Primer group and application thereof in construction of recombinant baculovirus-like particles of G5 type porcine rotavirus

文档序号：842700 发布日期：2021-04-02 浏览：17次中文

阅读说明：本技术 引物组及其在构建g5型猪轮状病毒的重组杆状病毒样颗粒中的应用 (Primer group and application thereof in construction of recombinant baculovirus-like particles of G5 type porcine rotavirus ) 是由杨鑫张兰付雪李豪王红宁于 2020-12-23 设计创作，主要内容包括：本发明公开了一种引物组及其在构建G5型猪轮状病毒的重组杆状病毒样颗粒中的应用,其中第一引物的上游引物的核苷酸序列如SEQ ID NO:1所示,第一引物的下游引物的核苷酸序列如SEQ ID NO:2所示,第二引物的上游引物的核苷酸序列如SEQ ID NO:3所示,第二引物的下游引物的核苷酸序列如SEQ ID NO:4所示；第三引物的上游引物的核苷酸序列如SEQ ID NO:5所示,第三引物的下游引物的核苷酸序列如SEQ ID NO:6所示；集中于同一病毒样颗粒中可表达式可以同时表达,具有多亚基蛋白结构,且其保留了自组装的能力,没有遗传物质,但模仿了天然病毒颗粒的整体结构且没有传染性。(The invention discloses a primer group and application thereof in constructing recombinant baculovirus-like particles of G5 type porcine rotavirus, wherein the nucleotide sequence of an upstream primer of a first primer is shown as SEQ ID NO. 1, the nucleotide sequence of a downstream primer of the first primer is shown as SEQ ID NO. 2, the nucleotide sequence of an upstream primer of a second primer is shown as SEQ ID NO. 3, and the nucleotide sequence of a downstream primer of the second primer is shown as SEQ ID NO. 4; the nucleotide sequence of the upstream primer of the third primer is shown as SEQ ID NO. 5, and the nucleotide sequence of the downstream primer of the third primer is shown as SEQ ID NO. 6; the expressible bodies, which are concentrated in the same virus-like particle, can be expressed simultaneously, have a multi-subunit protein structure, and retain the ability to self-assemble, are free of genetic material, but mimic the overall structure of a native virus particle and are not infectious.)

1. A primer set comprising a first primer, a second primer and a third primer;

wherein the nucleotide sequence of the upstream primer of the first primer is shown as SEQ ID NO. 1, and the nucleotide sequence of the downstream primer of the first primer is shown as SEQ ID NO. 2;

the nucleotide sequence of the upstream primer of the second primer is shown as SEQ ID NO. 3, and the nucleotide sequence of the downstream primer of the second primer is shown as SEQ ID NO. 4;

the nucleotide sequence of the upstream primer of the third primer is shown as SEQ ID NO. 5, and the nucleotide sequence of the downstream primer of the third primer is shown as SEQ ID NO. 6.

2. The use of the primer set of claim 1 in the construction of VP2 gene, VP6 gene and VP7 gene of porcine rotavirus G5.

3. The use of claim 2, wherein the specific steps for constructing the gene VP2, the gene VP6 and the gene VP7 are as follows:

s1: extracting RNA from the attenuated vaccine;

s2: carrying out PCR amplification reaction on the RNA;

s3: the genes for VP2, VP6, and VP7 were obtained by purification.

4. The use of the VP2 gene, VP6 gene and VP7 gene constructed in the application of claim 3 for constructing recombinant baculovirus-like particles of porcine rotavirus G5 containing target proteins expressed by VP2, VP6 and VP 7.

5. The use of claim 4, wherein the recombinant baculovirus-like particle comprising the gene VP2, the gene VP6 and the gene VP7 is constructed by the following steps:

h1: connecting the genes of VP2, VP6 and VP7 with a vector, and transforming to obtain respective recombinant plasmids;

h2: purifying the recombinant plasmid;

h3: carrying out enzyme digestion reaction on the recombinant plasmid to obtain respective gene fragments of VP2, VP6 and VP 7;

h4: the gene fragments of VP2, VP6, and VP7, respectively, were ligated to a vector with the same cohesive ends and transformed into a first competent bacterium;

h5: the identification result of the first competent bacterium is positive, and the first competent bacterium is the target recombinant plasmid of VP2, VP6 and VP 7;

h6: transforming target recombinant plasmids of VP2, VP6 and VP7 into second competent bacteria, and extracting respective recombinant plasmids which are positive by PCR;

h7: transfecting the cells in the logarithmic growth phase with the respective recombinant plasmids identified as positive to obtain respective first-generation recombinant baculovirus-like particles;

h8: carrying out subculture on the respective first-generation recombinant baculovirus-like particles, transfecting cells in logarithmic growth phase after the subculture is finished, carrying out subculture to the third generation, and collecting respective expression to obtain third-generation recombinant baculovirus-like particles;

h9: the third generation recombinant baculovirus-like particles obtained by respective expression are used for co-infecting cells in logarithmic phase, and the recombinant baculovirus-like particles simultaneously containing the VP2 gene, the VP6 gene and the VP7 gene are obtained.

6. The use of claim 5, wherein the second competent bacterium comprises a baculovirus shuttle vector and a helper plasmid having genes encoding transposase and tetracycline resistance.

7. The use according to claim 5, wherein the first competent bacterium is a DH5 a competent bacterium.

8. The use of the recombinant baculovirus-like particle of porcine rotavirus G5 containing target proteins expressed by VP2, VP6 and VP7 constructed in the use of claim 5 in preventing non-bacterial diarrhea.

9. The use of the recombinant baculovirus-like particle of porcine rotavirus G5 containing target proteins expressed by VP2, VP6 and VP7 constructed in the use of claim 5 in cell interaction research.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a primer group, which is applied to construction of a recombinant baculovirus-like particle simultaneously containing target proteins expressed by VP2, VP6 and VP7, wherein the expression of the recombinant baculovirus-like particle is based on a Bac-to-Bac baculovirus expression system.

Background

Porcine Rotavirus (Rotavirus RV) belongs to Reoviridae (Reoviridae) and Rotavirus (Rotavirus) members, which are non-enveloped icosahedral particles with a diameter of about 66-75nm and a genome size of 18,522bp, and consist of 11 segmented double-stranded rnas (dsrnas) and encode 6 structural proteins (VP1-VP4, VP6, VP7) and 5 or 6 non-structural proteins (NSP1-NSP5/NSP 6). Rotaviruses (RVs) are classified into 9 different serotypes (RVA-RVJ) based on the antigenicity of the VP6 protein, where RVB, RVC, RVE, RVH and RVI can infect various mammals, RVA, RVB, RVC, RVE and RVH can infect pigs, RVA, RVB, RVC, RVH can infect humans, and RVD, RVF and RVG can infect poultry, such as chickens and turkeys. Rotaviruses can be classified into different G and P genotypes, based on the two outer capsid proteins VP7 and VP4 containing neutralizing epitopes, 35G and 50P genotypes have been reported worldwide. RCWG recommends that rotavirus be named in the manner RV group/origin/isolation identified region/name/isolation identified time/G type P, based on different serotypes and VP7, VP4 genotypes.

The rotavirus is one of the important pathogens causing acute non-bacterial diarrhea of infants and young animals. The rotavirus infection host has wide range, including human, pig, cow, sheep, rabbit, dog, various livestock and poultry, etc. The porcine rotavirus can cause nonbacterial diarrhea in infants and young animals of various species and lead to higher mortality. Acute diarrhea and death from rotavirus in infants under 5 years of age account for over 50% of cases of pediatric intestinal infections. The data indicate that in developing countries, an average of 527000 children die each year from rotavirus infection. The hospitalization rate of children with diarrhea caused by rotavirus infection in China is up to 13 percent in each year. Causing serious harm to public health service in China. Piglets are prevalent within 8 weeks of age and porcine rotavirus infections are clearly seasonal in more than 12 months per year to 1-2 months next year, and outbreaks of the disease are also reported in summer. And pigs of various ages and sexes can be infected with rotavirus. Under the condition of no maternal antibody protection, the mortality rate of piglets within one week of age after infection with rotavirus is up to 100 percent, and the symptoms of the pigs infected with rotavirus are relieved along with the increase of age. The 20-day-old suckling piglets have less symptoms and lower death rate after being infected with rotavirus, and are always in a resistant state or invisibly infected. Piglet rotavirus diarrhea is an infectious disease characterized by diarrhea, anorexia, vomiting, dehydration, often outbreaks in two stages, the first, it is likely to occur in the suckling period, especially piglets produced by replacement sows lacking maternal antibodies. Secondly, rotavirus often causes diarrhea in weaned piglets, because the weaning causes the sudden loss of maternal antibodies and the resistance of the piglets is reduced to cause infection.

At least 11 different types of G were identified by the RVA strain, of which type G5 (45.8%) is the most prevalent genotype at present, followed by type G3 (11.2%) and type G4 (9.6%), the most prevalent of the 17 different types P being type P7 (47.4%), followed by type P6 (15.9%) and type P13 (3.2%), and the most prevalent of the 47 combinations of different types G and P being type G5P (37.4%) (Papp et al 2013). Of the 9 different rotaviral subtypes, RVA type G5 is considered to be the most prevalent and prevalent strain.

The main applications at present are traditional inactivated seedlings and attenuated seedlings. The inactivated vaccine has simple manufacturing process, stable property and easy storage and transportation. However, inactivated vaccines may damage or change the effective antigenic determinant during inactivation, and thus may require a large amount of antigen at a relatively high cost. The attenuated live vaccine can be added with value in the bodies of piglets and has better protection, but the attenuated live vaccine has potential risk of virulence reversion in the bodies of animals and can form potential infection or spread. The instability of the conventional vaccines makes it urgently necessary to develop novel vaccines and explore new immunological mechanisms to prevent and control the onset of RV infection.

Disclosure of Invention

Aiming at the problem that the traditional inactivated vaccine can damage or change effective antigenic determinants in the inactivation process, the antigen quantity is large and the cost is high; the attenuated vaccine has potential risk of virulence reversion in animals, and may form potential infection or spread problems.

The technical scheme of the invention is as follows: a primer set comprising a first primer, a second primer and a third primer;

wherein the nucleotide sequence of the upstream primer of the first primer is as follows: TCTAGAATGGCGTACAGGAAGCGYGG, the nucleotide sequence of the downstream primer of the first primer is: AAGCTTTTACAGTTCGTTCATDATGCGC, respectively;

the nucleotide sequence of the upstream primer of the second primer is as follows: TCTAGAATGGAGGTTCTGTACTCATTG, the nucleotide sequence of the downstream primer of the second primer is: AAGCTTTCACTTAATC AACATGCTTCTA, respectively;

the nucleotide sequence of the upstream primer of the third primer is as follows: TCTAGAATGTATGGTATTGAATATACCA, the nucleotide sequence of the downstream primer of the third primer is: AAGCTTCTAAACTCGRTARTARAATGC are provided.

Compared with the prior art, the invention has the beneficial effects that: the primer group has good specificity, so that the constructed VP2 gene, VP6 gene and VP7 gene can be accurately expressed to obtain corresponding target protein; the primer is used for the correct construction of the early plasmid, has certain influence on the successful expression of the later plasmid, and is the basis for the successful expression of the protein of the later shuttle plasmid infected cell.

The invention also provides application of the primer group in construction of a VP2 gene, a VP6 gene and a VP7 gene.

Further limiting, the specific steps for constructing the VP2 gene, the VP6 gene and the VP7 gene are as follows:

s1: extracting RNA from the attenuated vaccine;

s2: carrying out PCR amplification reaction on the RNA;

s3: the genes for VP2, VP6, and VP7 were obtained by purification.

The invention also provides application of the constructed VP2 gene, VP6 gene and VP7 gene in construction of recombinant baculovirus-like particles simultaneously containing VP2 gene, VP6 gene and VP7 gene.

Further limiting, the application of the recombinant baculovirus-like particle containing target proteins expressed by VP2, VP6 and VP7 is constructed, and the specific steps are as follows:

h1: connecting the genes of VP2, VP6 and VP7 with a vector, and transforming to obtain respective recombinant plasmids;

h2: purifying the recombinant plasmid;

h3: carrying out enzyme digestion reaction on the recombinant plasmid to obtain respective gene fragments of VP2, VP6 and VP 7;

h4: the gene fragments of VP2, VP6, and VP7, respectively, were ligated to a vector with the same cohesive ends and transformed into a first competent bacterium;

h5: the identification result of the first competent bacterium is positive, and the first competent bacterium is the target recombinant plasmid of VP2, VP6 and VP 7;

h6: transforming target recombinant plasmids of VP2, VP6 and VP7 into second competent bacteria, and extracting respective recombinant plasmids which are positive by PCR;

Further defined, the second competent bacterium comprises a baculovirus shuttle vector and a helper plasmid having genes encoding transposase and tetracycline resistance.

Further defined, the first competent bacterium is a DH5 a competent bacterium.

The invention also discloses application of the constructed recombinant baculovirus-like particles containing target proteins expressed by the VP2 gene, the VP6 gene and the VP7 gene in prevention of nonbacterial diarrhea.

The invention also discloses application of the constructed recombinant baculovirus-like particle containing the target protein expressed by the VP2 gene, the VP6 gene and the VP7 gene in cell interaction research.

Compared with the prior art, the invention has the beneficial effects that: the recombinant baculovirus-like particle containing target proteins expressed by the VP2 gene, the VP6 gene and the VP7 gene simultaneously can express the three genes in the same virus-like particle at the same time, has a multi-subunit protein structure, replaces the traditional triple vaccine, retains the self-assembly capacity, has no genetic material, imitates the integral structure of natural virus particles and has no infectivity; recombinant baculovirus-like particles containing both the VP2 gene, VP6 gene, and VP7 gene range in size from 20 to 100nm, are optimal for uptake of nanoparticles by dendritic cells and subsequent T cell activation and provide a spatial structure for display of conformational epitopes, and can be used as a platform for presentation of foreign epitopes or targeting molecules on chimeric VLPs.

Drawings

FIG. 1 is a transmission electron microscope image of a purified and dyed recombinant baculovirus-like particle containing target proteins expressed by VP2 gene, VP6 gene and VP7 gene;

FIG. 2 is an electrophoretogram of a target protein expressed from the first-generation recombinant baculovirus-like particles of VP2, VP6, and VP7, respectively;

FIG. 3 is a diagram showing the formation of recombinant baculovirus-like particles containing the target protein expressed by VP2 gene, VP6 gene and VP7 gene together with the purified protein.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the embodiments of the present invention are described in detail and completely, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

Example 1

Acquisition of strains

Coli DH 5. alpha. competent cells were purchased from Dalibao Biopsis (Takara); pGEM-TEAsy (pT) vector System was purchased from Promega; pFastbac-1 plasmid, E.coli DH10Ba competent cells, Sf9 insect cells were purchased from Invitrogen.

Example 2

Construction of VP2, VP6, and VP7 genes

1. The porcine rotavirus attenuated vaccine developed by Kazakh beasts in the market is purchased, is G5 type, and is a main porcine rotavirus type popular in China. Then the attenuated vaccine is placed in an ultra-clean workbench to extract virus RNA, and the operation steps are as follows:

2. mixing 300 μ L of attenuated vaccine liquid with 700 μ L of 4 deg.C pre-cooled RNAlso Plus, and standing at room temperature for 5 min;

3. adding 200 μ L chloroform, shaking, mixing, standing at room temperature for 15min,12000r/min, and centrifuging at 4 deg.C for 10 min;

4. removing the precipitate, transferring the supernatant to a new sterile centrifuge tube, adding 700 μ L of 4 deg.C pre-cooled isopropanol, and standing at room temperature for 10 min;

5.7500r/min, centrifuging at 4 ℃ for 10 min;

6. discarding the supernatant, resuspending with 1ml of 75% ethanol, washing, precipitating, centrifuging at 12000r/min at 4 ℃ for 15 min;

7. after the supernatant is discarded, the centrifugal tube is opened and dried and precipitated in a super clean bench;

8. dissolving the precipitate with appropriate amount of RNase-free water, and storing at-20 deg.C for use.

cDNA was synthesized using the total viral RNA obtained in the previous step as a template according to the standard procedures of PrimeScript RT reagent kit reverse transcription kit of Takara, Inc., reaction system:

total RNA	8.4μL
		5×PrimeScript Buffer	2μL
Oligo dT Primer	0.5μL
		Random 6mers	0.5μL
PrimeScript RT Enzyme Mix1	0.5μL

After being uniformly mixed in a PCR tube, the reaction system is placed in a PCR instrument under the conditions that: 15min at 37 ℃; 85 ℃ for 5 s. The cDNA product was stored at-20 ℃ for future use.

The VP2, VP6, and VP7 genes were amplified using the primers in Table 1, respectively.

TABLE 1 primers for amplifying the VP2, VP6, and VP7 genes

Note: underlined bases in the table are restriction enzyme recognition sites;

the PCR reaction was carried out using Q5 Ultrafidelity DNA polymerase from NEB as follows:

Q5 High-Fidelity 2×Master Mix	25μL
		upstream primer (10. mu.M)	2.5μL
Downstream primer (10. mu.M)	2.5μL
		cDNA	2μL
Nuclease-Free H₂O	18μL

And (3) PCR reaction conditions: pre-denaturation at 98 ℃ for 30 s; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 60s, and circulating for 30 times; extension at 72 ℃ for 5 min.

After preliminary identification of 5. mu.L of each of the above PCR products by 1% agarose gel electrophoresis was confirmed to match the expected results, the objective DNA fragment was purified and recovered by using an Omega agarose gel DNA recovery kit. The operation steps are as follows: after the DNA is separated by gel electrophoresis, cutting a target strip from the gel, transferring the cut target strip into a 1.5mL sterile centrifuge tube, weighing, adding a solution PN with the mass being 3 times of the volume of the sample PN, placing the sample in a water bath at 50 ℃ for 10min, and waiting until agar is completely melted; transferring the solution into adsorption column CA2 (CA2 placed in a collecting tube), standing at room temperature for 2min, centrifuging at 12000r/min for 1min, discarding waste liquid, adding 600 μ L of rinsing liquid PW into CA2, centrifuging at 12000r/min for 1min, and repeatedly rinsing once; after the waste liquid is discarded, the CA2 is put back into the collecting pipe, centrifuged at 12000r/min for 2min, the residual PW is discarded, and the collecting pipe is placed at room temperature for 5 min; transferring CA2 into a sterile 1.5mL centrifuge tube, adding 20 μ L deionized water, standing at room temperature for 2min, centrifuging at 12000r/min for 2min, and collecting the solution containing the corresponding target fragment; storing at-20 deg.C for use.

The VP2, VP6 and VP7 genes were then ligated to pGEM-T Easy (pT) Vector, respectively, in the following reaction:

2×T₄ DNA Ligase Buffer	5μL
		target gene	3μL
T₄ DNA Ligase	1μL
		pGEM-T Easy	1μL

Reaction conditions are as follows: after mixing, the mixture was ligated overnight at 4 ℃.

And (3) conversion reaction: mu.L of the ligation product was added to 100. mu.L of DH 5. alpha. competent bacteria; mixing, ice-cooling for 30min, heat-shocking at 42 deg.C for 2min, and ice-cooling for 2 min; adding 900 μ L LB liquid culture medium without antibiotic, culturing at 37 deg.C for 45min at 120r/min in constant temperature shaking table, centrifuging for 2min at 3000r/min, discarding part of supernatant, and suspending the rest 100 μ L bacterial solution; uniformly mixing, coating on a 50 mu g/mL ampicillin resistant LB plate containing X-gal and IPTG, placing in a constant temperature biochemical incubator at 37 ℃, culturing for about 16h, and screening out positive clones by blue-white spots; inoculating into 50 μ g/mL ampicillin-resistant LB culture solution, and shaking at 37 deg.C for about 16h at 200 r/min.

Example 3

Construction of recombinant baculoviruses VP2, VP6 and VP7

Extracting plasmids of pFastbac-1 vector, pGEM-T-VP2, pGEM-T-VP6 and pGEM-T-VP 7: the method comprises the following steps: collecting bacterial liquid in a clean 1.5mL centrifuge tube, centrifuging for 1min at 12000r/min, discarding the supernatant, adding 250 mu L of solution P1 into the precipitate, oscillating, mixing uniformly, resuspending the precipitate, adding 250 mu L of solution P2, gently turning for 6-8 times to fully crack the thalli, adding 350 mu L of solution P3, mixing fully, and centrifuging for 10min at 12000 r/min; collecting supernatant in adsorption column CP3 (placing adsorption column in collection tube), centrifuging at 12000r/min for 1min, discarding waste liquid in the collection tube, adding 600 μ L rinsing liquid PW into CP3, centrifuging at 12000r/min for 1min, and repeatedly rinsing once; putting CP3 into a collecting tube, centrifuging at 12000r/min for 2min, removing residual PW, transferring CP3 into a clean 1.5mL centrifuge tube, dripping 50 μ L sterile deionized water into CP3, standing at room temperature for 2min, centrifuging at 12000r/min for 2min, and collecting plasmid solution. The plasmid was stored at-20 ℃ for further use.

The pFastbac-1 vector, pGEM-T-VP2, pGEM-T-VP6 and pGEM-T-VP7 were digested simultaneously with Xba I and Hind III, respectively, as follows:

pFastbac-1/pGEM-T-VP2/pGEM-T-VP6/pGEM-T-VP7	8μL
		EcoR I	0.5μL
Hind III	0.5μL
		10×CutSmart Buffer	1μL

the enzyme digestion reaction conditions are as follows: and (3) uniformly mixing the reaction system in a centrifuge tube, and incubating for 2h at 37 ℃ in a metal bath.

After the enzyme-digested gene fragment and vector fragment gel are recovered and purified, the excised VP2, VP6 and VP7 are respectively connected with pFastbac-1 vectors with the same cohesive ends and transformed into DH5 alpha competent bacteria. The bacteria identified as positive were designated: pFB-VP2, pFB-VP6, and pFB-VP 7.

pFB-VP2, pFB-VP6, and pFB-VP7 plasmids were extracted, respectively, and DH10Bac competent cells containing a baculovirus shuttle vector (Bacmid) and a Helper plasmid (Helper) having genes encoding transposase and tetracycline resistance were transformed. The plasmid identified as positive by PCR is extracted by the following specific method: first, equilibrate the filtration column: 2ml of EQ1 solution in a filtration column, which is free-settling under the action of gravity; and (3) collecting thalli: centrifuging 1.5mL of bacterial solution at 9000r/min for 10min, and removing supernatant; adding 400 mu L of R3 solution containing RNase A, re-suspending, mixing evenly and precipitating; adding 400 μ L lysate L7, mixing gently, optionally shaking with vortex, standing at room temperature, and reacting for 5 min; adding 400 mu L N3 solution, immediately turning upside down, slightly mixing, centrifuging at room temperature at 15000r/min for 20 min; transferring the supernatant into a filter column which is subjected to equilibrium treatment, naturally flowing down the liquid under the action of gravity, and washing twice by using 2.5mL of rinsing liquid PW; the operation thereafter is transferred to the clean bench. Putting the filter column into a clean 1.5mL centrifuge tube, adding 900 mu L of eluent E4, naturally settling along with gravity, and eluting DNA; adding 630. mu.L of isopropanol into the eluate, mixing, ice-cooling for 10min, centrifuging at 4 deg.C and 12000r/min for 30min, and carefully removing the supernatant; resuspending the DNA precipitate with 1mL of 70% ethanol, centrifuging at 4 ℃ and 15000r/min for 5min, and discarding the supernatant; the tube was opened in a clean bench, air dried naturally for 10min, resuspended in 20. mu.L pyrogen-free water, DNA solubilized for 10min, stored at-20 ℃ and designated rB-VP2, rB-VP6 and rB-VP7, respectively, for use.

Each recombinant shuttle plasmid constructed was: rB-VP2, rB-VP6 and rB-VP7 transfect Sf9 insect cells in the logarithmic growth phase respectively, and the specific operation method is as follows: inoculating Sf9 insect cells growing in logarithmic phase to a six-well plate by using an Unsupplemental Grace culture medium without antibiotics and serum, wherein each hole is 2ml, and the insect cells adhere to the wall for 15min at room temperature; preparation of DNA and liposome mixture: diluting 8 mu L of liposome Cellffectin II and 1 mu g of recombinant shuttle vector by using 100 mu L of Unsupplement Grace cell culture solution for transfection, gently mixing the prepared Cellffectin II and plasmid diluent uniformly (the volume is about 210 mu L), and incubating for 30min at room temperature; adding about 210. mu.L of the mixture of the above DNA and liposomes dropwise to the cells in a 6-well plate; meanwhile, the same amount of transfection medium containing only 8. mu.L of transfection reagent without DNA was used as a negative control in Sf9 insect cells, one well, to exclude interference with the test results due to the toxic effect of the liposomes themselves on the cells; culturing at 27 deg.C for 4 h; removing the Unsupplement Grace cell culture solution in the 6-well plate, adding 2mL of complete culture medium sf-900IISFM into each well, and placing at 27 ℃ for continuous culture; the growth state of the cells is observed under a microscope every day until the cells show obvious pathological symptoms such as enlargement, agglomeration, wall separation and even rupture. At this point, the cell culture mixture in the 6-well plate was harvested and centrifuged at 500r/min for 5min to remove cells and debris from the mixture. After discarding the pellet, the supernatant was collected and dispensed into a clean centrifuge tube as the first generation recombinant baculovirus (P1) and stored at-80 ℃. The recombinant viruses are named rBV-VP2, rBV-VP6 and rBV-VP7 respectively.

Example 4

Western blot identification of protein expression

The first generation recombinant baculovirus was transferred to the 3 rd generation, and Sf9 insect cells grown in logarithmic phase in T25 cell culture flasks were infected with 5 MOIs alone, and after 3d infection, culture supernatant and cell pellet were collected, respectively. Resuspending the cell sediment sample by PBS, carrying out ultrasonic disruption and freeze-thawing treatment at-80 ℃, centrifuging at 4 ℃ and 12,000r/min for 10min to remove impurities, and collecting the supernatant. Then, the cell culture supernatant sample and the treated cell sediment sample which are collected before are respectively mixed with 2 xSDS loading buffer solution evenly, and the mixture is subjected to boiling water bath for 10 min. And then removing residues in the sample by centrifugation to prevent impurities in the sample from influencing the subsequent electrophoresis test. Then, 12% SDS-PAGE was performed. And after the electrophoresis is finished, detecting the target protein by using a Western-blot test, wherein the primary antibody is a murine HIS (human immunodeficiency Virus) tag antibody, and the secondary antibody is a goat anti-mouse IgG antibody.

The Western-blot test comprises the following specific steps: and transferring the protein onto the NC membrane by a semi-dry transfer method, and correspondingly adjusting the current and the membrane transfer time according to the area of the NC membrane and the molecular weight of the protein in a constant current mode. Putting the transferred NC membrane into 5% skimmed milk, and sealing overnight at 4 ℃; taking the NC membrane out of the sealing solution, washing the NC membrane for 3 times and 10 min/time by using 1 XPBS; adding primary antibody diluted at a ratio of 1:500, and incubating for 2h at room temperature in a shaking table; washing with 1 × PBS for 4 times (3 min/time); then adding a secondary antibody diluted by 1:1000, and incubating for 1h in a shaking table at room temperature; washing with 1 × PBS for 5 times, 3 min/time; the results of the color development with DAB are shown in FIG. 1, and WB identifies the expression of the respective proteins of interest in Sf9 cells, as shown in FIG. 1, note: m is a protein Marker, and the molecular weight of the protein corresponding to the strip is marked on the left part of the graph 1; lanes 1 to 3 correspond to Baculovir-vp 2, Baculovir-vp 6 and Baculovir-vp 7 infected cell samples, respectively.

Example 5

Purification of recombinant baculovirus-like particles containing target protein expressed by VP2 gene, VP6 gene and VP7 gene

After infecting SF9 insect cells in logarithmic phase with rBV-VP2, rBV-VP6 and rBV-VP7 for 96 hours at 5MOI, they were centrifuged at 4000r/min at 4 ℃ for 20 minutes. Taking the supernatant at 80000r/min, ultracentrifuging at 4 ℃ for 1.5 hours, resuspending the precipitate with PBS, preparing 20%, 30%, 40% and 50% sucrose gradients, performing sucrose density gradient centrifugation, and slowly adding the precipitate sample dissolved in PBS to the uppermost layer of the sucrose density gradient; 80000r/min, 4 ℃, and ultracentrifuging for 4 h; slowly sucking out the obvious white band between 30 percent and 40 percent of sucrose density gradient by using a long needle; adding the sample into another ultracentrifuge tube filled with PBS, mixing uniformly, centrifuging at 80000r/min at 4 ℃ for 4h, discarding the supernatant, resuspending the VLPs precipitate with PBS, and storing at-80 ℃ for later use.

The morphology of the recombinant baculovirus-like particles containing the target protein expressed by the VP2 gene, the VP6 gene and the VP7 gene was observed by a transmission electron microscope (JEM-100CX, JEOL, Japan) after sucrose density gradient centrifugation purification. The specific method comprises the following steps: dripping 1 μ L VLPs sample onto 200 mesh copper net, and adsorbing for 5 min; absorbing the redundant sample by using absorbent paper, and negatively dyeing the sample for 1min by using 2% phosphotungstic acid (PTA); after air drying, the glass is observed under an electron microscope, and the result is shown in FIG. 2.

rBV-VP2, rBV-VP6 and rBV-VP7 protein expression

After being properly treated, the sample is subjected to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) by 10 percent of separation gel, then transferred to a PVDF (polyvinylidene fluoride) membrane to be subjected to Western-blot detection test, and the expression condition of target protein in the virus simultaneously containing the VP2 gene, the VP6 gene and the VP7 gene is detected. And (3) displaying a detection result: corresponding specific immunoblots were detected in samples of cells infected with three viruses, rBV-VP2, rBV-VP6 and rBV-VP 7.

After the purified recombinant baculovirus-like particles containing the target proteins expressed by the VP2 gene, the VP6 gene and the VP7 gene, the formation of VLPs was observed by transmission electron microscopy after negative phosphotungstic acid staining. The results show that a typical spherical virus-like particle is observed at an MOI of 5, as shown in fig. 3: the size and morphology of the virus-like particle is close to that of a natural IBV virion, the diameter is about 100nm, and a layer of obvious crown-like protrusion surrounds the left particle in FIG. 3.

The invention is not limited to the above alternative embodiments, and any other various forms of products can be obtained by anyone in the light of the present invention, but any changes in shape or structure thereof, which fall within the scope of the present invention as defined in the claims, fall within the scope of the present invention.

Sequence listing

<110> Sichuan university

<120> primer group and application thereof in construction of recombinant baculovirus-like particles of G5 type porcine rotavirus

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 26

<212> DNA