunming female mice of 6-8 weeks old to obtain antibody serum, and detecting the corresponding antibody titer by using indirect ELISA. The results show that: the nsp10 antibody titer of the nsp10 protein immunized mice reaches 1:13000, and the nsp10 antibody titer of the MBP-nsp10 protein immunized mice reaches 1:42000, which indicates that the MBP possibly plays an immune enhancement function and increases the antibody titer induced by the nsp 10. The PEDV pig positive serum is used as a primary antibody to carry out immunoblot analysis, and the result shows that: pig positive serum can specifically recognize the nsp10 protein, and further confirms the immunogenicity of the recombinant protein nsp 10. Humoral immunity and cellular immunity play an important role in host antiviral processes, and in order to evaluate the humoral and cellular immunity levels after mice are immunized with antigens, the invention detects the expression of cytokines in spleen lymphocytes and serum of the mice. Since cytokines are usually soluble proteins and secreted in body fluids, the sandwich ELISA method measures the expression of mouse spleen lymphocytes TNF-alpha and serum IL-4. Next, IL-2, IL-4, IL-10, TNF- α and IFN- γ in mouse spleen lymphocytes were subjected to Real-time PCR at mRNA level. The results show that: the expression levels of IL-2, IL-4, IL-10, TNF-alpha and IFN-gamma of the recombinant protein nsp10 immune group are obviously increased compared with those of a control group, and the nsp10 protein induces the immune response of a mouse after the mouse is immunized.

The amino acid sequence of nsp10 protein of PEDV is shown in SEQ ID NO. 1, and the amino acid sequence of MBP protein is shown in SEQ ID NO. 2.

The invention has the beneficial effects that:

1. the invention discovers that the recombinant nsp10 can induce active humoral and cellular immunity in mice, and MBP can enhance the immunity induction capability of nsp10, specifically, the nsp10 antibody titer of nsp10 protein immunized mice reaches 1:13000, and the nsp10 antibody titer of MBP-nsp10 protein immunized mice reaches 1: 42000. Therefore, the recombinant proteins nsp10 and MBP-nsp10 can be involved in preparing PEDV vaccines. MBP can also be used as an immunoadjuvant for nsp 10.

2. The recombinant proteins nsp10 and MBP-nsp10 can be specifically combined with PEDV-infected pig positive serum, so that the recombinant proteins nsp10 and MBP-nsp10 can be used for preparing PEDV diagnostic reagents.

Drawings

FIG. 1 shows the RNA extraction result (A) of PEDV virus CV777 and the amplification result (B) of PEDV nsp10 gene. In FIG. 1A, M is DNA mark 2000, Lane 1-3 is Total RNA; in FIG. 1B, M is DNA marker2000, Lane1 is the amplified band of PEDV nsp10 gene, and Lane2 is the negative control.

FIG. 2 shows the PCR identification result of the bacterial liquid of pMAL-c2x-MBP-nsp10 and pET-28a-nsp10 recombinant plasmids. DNA marker2000, Lane1, 2 and 3 are pMAL-c2x-MBP-nsp10, 5 and 6 are pET-28a-nsp10 positive samples, Lane4 and 7 are negative samples, and Lane8 is negative control.

FIG. 3 shows the results of double-restriction enzyme identification of pMAL-c2x-MBP-nsp 10. In FIG. 3A, M is DNA marker 15000, Lane1, pMAL-c2x-MBP-nsp10(7062bp), in FIG. 3B, M is DNA marker 15000, Lane1, pMAL-c2x-MBP-nsp10 double-cleaved band.

FIG. 4 shows the PCR identification result of pET-28a-nsp 10. In FIG. 4A, M: DNA marker 15000, Lane1: pET-28a-nsp10(5740bp), in FIG. 4B, M: DNA marker2000, Lane1 and 2 are nsp10 amplification products (408bp), Lane4 and 5 are T7 universal primer amplification products (618bp), and Lane3 is a negative control.

FIG. 5 shows the expression of recombinant proteins. Lane1 and Lane2 in FIG. 5A are MBP-_TFFBefore and after induction, the protein size is 60 kDa. In FIG. 5B, Lane1 is the protein MBP-nsp10 before induction, and Lane 2-6 is after induction, and the protein size is 56 kDa. In FIG. 5C, Lane1 was before induction of nsp10, Lane 2-5 was after induction, and the protein size was 16 kDa.

FIG. 6 is a drawing showingMBP-_TFFAnd (5) inducing temperature optimization results. Lane1 is before induction, Lane 2-7 is after induction at 28 ℃, 30 ℃, 34 ℃, 37 ℃, 39 ℃ and 40 ℃.

FIG. 7 shows the MBP-nsp10 induced temperature optimization results. Lane1 is before induction, Lane 2-7 is after induction at 14 ℃, 15 ℃, 16 ℃, 18 ℃, 20 ℃ and 24 ℃.

Fig. 8 shows the nsp10 induced temperature optimization results. Lane1 is before induction, Lane 2-7 is after induction at 34 deg.C, 37 deg.C, 39 deg.C, 40 deg.C, 42 deg.C, 43 deg.C.

FIG. 9 shows MBP-_TFFAnd optimizing the result of the induction time. Lane1 is before induction, Lane 2-7 is after 16h, 18h, 20h, 24h, 28h and 30h of induction.

FIG. 10 shows the result of the MBP-nsp10 optimization of induction time. Lane1 was before induction, Lane 2-7 were after 16h, 18h, 20h, 24h, 28h, and 30h induction, respectively.

Fig. 11 shows the results of nsp10 induction time optimization. Lane1 and Lane2 are before induction and after induction of unloaded pET-28a, Lane 3-8 are 4h, 6h, 8h, 10h, 12h and 16h after induction, and Lane9 is nsp10 and is not induced.

FIG. 12 shows MBP-_TFFAnd (5) inducing an IPTG concentration optimization result. Lane1 was pre-induction, Lane 2-7 was post-induction at 0.5mM, 0.8mM, 1.0mM, 1.4mM, 1.8mM, 2.0 mM.

FIG. 13 shows the result of optimization of the concentration of IPTG induced by MBP-nsp 10. Lane1 was pre-induction, Lane 2-7 was post-induction at 0.5mM, 0.8mM, 1.0mM, 1.4mM, 1.8mM, 2.0 mM.

FIG. 14 shows the result of optimization of concentration of IPTG induced by nsp 10. Lane1 and Lane2 were pre-and post-induction with no-load pET-28a, respectively, Lane 3-8 were 0.5mM, 0.8mM, 1.0mM, 1.4mM, 1.8mM, and 2.0mM post-induction, and Lane9 was nsp10 non-induced.

FIG. 15 shows the recombinant protein MBP-_TFFMBP-nsp10, nsp 10. Lane1 and Lane4 in FIG. 15A are MBP-_TFFProteins were not induced, Lane2 and Lane5 are MBP-_TFFLane3 and Lane6 are MBP-_TFFProtein 250mm imidazole eluted purified band. Lane1 in FIG. 15B is a purified band eluted by 100mm, 200mm, 250mm, 300mm imidazole, and Lane 2-5 is not induced by the recombinant protein MBP-nsp 10. In FIG. 15CLane1 and Lane2 are purified proteins eluted by 200mm and 250mm imidazole, after no-load pET-28a is induced and induced, Lane3 and Lane4 are nsp10 are not induced and induced, Lane5 is flow-through liquid, Lane6 is washing liquid, and Lane 7-8 are purified proteins eluted by 200mm and 250mm imidazole.

FIG. 16 shows the Western Blot identification of the recombinant proteins MBP-nsp10, nsp 10. MBP-nsp10 is before induction from left to right Lane1, Lane2 is after induction, Lane3 is NI column purified protein. nsp10 was obtained before induction of the cells by Lane1 from left to right, and after induction by Lane2, purified protein was obtained by Lane 3.

FIG. 17 shows MBP-_TFFELISA test results of serum antibody levels of MBP-nsp10, nsp10 immunized mice.

FIG. 18 shows the result of identifying the specificity of the serum antibody by Western Blot. The recombinant proteins pMAL-c2x-MBP-nsp10 and pET-28a-nsp10 are before induction from left to right Lane1, and Lane2 is after induction, and Lane3 is a purified protein.

FIG. 19 shows the results of ELISA expression of TNF-. alpha.in mouse spleen.

FIG. 20 shows the results of ELISA assay of IL-4 expression in mouse serum.

FIG. 21 shows the results of Real-time PCR detection of IL-2, IL-4, IL-10, TNF- α, IFN- γ expression in mouse spleen.

FIG. 22 is a diagram showing the results of detection of the recombinant protein MBP-nsp10 ELISA and a commercial ELISA kit.

Detailed Description

The present invention will be described in more detail with reference to the following embodiments for understanding the technical solutions of the present invention, but the present invention is not limited to the scope of the present invention.

The Vero cells infected with PEDV virus CV777 in this example were stored in the laboratory of the inventors;

kunming female mice were purchased from Jiangxi university of traditional Chinese medicine.

Construction of recombinant expression vector of PEDV nsp10 protein

1. Extraction of PEDV RNA

(1) Preparing a reagent: trizol, chloroform, isopropanol, RNase-Free water, made up with RNase-Free water in 75% ethanol.

(2) Quickly thawing venom (Vero cells infected with PEDV virus CV 777) stored at-80 ℃, centrifuging to remove supernatant to obtain Vero cells infected with PEDV, adding 1ml of guanidinium isothiocyanate/phenol into an EP tube, repeatedly blowing and uniformly mixing by using a gun, breaking cells to release RNA, allowing Trizol to have the function of cutting DNA, and standing at room temperature for 5min to completely separate nucleic acid and protein.

(3) Adding 0.2mL of chloroform, oscillating vigorously for 15-30 s, and standing for 2-3 min.

(4) Centrifugation is carried out at 4 ℃ for 12000g × 15 min. Sample RNA was seen in the EP tube predominantly in the upper aqueous layer.

(5) DEPC water treatment 1.5mLEP tube, suction RNA, abandon the lower organic phase, ensure RNA purity, add 500 u LIsopropanol (isopropanol), after mixing, at room temperature to stand for 10 min.

(6) The mixture was centrifuged at low temperature for 10min, and the RNA was precipitated at the bottom of the EP tube as a transparent gel.

(7) The supernatant was discarded, the precipitated RNA was dissolved in 1mL of precooled 75% ethanol, and the RNA was washed thoroughly by gently tapping the wall of the EP tube.

(8) Centrifuge at 12000g for 5min at 4 ℃.

(9) And pouring out the upper ethanol layer, reversely buckling the EP pipe on the table top to completely drain the liquid, and standing at room temperature for 5-10 minutes until the ethanol is basically volatilized.

(10) Adding 20-50 μ L DEPC water (RNase Free) to dissolve RNA (if insoluble, can promote dissolution at 65 ℃ for 10-15 min).

(11) Taking 2 mu L for electrophoresis, and storing the rest at-80 ℃ for a long time.

The RNA electrophoresis results showed clear bands of 28s, 18s and 5s, respectively, indicating successful total RNA extraction, as shown in FIG. 1A.

2. Synthesis of Nsp10cDNA

And (3) performing RT reaction by taking the extracted RNA of the PEDV as a template, wherein the specific reaction system is as follows:

RNA	1μg
		5xRT Mix	4μL
Water(Nuclease-free)	up to 20μL
		Total volume	20μL

the reaction procedure was as follows:

temperature of	Time
		37℃	5s
85℃	15min
		4℃	Until

3. PCR primer design and Synthesis

Primers were designed using Primer 6.0 software based on the sequence of PEDV nsp10(KT323979.1) queried in GENBANK, as detailed in table 1.

TABLE 1 primer sequences for PEDV nsp10 amplification

4. Amplification of the PEDV nsp10 Gene

Nsp10 was PCR amplified, template cDNA was synthesized in step 2, and the PCR reaction was as follows:

the PCR reaction procedure was as follows:

the amplification products were all detected by electrophoresis on a 1% agarose gel. The results show that in FIG. 1B, there is a significant band near the position 500bp below DNAmarker, which is consistent with the expected band of 408bp, indicating that the PCR successfully amplified the target band.

5. Gel recovery and purification of PEDV nsp10

(1) The running agarose gel is taken, the band of the required purpose is cut off as accurately as possible, and is added into GSB solution with the volume 3 times of the volume of the gel, and 1g of the gel is about 1 mL.

(2) And melting the glue in a water bath kettle at 55 ℃ for 10 minutes, and slightly shaking the EP pipe during the melting process to uniformly heat the pipe wall and ensure that the glue blocks are completely melted.

(3) The EP tube was removed and the solution was allowed to cool to room temperature. Adding the dissolved solution into an adsorption column, standing for 1min, centrifuging at 10000g for 1min, and repeatedly centrifuging to improve the recovery efficiency of the gel.

(4) Add 650. mu.L of washing solution to the adsorption column and centrifuge at 10000g for 1 min.

(5) Centrifuging at 10000g for 2min, pouring out the EP tube, draining off water, and airing at room temperature for 5-10 min.

(6) Adding 30-50 mu L of eluent into an adsorption column, standing for 1-2 min at room temperature to enable the eluent to completely infiltrate the bottom, centrifuging 10000g for 1min to elute DNA, increasing recovery efficiency through multiple elutions, and improving DNA concentration

6. Extraction of plasmid pMAL-c2x-MBP, pET-28a (+) (plasmid)

(1) The DH5a E.coli containing the vector plasmids pMAL-c2x-MBP, pET-28a was inoculated in LB liquid medium containing antibiotics ampicillin (Amp) or kanamycin (Kana), and cultured overnight with shaking at 37 ℃.

(2) A1.5 mLEP tube was centrifuged at 12000g for 1min to collect the cells, the supernatant was discarded, all the cells were collected by centrifugation many times, the EP tube was inverted and the water was blotted on filter paper, and the pellet was washed with 1 XPBS.

(3) The precipitate was added with 250. mu.L of solution 1, and shaken for 30 seconds to sufficiently lyse the cells.

(4) 250 μ LP2 solution was added and mixed by inversion from top to bottom.

(5) Adding 350 mu LP3 solution, gently inverting the solution for 6-8 times, and centrifuging the solution for 10min at 13000 g. The P1 solution, P2 solution, and P3 solution were from a plasmid miniprep kit (tiangen high purity plasmid miniprep kit (DP 107)).

(7) Transferring the supernatant to a collection tube, absorbing the white mixture at the lower layer as little as possible to prevent protein from polluting DNA, and centrifuging for 1min at 10000 rmp. Here the DNA can be collected repeatedly to increase the plasmid concentration.

(8) Add 600. mu.L DNA Wash Buffer, 10000rmp centrifugation for 1min, can repeat the washing once.

(9) Air-drying at room temperature for 10-20 min, adding 50 mu LEB buffer solution, and dissolving DNA.

(10) The samples were stored in a-20 ℃ freezer for further use.

7. Preparation of competent cells of Escherichia coli

(1) The previous day: the preserved glycerol bacterial liquid is taken out at the temperature of minus 20 ℃,100 mu L of escherichia coli is sucked and inoculated in an LB liquid culture medium, and the culture is carried out overnight.

(2) The next day: 50 μ L of overnight culture broth was inoculated to 5mLLB broth at a ratio of 1: 100.

(3) Culturing until the growth logarithmic phase of Escherichia coli, and detecting OD of bacterial liquid₆₀₀The value is 0.4-0.6.

(4) Subpackaging the bacterial liquid into EP tubes, adding 1mL of bacterial liquid into each EP tube, and carrying out ice bath for 10 min.

(5) Centrifuge at 4000rpm for 10min at 4 ℃ and pour off the clear solution.

(6)0.1M CaCl₂After precooling, 1mL of the resuspended thallus is added into an EP tube, and the mixture is evenly blown and stirred and then is subjected to ice bath for 10 min.

(7) Centrifuging at 4000rpm for 10min at 4 deg.C, and removing supernatant.

(8) Add 200. mu.L of 0.1M CaCl per tube₂Resuspending the cells, dispensing competent cells at 50-100. mu.L/tube, storing at 4 ℃ for a week (or adding 240. mu. L0.1M CaCl/tube)₂And 60 μ L of 50% glycerol, 50-100 μ L/tube of competent cells, storage at-80 deg.C for long-term use)

8. Ligation of vector to target Gene fragment

(1) pMAL-c2x-MBP was double digested with EcoRI, HindIII, pET-28a with BamHI, XhoI, double digestion system:

name of reagent	Volume of
		10×Buffer	2μL
pMAL-c2x-MBP/pET-28a(+)	1μg
		EcoR I/BamH I	1μL
Or Hind III/Xho I	1μL
		Water	Up to 20μL

The DNA fragment was recovered by agarose electrophoresis in a water bath at 37 ℃ for 1 h.

(2) DNA fragment and vector ligation system:

name of reagent	Volume of
		10×T4 DNA ligase Buffer	2.5μL
DNA fragment	0.3pmoL
		Vector DNA	0.03pmoL
T4 DNA ligase	1μL
		dH2O	Up to 25μL

After the connection, recombinant plasmids pMAL-c2x-MBP-nsp10 and pET-28a-nsp10 are obtained.

(3) Reacting at 16 ℃ for 12-16 h, and transforming competent cells by 10 mu L.

9. Transformation of recombinant plasmids

(1) Heating the sterilized LB solid culture medium to boiling, cooling to 50-60 ℃ at room temperature, adding antibiotics Amp or Kana, pouring a plate on an ultra-clean bench, and preparing the LB solid culture medium without antibiotics as a control.

(2) The dispensed competent cells of Escherichia coli DH5 alpha were removed from-80 deg.C and thawed in ice bath for 5 min.

(3) Add 10. mu.L of ligation product and ice-wash for 30 min.

(4) Heating in 42 deg.C water bath for 1min for 30 s.

(5) Ice-bath was immediately carried out for 3 min.

(6) Adding 600 μ L antibiotic-free culture medium, and shake culturing at 37 deg.C for 45 min.

(7) The cells were centrifuged at 8000rmp for 30s, and 300. mu.L of the cells were plated on a solid medium containing antibiotics and cultured overnight in an inverted state at 37 ℃. If necessary, a control group without antibiotics can be prepared.

10. Identification of recombinant plasmids

(1) And (3) PCR identification of bacterial liquid: and (2) selecting recombinant plasmid pMAL-c2x-MBP-nsp10 and pET-28a-nsp10 bacterial solutions to carry out PCR identification respectively, wherein a PCR system comprises the following components:

name of reagent	Volume of
		5×PCR Mix	2μL
Forward primer(10μM)	0.2μL
		Reverse primer(10μM)	0.2μL
Single colony bacterial liquid	0.2μL
		Water Nuclease-free	Up to 10μL

The PCR reaction procedure was as described above in step 4.

The PCR amplification of the bacterial liquid shows that the product band is positioned near the bottom of 500bp of DNAmarker and is consistent with the expected band (408bp), as shown in FIG. 2.

(2) Enzyme digestion identification: extracting recombinant plasmid pMAL-c2x-MBP-nsp10, and identifying by EcoRI and Hind III double enzyme digestion, wherein the system is as follows:

name of reagent	Volume of
		10×Buffer	2μL
Recombinant plasmid	1μg
		EcoRI	1μL
HindIII	1μL
		Water	Up to 20μL
Total volume	20μL

The samples were subjected to water bath at 37 ℃ for 1 hour, and then subjected to 1% agarose electrophoresis, and the results are shown in FIG. 3. The recombinant plasmid pMAL-c2x-MBP-nsp10 after double digestion obtains two bands, one is a linear fragment of a pMAL-c2x-MBP vector, and the other band is at the position of 408bp and is consistent with an expected band (408 bp). The success of the construction of the recombinant plasmid pMAL-c2x-MBP-nsp10 was preliminarily determined.

(3) And (3) PCR identification: the recombinant plasmid pET-28a-nsp10 was extracted. pET-28a-nsp10 can be directly identified by PCR through a T7 universal primer and a PCR primer, the identification result is shown in figure 4, a band amplified by an nsp10 forward and reverse primer suitable for pET-28a is near the lower part of 500bp of a DNA marker, and a band amplified by a T7 universal primer is between 500bp and 750bp of the DNA marker and is consistent with an expected band (408bp and 621 bp). The success of the construction of the recombinant plasmid pET-28a-nsp10 was preliminarily determined.

11. Sequencing identification

The recombinant plasmids pMAL-c2x-MBP-nsp10 and pET-28a-nsp10 identified by PCR and double digestion are sent to a sequencing company for sequencing, and the sequencing result shows that no base deletion or mutation exists through NCBI Blast comparison. The results show that 2 prokaryotic recombinant plasmids pMAL-c2x-MBP-nsp10 and pET-28a-nsp10 are successfully constructed.

Secondly, expression and purification of recombinant nsp10 protein

1. Expression identification of recombinant proteins

(1) The recombinant plasmid expression strain was inoculated in LB liquid medium containing antibiotics the previous day, and cultured overnight at 37 ℃.

(2) Transferring to new culture medium at 2 days at a ratio of 1:100, and continuously culturing to OD₆₀₀0.6 to 0.8.

(3) According to the expression condition range: MBP-_TFF: the temperature is 28-40 ℃, the IPTG concentration is 0.5-2.0 mM, and the induction time is 15-30 h; MBP-nsp 10: inducing for 16-30 h at 15-24 ℃ and IPTG concentration of 0.5-2.0 mM; ③ nsp 10: 34-43 ℃ and IPTG concentration of 0.5-2.0 mM, and inducing for 4-16 h. The bacteria solution without inducer is used as negative control.

(3) The bacterial liquid before and after induction is collected into a 1.5mL centrifuge tube respectively, centrifuged at 12000rmp for 1min, supernatant is removed, the precipitate is resuspended by 500 muL of 1 XPBS, 50 muL of the resuspension liquid is taken, 10 muL of 5 xSDS-PAGE Loading Buffer is added, and boiling is carried out for 10 min. A10. mu.L sample was subjected to SDS-PAGE and stained with Coomassie Brilliant blue for characterization.

The results are shown in FIG. 5, MBP-_TFFThe size of the protein after induction is about 60kDa, the size of the protein after MBP-nsp10 induction is about 56kDa, the size of the protein after nsp10 induction is about 16kDa, MBP-_TFFMBP-nsp10 and nsp10 induced successfully. Thus, the range of conditions for protein expression is:

①MBP-_TFF: the temperature is 28-40 ℃, the IPTG concentration is 0.5-2.0 mM, and the induction time is 15-30 h;

MBP-nsp 10: inducing for 16-30 h at 15-24 ℃ and IPTG concentration of 0.5-2.0 mM;

③ nsp 10: 34-43 ℃ and IPTG concentration of 0.5-2.0 mM, and inducing for 4-16 h.

2. Induction temperature optimization

(1) Ampicillin or kanamycin was added to 5mLB liquid medium to a final concentration of 50. mu.g/. mu.L, and the recombinant plasmid expression strain was inoculated and cultured overnight at 37 ℃.

(2) On day 2, the recombinant plasmid expression strain was transferred to a new medium at a ratio of 1:100, and OD was measured with a microplate reader₆₀₀When the value is 0.6-0.8, IPTG is added, and the final concentration is 1 mmoL/L.

(3) Respectively in MBP-_TFF: 28 ℃, 30 ℃, 32 ℃, 34 ℃, 37 ℃ and 40 ℃; MBP-nsp 10: 14 ℃, 15 ℃, 16 ℃, 18 ℃, 20 ℃ and 24 ℃; nsp 10: 34 ℃, 37 ℃, 39 ℃, 40 ℃, 42 ℃ and 43 ℃; induction was carried out for 8h, and no inducer was added to the negative control.

(4) The thalli is centrifuged for 1min at 12000g, washed by PBS and then resuspended, then 5 Xprotein loading buffer is added, the temperature is heated for 10min at 100 ℃, and the optimum induction temperature is determined by 12 percent SDS-PAGE analysis after centrifugation.

For the recombinant protein MBP-_TFFThe results are shown in FIG. 6, MBP-_TFFThe protein size is about 60kDa, MBP-_TFFThe expression of the protein starts from Lane2, the expression is firstly increased and then decreased along with the increase of the temperature, and the MBP protein expression reaches the maximum value at 37 ℃, thereby obtaining the MBP-_TFFThe optimal protein expression temperature was 37 ℃.

As for the recombinant protein MBP-nsp10, the result is shown in FIG. 7, the size of the MBP-nsp10 protein is about 56kDa, the MBP-nsp10 protein starts to express from 15 ℃ of Lane3, the protein is expressed at 16 ℃ in Lane3 and Lane4, namely 15 ℃ with the increase of temperature, the expression of the MBP-nsp10 protein is induced at 16 ℃ to be the maximum, and the expression of the MBP-nsp10 protein is not expressed or is smaller at other temperatures, so that the optimal expression temperature of the MBP-nsp10 protein is 16 ℃.

For the recombinant protein nsp10, the result is shown in fig. 8, the size of the nsp10 protein is about 16kDa, the expression of the nsp10 protein is started from 34 ℃ of Lane2, the protein expression level is increased firstly and then decreased with the increase of temperature, and the maximum expression of the nsp10 protein is reached at 40 ℃, so that the optimal expression temperature of the nsp10 protein is 40 ℃.

3. Induction time optimization

(1) The recombinant plasmid expression strain is cultured in the same manner as described above.

(2) Respectively induces MBP-_TFF: 16h, 18h, 20h, 24h, 28h and 30 h; induction of MBP-nsp10 at 16 ℃: 16h, 18h, 20h, 24h, 28h and 30 h; induction of nsp10 at 40 ℃: 4h, 6h, 8h, 10h, 12h and 16 h. The inoculum without inducer was used as a negative control.

(3) Centrifuging at 12000g for 1min to collect thallus, washing with PBS once, re-suspending with PBS, adding 5 Xprotein sample buffer, heating at 100 deg.C for 10min, centrifuging, and analyzing with 12% SDS-PAGE to obtain optimal induction time.

For the recombinant protein MBP-_TFFThe results are shown in FIG. 9, MBP-_TFFThe protein size is about 60kDa, the expression is started from Lane2MBP protein, the protein expression quantity is increased and then reduced along with the increase of time, and MBP-_TFFThe protein amount reaches the maximum, and then the MBP-_TFFThe optimal expression time of the protein is 24 h.

As for the recombinant protein MBP-nsp10, the result is shown in FIG. 10, the size of the MBP-nsp10 protein is about 56kDa, the MBP-nsp10 protein starts to be expressed from Lane2, the protein has higher expression level when Lane2, Lane3 and Lane5 express for 16h, 18h and 24h along with the increase of time, and the MBP-nsp10 protein has the maximum expression level when the protein is induced to express 24h, so that the optimal expression time of the MBP-nsp10 protein is 24 h.

As for the recombinant protein nsp10, the result is shown in FIG. 11, the size of the nsp10 protein is about 16kDa, the nsp10 protein is expressed from Lane3, the protein expression amount is increased firstly and then reduced along with the increase of time, the amount of the nsp10 protein reaches the maximum when the expression is induced for 8 hours, and the optimal expression time of the nsp10 protein is 8 hours.

4. IPTG induced concentration optimization

(1) The recombinant plasmid expression strain is cultured by the same method as above until OD is reached₆₀₀When the concentration is 0.6 to 0.8, MBP-_TFFAdding IPTG: 0.5mM, 1.0mM, 1.2mM, 1.4mM, 1.8mM, 2.0mM induced at 37 ℃ for 24 h; MBP-nsp10 IPTG was added: 0.5mM, 1.0mM, 1.2mM, 1.4mM, 1.8mM, 2.0mM induced at 16 ℃ for 24 h; nsp10 addition of IPTG: 0.5mM, 1.0mM, 1.2mM, 1.4mM, 1.8mM, 2.0mM induced at 40 ℃ for 8 h. The bacteria solution without inducer is used as negative control.

(2) Centrifuging at 12000g for 1min to collect thallus, washing with PBS once, re-suspending with PBS, adding 5 Xprotein sample buffer, heating at 100 deg.C for 10min, centrifuging, and analyzing with 12% SDS-PAGE to obtain the optimal IPTG final concentration.

For the recombinant protein MBP-_TFFThe results are shown in FIG. 12, MBP-_TFFThe protein has a size of about 60kDa from Lane2MBP-_TFFThe expression of the protein begins, the expression amount of the protein is increased and then reduced along with the increase of time, and the MBP-activated protein is induced and expressed at the concentration of IPTG of 1.0mM_TFFThe protein amount reaches the maximum, and then the MBP-_TFFThe optimum IPTG concentration for the protein is 1.0 mM.

As for the recombinant protein MBP-nsp, the result is shown in FIG. 13, the size of the MBP-nsp10 protein is about 56kDa, the MBP-nsp10 protein starts to express from Lane2, the protein has higher expression level at Lane5 and Lane6, namely at IPTG concentration of 1.4mM and 1.8mM, and the expression level of the MBP-nsp10 protein is maximum at 1.4mM, namely the optimal IPTG concentration of the MBP-nsp10 protein is 1.4 mM.

As for the recombinant protein nsp10, the result is shown in FIG. 14, the size of the nsp10 protein is about 16kDa, the nsp10 protein is expressed from Lane3, the protein expression amount is increased and then decreased along with the increase of time, the protein expression amount of the nsp10 protein is induced and expressed at 1.4mM to reach the maximum, and the concentration of IPTG (isopropyl thiogalactoside) expressed by the nsp10 protein is 1.4 mM.

5. Purification of recombinant proteins

(1) Large scale expression of recombinant proteins MBP-nsp10, nsp10 and MBP-_TFFAnd (3) centrifuging 12000g for 1min to collect thalli, resuspending by PBS, ultrasonically crushing, adding PMSF to enable the final concentration to be 1mM, and crushing the thalli until bacterial liquid is clear and transparent.

(2) Centrifuging to remove supernatant, adding 2mL of inclusion body dissolving solution (8M urea or 6M guanidine hydrochloride) per 100mg of precipitate, and centrifuging at high speed for 1h at 10000g to obtain supernatant.

(3)12000g of the dissolved inclusion bodies are centrifuged for 20-30 min, supernatant liquid is firstly coarsely filtered by a filter tip with the diameter of 0.45 mu m to prevent a large amount of impurities from blocking the filter tip, and then is finely filtered by the filter tip with the diameter of 0.22 mu m.

(4) The nickel column was equilibrated with 10 column volumes of equilibration buffer.

(5) Adding the filtered protein into nickel column, and allowing it to flow down naturally by gravity

(6) After the loading, the impurity-washing buffer solution with the volume 5 times that of the column is added, and the impurity protein bound on the nickel column is washed away.

(5) And after the impurity washing is finished, eluting the target protein by using imidazole with 2-3 times column volume gradient.

(6) And (3) washing the residual protein on the nickel column by using 500mM imidazole in 10 times of the column volume after elution, washing the nickel column by using an equilibrium buffer solution in 10 times of the column volume, and washing the nickel column by using deionized water in 10 times of the column volume.

(7) After the cleaning, 20% ethanol was added, the upper and lower ends of the nickel column were sealed, and the mixture was stored in a refrigerator at 4 ℃.

(8) mu.L of the eluate was added to 10. mu.L of 5 XSDS-PAGE protein loading buffer and boiled for 10 min. A10. mu.L sample was subjected to SDS-PAGE and identified by staining with Coomassie Brilliant blue.

Recombinant protein MBP-_TFFThe results of MBP-nsp10 and nsp10 purification are shown in FIG. 15.

6. Western Blot identification of recombinant proteins

(1) SDS-PAGE gels were prepared.

(2) mu.L of protein purification eluate was added to 10. mu.L of 5 XSDS-PAGE protein loading buffer and boiled for 10 min. Taking 10 mu L of sample to carry out SDS-PAGE, taking out SDS-PAGE gel, and soaking in freshly prepared 1 x membrane transfer liquid for 5-10 min.

(3) Appropriate PVDF membrane was cut according to the desired lane size and soaked in methanol for 2 minutes.

(4) A gasket, 3 layers of filter paper, PAGE glue, a PVDF membrane, 3 layers of filter paper and a gasket are sequentially placed on a black transfer template, and the black transfer template is covered and carefully pressed. The current is set to 300mA during membrane transfer, and the membrane transfer time is set for 1min per 1kDa membrane transfer according to the size of the membrane transfer protein.

(5) After the mold transfer was completed, the PVDF membrane was removed, sealed with 5% skimmed milk powder at 37 ℃ for 1 hour, and rinsed twice with 1 × TBST on a shaker for 10min each time.

(6) PVDF membrane was incubated in primary antibody (6 XHis-Tag murine antibody or MBP murine antibody) overnight at 4 ℃. The following day, rinse 3 times with 1 × TBST on a shaker for 10min each time.

(7) The secondary antibody goat anti-mouse-HRP was diluted 1:5000 with 1 XPBS, and PVDF membrane soaked in the antibody was incubated at low shaker speed for 2h at room temperature, and rinsed 3 times with 1 XPTBST for 10min each.

(8) The chemiluminescent solution was prepared at 1:1, and the Western Blot bands were analyzed on a gel imaging system.

MBP-_TFFProtein, identification of MBP using MBP-tagged antibody as a primary antibody_TFFExpression of (2). The results are shown in fig. 16, and show that: lane1 was no specific band before induction, Lane2 was after induction, Lane3 was NI column purified protein, and there were distinct specific bands after induction and after purification.

Third, recombinant protein polyclonal antibody titer determination and specificity analysis

1. Animal immunization

(1) Adding a quantitative antigen (recombinant protein) and Freund's complete adjuvant 1:1 into the mixture for full emulsification, uniformly mixing the mixture for 20-30 min by using a rotary mixing machine until the liquid is dripped on the water surface and does not disperse, thus obtaining stable emulsion, and inoculating 4 Kunming female mice with the age of 6-8 weeks by subcutaneous multipoint injection or intraperitoneal injection at the dose of 50 mu g per mouse.

(2) After 7d, adding the antigen (recombinant protein) and Freund's incomplete adjuvant 1:1, uniformly mixing by a uniform speed mixer until the mixed solution is dropped into water and is not dispersed, namely fully emulsifying, and performing boosting immunization in the same immunization mode as the primary immunization and half dosage of the primary immunization.

(3) And in the third week of first immunization, cutting off the tail of the mouse by 0.2-0.3 cm by using sterile scissors or a razor blade to collect blood. 100-200 mu L of blood is collected and filled in a 1.5mL microcentrifuge tube. The tail of the mouse is placed in warm water at 37 ℃ for 2-3 min, or the tail of the mouse is rubbed with the outside of the thumb and the forefinger for 1-2 min, so that the tail of the mouse is congested and blood is conveniently collected.

(4) The first drop of blood is discarded during blood collection, 200-400 uL of blood is taken from each mouse, the blood is placed for 1h at 37 ℃, and then the blood is centrifuged at low speed for 15-20 min.

(5) And (4) slightly sucking the centrifuged upper serum out, and storing at-20 ℃ for 1-3 months. Thereafter, blood was collected at intervals of 7d to isolate serum, and the serum antibody titer was determined by ELISA.

TABLE 2 immunization protocols for different immunogens

Immunogens	Number of immunizations	The week of the year	Number of mice	Immunization regimen	Amount of immunity (μ g)
						MBP-nsp10	3	6～8	4	Under the skin	50
MBP-TFF	3	6～8	4	Under the skin	50
						PBS	3	6～8	4	Under the skin	50
nsp10	3	6～8	4	Abdominal cavity	50
						PBS	3	6～8	4	Abdominal cavity	50

2. Indirect ELISA for detecting antibody titer

(1) And (3) measuring the antigen concentration by BSA, diluting the coating solution to 1-10 mu g/mL, adding 100 mu L of the coating solution into each hole of the ELISA plate, and standing overnight at 4 ℃.

(2) The following day was washed 3 times with 1 × TBST, 3min each time.

(3) Add blocking solution and incubate at 37 ℃ for 1 h. And (6) washing.

(4) A diluted serum sample (300. mu.L) was added, the antibody titer was measured by gradient dilution, and the plate was exposed to an enzyme at 37 ℃ for 2 hours. The washing was 1 × TBST, as many times as possible, to prevent affecting binding to enzymatically-linked secondary antibodies in serum.

(5)1, 50001 XPBS or 1 XPSS diluted goat anti-mouse-HRP, 100 mu L of enzyme label plate is added into each hole, and the mixture is placed at room temperature for 2 hours. Washing with 1 × TBST for 6-8 times, 3min each time

(6) Quickly adding 100 mu LTMB substrate reaction liquid into each hole of the ELISA plate by using a discharging gun, slightly beating the ELISA plate to mix evenly when necessary, and developing for 15 minutes at room temperature in a dark place.

(7) The reaction was stopped with 2M sulfuric acid, 100. mu.L per well.

(8) And detecting the OD value of the mouse serum antibody by using a microplate reader.

The recombinant protein nsp10 coats the ELISA plate, serum immunized with the recombinant protein MBP-nsp10 is used as a primary anti-incubation ELISA plate, goat anti-mouse-HRP (horse radish peroxidase) is used as a secondary anti-incubation, the antibody titer for detecting the nsp10 protein is 1:42000 (figure 17A), the single nsp10 protein coats the plate, serum immunized with the recombinant protein nsp10 is used as a primary anti-incubation ELISA plate, and the antibody titer for detecting the nsp10 protein is 1:13000 (figure 17C). FIG. 17B is MBP-_TFFThe protein alone immunizes the mice to generate immune effect. From the results shown in FIG. 17, the immunogenicity of the recombinant protein nsp10 was significantly increased in the presence of MBP protein, i.e., the MBP protein plays a role in enhancing immunity during the fusion expression with nsp10, enhancing the immune effect of nsp10 protein, but simultaneously MBP alone_TFFImmunization of mice with proteins also results in the production of antibodies in the mice. ELISA results show that the recombinant proteins nsp10 and MBP-nsp10 have good immunogenicity.

3. Western Blot antibody specificity detection

(1) SDS-PAGE gels were prepared.

(2) Sampling a bacterial liquid, a purified Protein and a Protein Marker, running SDS-PAGE gel, concentrating the gel at 80V, and separating the gel at 120V. (3) Taking out SDS-PAGE gel, and transferring the SDS-PAGE gel to a mold.

(4) The PVDF membrane is taken out and sealed by 5 percent of skimmed milk powder for 1 hour.

(5) PVDF membrane incubation antibody: a primary antibody of mouse serum and a secondary antibody of goat anti-mouse-HRP; or the pig serum is a primary antibody, and the sheep anti-pig-HRP secondary antibody.

(6) PVDF membrane was rinsed 3 times in 1 XTSST and 10min each time on a shaker.

(7) High sensitivity chemiluminescence liquid color development.

The detection result shows that the antibodies in the serum can be specifically combined with the corresponding recombinant proteins, as shown in FIG. 18, no specific band can be seen in FIG. 18 before induction, specific bands can be seen after induction and purified proteins, an obvious band can be seen in the specific combination of the mouse positive serum, and no obvious band can be seen in the mouse negative serum, which indicates that the mouse generates specific antibodies aiming at antigens MBP-nsp10 and nsp 10. And the nsp10 recombinant protein can be specifically combined with the porcine positive serum infected with PEDV, has an obvious strip and has no obvious reaction with the porcine negative serum, which indicates that the recombinant protein can interact with the porcine positive serum infected with the natural PEDV. By MBP-_TFFAgain, the specificity of the sera was identified, again with a clear band for the mouse positive sera, while the negative sera were not and could not bind to PEDV infected porcine sera, indicating that the mice produced both MBP-nsp10 and MBP-_TFFThe specific antibody of (1).

Fourth, recombinant protein induced cell factor

1. Detection of TNF-alpha expression difference in mouse spleen by sandwich ELLISA method

1.1 obtaining lymphocytes

(1) All the tools are sterilized, the experiment is carried out on a clean bench, the experiment at room temperature is removed, and other operations are carried out at low temperature;

(2) taking the spleen of a nsp10 protein immunized mouse, putting the spleen into Hanks/PBS solution (added with 2% cell culture solution), grinding the spleen by using a frosted surface of a glass slide to obtain a mixed solution containing lymphocytes and blood cells, and centrifuging at 1500rmp for 4-5 min to obtain a precipitate which is scraped to be loosened by force;

(3) adding 3-5 mLRBC NH into the precipitate at room temperature₄Suspending the cells in Cl Buffer for 3-5 min, adding 5-10 mL of 2% cell culture solution for cleaning, and centrifuging to remove the supernatant; putting the suspension cells into a culture bottle for culturing at 37 ℃ for incubation for 1-2 h;

(4) 20 mug, 40 mug and 60 mug of purified target protein are added into a culture medium to stimulate lymphocytes in vitro to keep the activity for 2-3 days, and culture solution is collected in centrifugation for 24h, 48h and 72h respectively to carry out sandwich ELISA experiments. The control group is PBS group lymphocyte culture solution.

1.2 Sandwich ELISA

(1) Selecting TNF-alpha rabbit polyclonal antibody as a capture antibody, diluting the capture antibody to the concentration of 1-10 mu g/mL by using a coating solution, and incubating overnight at 4 ℃.

(2) And (3) turning the ELISA plate over on a paper towel the next day, pouring the coating solution, washing for 5-6 times by 1 × TBST, and slightly beating the ELISA plate for 3min each time.

(3) 5% skimmed milk powder is added into the ELISA plate, 100 μ L of the skimmed milk powder is added into each well, and the microplate is sealed at 37 ℃ for 1h or incubated overnight at 4 ℃ in a refrigerator.

(4) Washing is carried out 6-8 times by 1 xTBST, each time is 3min, and the volume of the washing liquid is higher than that of the added sample so as to remove the redundant confining liquid.

(5) The test sample, mouse serum, was added to the microplate in an amount of 100. mu.L per well and incubated at 37 ℃ for 90 minutes or at 4 ℃ overnight.

(6) The sample was discarded and washed as above.

(7) mu.L of diluted murine monoclonal antibody TNF-alpha was added to each well as the detection antibody and incubated for 2 hours at room temperature.

(8) The microplate was inverted on a dry paper towel, and the incubated antibody in the wells drained, the sample discarded, and washed with 1 × TBST as above.

(9) Adding 100 mu L of detection antibody goat anti-mouse-HRP, and incubating for 2-4 h at room temperature.

(10) The 1 × TBST wash was as above.

(11) Adding 100 mu L of TMB color development liquid into each hole, reacting for 15-30 minutes in a dark place at room temperature, and reacting for 2M H₂SO₄The reaction is stopped by the stop solution, and the OD value of 450nm is measured by an enzyme-linked immunosorbent assay.

1.3 detection of IL-4 expression differences in mouse serum by Sandwich ELLISA

ELISA procedure was as above.

The results are shown in FIG. 19, and the expression level of TNF-alpha in the mouse lymphocyte culture solution after the recombinant protein nsp10 is immunized is obviously higher than that in the PBS immunized group.

1.4 detection of mouse lymphocytes IL-2, IL-4, IL-10, TNF-alpha, IFN-gamma cytokines

1.5 RNA extraction as above

Extracting by using a Trizol reagent one-step method: RNA was extracted from spleen lymphocytes of mice immunized with the recombinant protein nsp10, the RNA concentration was measured, and cDNA was synthesized by reversing the procedure of the kit. Reverse transcription of cDNA, reverse transcription system:

name of reagent	Volume of
		SYBR Premix Ex TaqT M(2×)	10.0μL
ROX refer-ence dyeⅡ(10×)	0.4μL
		cDNA	2.0μL
Upstream primer (10. mu. moL/L)	0.4μL
		Downstream primer (10. mu. moL/L)	0.4μL
RNasefreed H2O	Up to 20μL

The reverse transcription reaction system is as follows:

1.6 primer design

Primers for amplifying 5 cytokines, IL-2, IL-4, IL-10, TNF-alpha and IFN-gamma, from mice in PrimerBank were cited. The accession number and the length of the amplified fragment of the amplification primer are shown in the table 3, and the internal reference is a murine GAPDH gene. The data were analyzed using the following formula: 2^-ΔΔCT(Δ CT is the Δ CT value of the target gene-reference Δ CT value).

The amplification reaction system is as follows:

TABLE 3 amplification primers for cytokines

The result of IL-4 expression in serum of mice detected by Real-timePCR is shown in FIG. 20, and the expression level of IL-4 in the nsp10 immune group is obviously higher than that in the control group.

The results of Real-time PCR detection of the changes in the expression levels of IL-2, IL-4, IL-10, TNF-alpha and IFN-gamma in the spleen of the mice are shown in FIG. 21, and compared with the PBS group, the expression levels of IL-2, IL-4, IL-10, TNF-alpha and IFN-gamma in the recombinant protein nsp10 immune group are obviously increased. This is consistent with previous validation at the protein level, suggesting that the recombinant protein induces an immune protective mechanism in mice.

Fifth, recombinant protein MBP-nsp10 ELISA and commercial ELISA kit contrast

The MBP-Nsp10 antigen-coated ELISA assay plate was compared to a commercial PEDV-Ab ELISA assay kit (available from Shanghai, preferably Biotech, Inc.). Wherein the pig serum sample is collected in a pig farm in Jiangxi province in China.

30 porcine PEDV antibody positive or negative serum samples from the farm were randomly pooled (sample number n 30) and ELISA tests were performed using an ELISA test plate coated with MBP-Nsp10 (FIG. 22 left) and a commercial kit antigen (FIG. 22 right). Each point in the graph represents a sample, the solid line represents the cut-off value, which is 0.2, OD, as determined by the kit instructions and experimental results₄₅₀A value higher than 0.2 is judged to be positive. The results showed that the pig serum samples showed different reactions with MBP-Nsp10 as antigen and the antigen coated in the commercial kit, and the samples with differences were indicated by arrows. By comparison, the detection result of 29 samples of 30 samples of MBP-Nsp10 as antigen is consistent with that of the commercial kit, and the coincidence rate is 96.67%. The result of the ELISA method using the recombinant MBP-Nsp10 as the coating antigen is stable and reliable, and the ELISA method can be used for detecting clinical samples.

The above-described embodiments are merely preferred embodiments of the present invention, and not intended to limit the scope of the invention, so that equivalent changes or modifications in the structure, features and principles described in the present invention should be included in the claims of the present invention.

SEQUENCE LISTING

<110> university of agriculture in Jiangxi

<120> porcine epidemic diarrhea virus Nsp10 protein, fusion protein containing Nsp10 protein, preparation method and application thereof

By using

<130> do not

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 128

<212> PRT

<213> porcine epidemic diarrhea virus

<400> 1

Met Ile Asn Ser Ser Leu Leu Thr Leu Cys Ala Phe Ala Val Asp Pro

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Ala Lys Thr Tyr Ile Asp Ala Val Lys Ser Gly His Lys Pro Val Gly

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Asn Cys Val Lys Met Leu Ala Asn Gly Ser Gly Asn Gly Gln Ala Val

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Thr Asn Gly Val Glu Ala Ser Thr Asn Gln Asp Ser Tyr Gly Gly Ala

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Ser Val Cys Leu Tyr Cys Arg Ala His Val Glu His Pro Ser Met Asp

65 70 75 80

Gly Phe Cys Arg Leu Lys Gly Lys Tyr Val Gln Val Pro Leu Gly Thr

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Val Asp Pro Ile Arg Phe Val Leu Glu Asn Asp Val Cys Lys Val Cys

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Gly Cys Trp Leu Ala Asn Gly Cys Thr Cys Asp Arg Ser Ile Met Gln

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<210> 2

<211> 671

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys

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Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr

20 25 30

Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe

35 40 45

Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala

50 55 60

His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile

65 70 75 80

Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp

85 90 95

Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu

100 105 110

Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys

115 120 125

Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly

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Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro

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Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys

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Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly

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Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp

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Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala

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Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys

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Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser

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Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro

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Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp

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Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala

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Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala

305 310 315 320

Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln

325 330 335

Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala

340 345 350

Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn

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Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly Ile

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Glu Gly Arg Ile Ser Glu Phe Met Arg Asn Val Leu Leu Val Gly Ser

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Phe Leu Thr Phe Phe Trp Ser Glu Leu Val Ser Tyr Thr Lys Phe Phe

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Trp Val Asn Pro Gly Tyr Val Thr Pro Met Phe Ala Cys Leu Ser Leu

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Leu Ser Ser Leu Leu Met Phe Thr Leu Lys His Lys Thr Leu Phe Phe

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Gln Val Phe Leu Ile Pro Ala Leu Ile Val Thr Ser Cys Ile Asn Leu

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Ala Phe Asp Val Glu Val Tyr Asn Tyr Leu Ala Glu His Phe Asp Tyr

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His Val Ser Leu Met Gly Phe Asn Ala Gln Gly Leu Val Asn Ile Phe

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Val Cys Phe Val Val Thr Ile Leu His Gly Thr Tyr Thr Trp Arg Phe

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Phe Asn Thr Thr Val Ser Ser Val Thr Tyr Val Val Ala Leu Leu Thr

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Ala Ala Tyr Asn Tyr Phe Tyr Ala Ser Asp Ile Leu Ser Cys Ala Met

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Thr Leu Phe Ala Ser Val Thr Gly Asn Trp Phe Val Gly Ala Val Cys

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Tyr Lys Ala Ala Val Tyr Met Ala Leu Arg Phe Pro Thr Phe Val Ala

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Ile Phe Gly Asp Ile Lys Ser Val Met Phe Cys Tyr Leu Val Leu Gly

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Tyr Phe Thr Cys Cys Phe Tyr Gly Ile Leu Tyr Trp Phe Asn Arg Phe

595 600 605

Phe Glu Val Gly Val Gly Val Tyr Asp Tyr Thr Val Ser Ala Ala Glu

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Phe Lys Tyr Met Ala Ala Asn Gly Leu Arg Ala Pro Thr Gly Thr Leu

625 630 635 640

Asp Ser Leu Leu Leu Ser Ala Lys Leu Ile Gly Ile Gly Gly Glu Arg

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Asn Ile Lys Ile Ser Ser Val Gln Ser Lys Leu Thr Asp Ile Lys

660 665 670