阅读说明：本技术 一种柔嫩艾美耳球虫重组多肽疫苗vkvq的制备及其在抗鸡球虫病中的应用方法 (Preparation method of Eimeria tenella recombinant polypeptide vaccine VKVQ and application method thereof in chicken coccidiosis resistance ) 是由 戚南山 孙铭飞 廖申权 吕敏娜 吴彩艳 李娟� 林栩慧 胡俊菁 蔡海明 肖文婉 于 2020-07-30 设计创作，主要内容包括：本发明公开了一种柔嫩艾美耳球虫重组多肽疫苗VKVQ的制备方法,制备方法包括：步骤一,VKVQ编码基因的克隆,根据鸡球虫基因序列设计上游引物SEQ ID NO:3和下游引物SEQ ID NO:4,进行PCR扩增、电泳得到一条约1200bp的片段,将该产物连接转化后得到序列全长为1245bp的VKVQ,其序列为SEQ ID NO:1以及后续相关步骤。该方法制备的疫苗通过免疫柔嫩艾美耳球虫重组多肽疫苗VKVQ真核表达质粒和原核表达蛋白,能有效控制鸡体感染柔嫩艾美耳球虫,大大降低养鸡场抗球虫药的使用量,有效控制鸡球虫病。(The invention discloses a preparation method of an Eimeria tenella recombinant polypeptide vaccine VKVQ, which comprises the following steps: cloning VKVQ coding genes, designing an upstream primer SEQ ID NO. 3 and a downstream primer SEQ ID NO. 4 according to a coccidium gene sequence, carrying out PCR amplification and electrophoresis to obtain a fragment of about 1200bp, connecting and converting the product to obtain VKVQ with a sequence total length of 1245bp, wherein the sequence is SEQ ID NO. 1, and carrying out subsequent related steps. The vaccine prepared by the method can effectively control chicken body infection with Eimeria tenella through immunizing VKVQ eukaryotic expression plasmid and prokaryotic expression protein of the Eimeria tenella recombinant polypeptide vaccine, greatly reduce the using amount of coccidiostat in chicken farms, and effectively control chicken coccidiosis.)

1. A preparation method of an Eimeria tenella recombinant polypeptide vaccine VKVQ is characterized in that: the preparation method comprises the following steps:

cloning VKVQ coding genes, designing an upstream primer SEQ ID NO. 3 and a downstream primer SEQ ID NO. 4 according to a coccidium gene sequence, carrying out PCR amplification and electrophoresis to obtain a fragment of about 1200bp, and connecting and converting the product to obtain VKVQ with the sequence total length of 1245bp and the sequence of SEQ ID NO. 1;

step two, preparing VKVQ eukaryotic plasmids, designing primer sequences aiming at target genes, obtaining PCR products through PCR amplification and electrophoresis, recovering target fragments and carrying out enzyme digestion reaction, constructing VKVQ recombinant eukaryotic expression plasmids through treatment of expression vectors and connection of the enzyme digestion vectors and the enzyme digestion fragments, identifying transformation clones through a connection product transformation and colony PCR method and carrying out sequencing verification of positive clones, and finally carrying out plasmid minification and expression verification of the VKVQ eukaryotic plasmids through a plasmid minipill kit;

and step three, preparing VKVQ protein, designing an upstream primer SEQ ID NO 7 and a downstream primer SEQ ID NO 8 aiming at a target gene, recovering a target fragment after enzyme digestion by using a DNA gel recovery kit through gene amplification, then carrying out treatment on an expression vector and connection of the enzyme digestion vector and the enzyme digestion fragment to construct VKVQ recombinant prokaryotic expression plasmid, identifying transformed clones by a ligation product transformation method and a colony PCR method, and finally carrying out plasmid miniextraction and induced expression of VKVQ in an expression bacterium by using a plasmid miniextraction kit.

2. An application method of Eimeria tenella recombinant polypeptide vaccine VKVQ in chicken coccidiosis resistance is characterized in that: the application method comprises the following steps:

step one, emulsifying VKVQ recombinant protein, namely, taking VKVQ recombinant protein obtained after purification in the step three of claim 1 and Freund's adjuvant (FCA) according to the ratio of 1: 1, repeatedly sucking by a No. 7 needle syringe until the mixture is dripped on water and does not diffuse within 5 min;

step two, intramuscular injection of the VKVQ eukaryotic plasmid in the step two of the claim 1 into the legs of the chicken or subcutaneous injection of the VKVQ recombinant protein in the step one into the chicken respectively for immunization application.

Technical Field

The invention relates to the technical field of veterinary biological products, in particular to a preparation method of an Eimeria tenella recombinant polypeptide vaccine VKVQ and an application method thereof in chicken coccidiosis resistance.

Background

Eimeria tenella (e.tenella) is a species of obligate intracellular parasitic apicomplexa that can cause coccidiosis in chickens that seriously jeopardize the production of intensive chicken farming. Under the condition of no preventive measures or failure of prevention (such as drug ineffectiveness caused by drug resistance problems), the morbidity of the chickens can reach 30-100%, and the mortality can reach 80%. The global economic loss caused by the coccidiosis of the chickens exceeds more than 30 billion dollars each year. At present, the prevention and control of chicken coccidiosis are still mainly implemented by technical methods of adding various anticoccidial drugs into feed for drug prevention and control and vaccine prevention and control of live oocyst vaccines. However, the wide and serious drug resistance of chicken coccidia and the potential virus-dispersing risk of live oocyst vaccine make the prevention and control of chicken coccidia face a serious challenge, and new anticoccidial drugs and vaccines are developed as problems to be solved urgently. However, the detailed interaction mechanism between coccidia and host cells is not systematically known so far, and the development of novel anticoccidial drugs and molecular vaccines is faced with great difficulty.

The Apical Membrane Antigen (AMA) is a highly conserved microwire secreted protein in the phylum apicomplexa, and can form a 'Moving Junction' together with the neck protein secreted by the rod-shaped body, so that the adhesion of the polypide and the host cell is completed together, and the Apical Membrane Antigen (AMA) is a key substance for assisting the polypide to enter the host cell. In the proteomics of Bromley et al, it was found that Eimeria tenella is more than one apical membrane antigen and occurs at different developmental stages, so it was possible that these proteins would exert similar effects at different invasive stages, respectively. In the course of cloning the cDNA of the E.tenella sporulated oocyst of the Guangdong strain, a plurality of genes similar to apical membrane antigen were obtained, and the functions of the genes were respectively studied, and it was found that one of the proteins (named VKVQ, hereinafter, the following parts are used) can effectively prevent chicken coccidiosis from infecting chicken. To date, however, there has been no other study on the immunoprotection of this protein against Eimeria.

Disclosure of Invention

The invention aims to provide a preparation method of an Eimeria tenella recombinant polypeptide vaccine VKVQ and an application method thereof in chicken coccidiosis resistance.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a preparation method of an Eimeria tenella recombinant polypeptide vaccine VKVQ comprises the following steps:

cloning VKVQ coding genes, designing an upstream primer SEQ ID NO. 3 and a downstream primer SEQ ID NO. 4 according to a coccidium gene sequence, carrying out PCR amplification and electrophoresis to obtain a fragment of about 1200bp, and connecting and converting the product to obtain VKVQ with the sequence total length of 1245bp and the sequence of SEQ ID NO. 1;

step two, preparing VKVQ eukaryotic plasmids, designing primer sequences aiming at target genes, obtaining PCR products through PCR amplification and electrophoresis, recovering target fragments and carrying out enzyme digestion reaction, constructing VKVQ recombinant eukaryotic expression plasmids through treatment of expression vectors and connection of the enzyme digestion vectors and the enzyme digestion fragments, identifying transformation clones through a connection product transformation and colony PCR method and carrying out sequencing verification of positive clones, and finally carrying out plasmid minification and expression verification of the VKVQ eukaryotic plasmids through a plasmid minipill kit;

and step three, preparing VKVQ protein, designing an upstream primer SEQ ID NO 7 and a downstream primer SEQ ID NO 8 aiming at a target gene, recovering a target fragment after enzyme digestion by using a DNA gel recovery kit through gene amplification, then carrying out treatment on an expression vector and connection of the enzyme digestion vector and the enzyme digestion fragment to construct VKVQ recombinant prokaryotic expression plasmid, identifying, transforming and cloning by a ligation product transformation and colony PCR method, and finally carrying out plasmid miniextraction and induced expression of VKVQ in an expression bacterium by using a plasmid miniextraction kit.

The application also discloses an application method of the Eimeria tenella recombinant polypeptide vaccine VKVQ in resisting chicken coccidiosis, which comprises the following steps:

step one, emulsifying VKVQ recombinant protein, namely, taking VKVQ recombinant protein obtained after purification in the step three of claim 1 and Freund's adjuvant (FCA) according to the ratio of 1: 1, repeatedly sucking by a No. 7 needle syringe until the mixture is dripped on water and does not diffuse within 5 min;

step two, intramuscular injection of the VKVQ eukaryotic plasmid in the step two of the claim 1 into the legs of the chicken or subcutaneous injection of the VKVQ recombinant protein in the step one into the chicken respectively for immunization application.

Compared with the prior art, the invention has the following beneficial effects:

according to the vaccine prepared by the method, through immunizing the Eimeria tenella recombinant polypeptide vaccine VKVQ eukaryotic expression plasmid and prokaryotic expression protein, the infection of Eimeria tenella on chicken bodies can be effectively controlled, the using amount of coccidiostat in chicken farms is greatly reduced, and the coccidiosis of chicken is effectively controlled.

Drawings

FIG. 1 is WB analysis of Eimeria tenella recombinant polypeptide vaccine Et-VKVQ eukaryotic expression plasmid expressed in DF-1 cells (in the figure, M. protein molecular mass standard; 1.DF-1 cell total protein; 2.pCDA3.1-Et-VKVQ transfected into the expression product in DF-1 cells; 3. prokaryotic expression positive control);

FIG. 2 is an SDS-PAGE (SDS-PAGE) electrophoretic analysis chart of an Eimeria tenella recombinant polypeptide vaccine Et-VKVQ expression product (in the figure, M. protein molecular mass standard; 1. non-induced bacterial liquid; 2.37 ℃ induced bacterial liquid; 3-4. purified recombinant expression protein);

FIG. 3 is a WB diagram of an E.tenella recombinant polypeptide vaccine Et-VKVQ expression product (in the figure, M. protein molecular mass standard; 1-2.pET30a-Et-VKVQ recombinant purified protein).

Detailed Description

In order to make the technical means, the creation characteristics, the achievement objects and the effects of the invention easy to understand, the invention is further described below with reference to the specific figure, and the molecular biology experimental techniques used in the following examples, including PCR amplification, plasmid extraction, plasmid transformation, DNA fragment ligation, enzyme digestion, gel electrophoresis, etc., all adopt conventional methods, which can be specifically referred to "molecular cloning experimental guidelines" (third edition) (Sambrook J, Russell DW, Janssen K, Argentine J. Huangpeyer, 2002, Beijing: scientific Press). And "modern molecular biology laboratory techniques" (edited by lou-saint-a-de, 2 nd edition, beijing, published by china cooperative medical university, 1999).

A preparation method of an Eimeria tenella recombinant polypeptide vaccine VKVQ comprises the following steps:

cloning of the Et-VKVQ-encoding Gene:

1.1 primer design

Gene sequences were annotated according to coccidia in chicken ETH _00017730 and were designed using Premier Primer 5.0 software, synthesized by ThermoFisher Scientific inc:

upstream primer Et-VKVQ-F:5 'GTCAAAGTTCAGCAGCAGCAGCAGC 3' (SEQ ID NO: 3);

the downstream primer Et-VKVQ-R:5 'CGGGAGGGCCTCCTCCGCCAAAGAA 3' (SEQ ID NO: 4).

1.2 Guangdong strain E.tenella oocysts

Stored in a biological research room parasitized by the animal health research institute of the academy of agricultural sciences of Guangdong province;

1.3 E. extraction of Tenella oocyst Total RNA

The method refers to the specification of HiPure Total RNA Plus Micro Kit of Beijing Meiji Biotechnology company.

1.4 RT-PCR amplification of Et-VKVQ encoding Gene sequences

Using the total RNA of E.tenella oocysts extracted in the above procedure as a template, cDNA was synthesized using the PrimeScript 1st Strand cDNA Synthesis Kit from Takara; taking cDNA as a template, Et-VKVQ-F and Et-VKVQ-R as primers, carrying out PCR amplification by TaKaRa LA TaqTM enzyme, and carrying out reaction conditions: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 2min, extension at 72 ℃ for 10min, and 35 cycles. The product was analyzed by 1% agarose gel electrophoresis, resulting in amplification of a fragment of about 1200bp (see FIG. 1). The PCR product was purified and recovered, and ligated to pMD18-T vector (Takara Co.) according to the kit instructions, and the ligation product was transformed into E.coli DH 5. alpha. competent cells to obtain E.coli DH 5. alpha. (pMD 18-T-Et-VKVQ). The positive bacteria are sent to Shanghai Yingjun biotechnology limited for sequencing, and the total length of an Et-VKVQ sequence is 1245bp, which is shown as SEQ ID NO. 1.

Preparation of Et-VKVQ eukaryotic plasmid:

2.1 primer design

Designing primer sequences aiming at the upstream and the downstream of a target gene:

upstream primer Et-VKVQ-EuF:5 'CTTGGTACCGAGCTCGGATCCGCCACCGTCAAAGTTCAGCAGCAGCAGCAGC 3' (SEQ ID NO: 5);

the downstream primer Et-VKVQ-EuR:5 'GGTTTAAACGGGCCCTCTAGACGGGAGGGCCTCCTCCGCCAAAGAA 3' (SEQ ID NO: 6).

2.2 Gene amplification

The PCR reaction system and the amplification conditions use pMD18-T-Et-VKVQ plasmid as a template, and PrimeSTAR high fidelity enzyme is used for amplifying a gene target fragment, and the specific reaction system and the conditions are as follows (Table 1):

TABLE 1 PCR reaction System (unit: μ L) of Et-VKVQ

PCR reaction procedure: pre-denaturation at 94 ℃ for 5min → denaturation at 94 ℃ for 30sec → annealing at 60 ℃ for 30sec → extension at 72 ℃ for 2min → extension at 72 ℃ for 10min → 4 ℃; a total of 30 cycles;

electrophoresis: and (3) carrying out 1% agarose gel electrophoresis on the PCR product, and observing the result under an ultraviolet detector. The size of the product was approximately 1200bp, consistent with the expected product size.

And (3) recovering a PCR product: the target fragment was recovered with DNA gel recovery kit and subjected to enzyme digestion (Table 2)

TABLE 2 Et-VKVQ cleavage reaction system (unit: μ L)

After incubation for 3h at 37 ℃, 1% agarose gel electrophoresis is carried out, and the target fragment after enzyme digestion is recovered by a DNA gel recovery kit.

2.3 treatment of expression vectors:

mu.L (1. mu.g/. mu.L) of pCDNA3.1 plasmid was digested with BamHI and XbaI to create the following (Table 3) digestion reactions which digested the plasmid:

TABLE 3 digestion reaction System of plasmid (unit: μ L)

After incubation at 37 ℃ for 3h, electrophoresis was performed on a 1% agarose gel. Recovering the plasmid after enzyme digestion by using a DNA gel recovery kit;

2.4 connection of the enzyme digestion vector and the enzyme digestion fragment:

the enzyme-digested Et-VKVQ and pCDNA3.1 are combined according to the composition in the table 4 to establish a connection reaction (table 4), and a pCDNA3.1-Et-VKVQ recombinant eukaryotic expression plasmid is constructed;

TABLE 4 ligation reaction System of enzyme digestion vector and fragment (Unit:. mu.L)

2.5 ligation product transformation E.coli DH5 α:

(1) add 10 μ L of ligation product to 100 μ L e.coli DH5 α competent cells in an ice bath. Mix by gentle rotation and ice-bath for 30 min.

(2) Placing the centrifuge tube into a water bath preheated to 42 ℃, and standing for 90 s.

(3) The tube was quickly transferred to an ice bath to cool the cells for 1-2 min.

(4) Add 800. mu.L SOC medium to each tube, incubate 45min at 37 ℃ with slow shaking.

(5) The culture was spread on LB agar plates (containing 100. mu.g/mL Amp), the plates were left at room temperature until the liquid was absorbed, the plates were inverted and incubated overnight at 37 ℃ (about 12-16 h).

2.6 colony PCR method for identifying transformed clones:

selecting some colonies, inoculating in 10ml LB culture medium (containing 100. mu.g/ml ampicillin), shaking at 37 deg.C overnight culturing, taking out culture, and establishing the following (Table 5) PCR reaction to screen positive clone;

TABLE 5 PCR reaction System (unit: μ L) for identifying transformed clones

PCR reaction procedure: and taking 10 mu L of PCR reaction product, performing 1% agarose gel electrophoresis, selecting positive bacterial colony, shaking bacteria and sequencing.

2.7 sequencing verification of positive clones:

and (3) sending the positive clone obtained by colony PCR identification to a sequencing company for sequencing verification, after sequencing is completed, comparing sequencing results by software, wherein sequencing primers are shown in the following table (table 6):

TABLE 6 sequencing primers

Sequencing primer name	Sequencing primer sequence (5 '-3')
		pCDNA3.1F	CTAGAGAACCCACTGCTTAC
pCDNA3.1R	TAGAAGGCACAGTCGAGG

2.8 plasmid miniprep:

the positive clone is verified by sequencing, and the plasmid is arranged to be extracted in small scale.

2.9 Et-VKVQ eukaryotic plasmid expression verification:

cell transfection: (1) one day before transfection, digesting and counting DF-1 cells, inoculating the cells into a 6-well plate according to the cell amount of 1.0x106 cells/well, ensuring that the confluence degree of the cells after 24 hours is between 70 and 90 percent, and culturing the cells in 2ml of complete culture medium per well; (2) on the day of transfection, cells were changed to a double-antibody-free complete medium and incubated at 37 ℃ in a 5% CO2 incubator; (3) preparation before transfection: a. diluting the plasmid DNA with 250 mu L of serum-free DMEM, and gently mixing; b. uniformly mixing lipofectamin reagent, taking a proper amount of lipofectamin reagent, diluting the lipofectamin reagent by 250 mu L of serum-free DMEM, slightly and uniformly mixing, and standing for 5 minutes at room temperature; c. mixing the DNA diluted in the first two steps with lipofectamin reagent, gently mixing the mixture evenly, and standing the mixture for 20 to 30 minutes at room temperature; (4) adding the mixed solution obtained in the step 3 into each hole of the cells; (5) after 4-6h of transfection, the complete culture medium can be replaced; (6) after incubation of the cells at 37 ℃ for 48-72h in a 5% CO2 incubator, post-transfection assays were performed.

Carrying out Western blot verification, and then carrying out sample preparation: (1) removing the cell culture medium, gently washing the cells with PBS, scraping the cells from the culture dish with a scraper, transferring the cells into a 1.5ml EP tube, centrifuging for 5min at 1000rpm, washing for 3 times with PBS, centrifuging for 5min at 1000rpm, and discarding the supernatant; (2) adding 100 μ L of lysis solution into each tube, and performing lysis on ice for 10 min; (3) centrifuge at 12000rpm for 10min at 4 ℃ and transfer the supernatant to a new 1.5ml EP tube.

For protein quantification, standard curves were prepared using 5 (1. mu.L), 10 (2. mu.L), 15 (3. mu.L), 20 (4. mu.L), 25 (5. mu.L), 30 (6. mu.L) and 35 (7. mu.L) of BSA (5 ug/. mu.L), and 2. mu.L of sample was taken, and the mean value was obtained by three-tube assay. Adding 1mLBradford into each branch pipe for dyeing, performing vortex oscillation for 20s, and fully and uniformly mixing to determine the light absorption value, wherein the operation interval between two samples is about 20s during determination. The liquid is injected uniformly to avoid the generation of bubbles.

SDS-PAGE gel electrophoresis, according to the quantitative result, 20ug of each sample was taken, ddH2O was added to make up to 18. mu.L, 6. mu.L of 4 × Loading buffer was added, boiling was performed at 100 ℃ for 5min, centrifugation was performed at 12000rpm at 4 ℃ for 3min, and Loading and electrophoresis were started. Constant pressure 130V/gel electrophoresis, until bromophenol blue runs out of the bottom of the gel, laminated gel concentration is 4%, and separation gel is 10%.

And (3) electrophoresis membrane transfer: (1) NC membrane is prepared, and gloves are worn when membrane cutting is carried out. (2) The clamp is opened to keep the black side horizontal. A sponge cushion is arranged on the upper surface of the bag body, and a glass rod is used for rolling for several times to roll away air bubbles inside. Two layers of filter paper are padded on the sponge pad, the filter paper is fixed by one hand, and air bubbles in the filter paper are rolled away by a glass rod by the other hand. (3) The sample glue and the film are put into a film rotating clamping plate marked with a positive electrode and a negative electrode: from the cathode side, the sponge pad → 2 layers of filter paper → sample gel → NC membrane → 2 layers of filter paper (note: air bubble removal) → sponge pad were fastened to the transfer nip plate, and the transfer nip plate was placed in a transfer electrophoresis tank containing a transfer buffer. (4) The membrane transfer time is 1 hour and 30 minutes, and the constant current is as follows: 300 mA.

Blocking, 5% skimmed milk powder dissolved in 1 × TBST, blocking at room temperature for 1 h.

Incubation of the antibody: (1) the primary antibody (rabbit anti-Et-VKVQ polyclonal antibody, accession KHD2016165, Shanghai Productions) was diluted with 1 XTBST to appropriate concentration and incubated at 4 ℃ for the primary antibody overnight. (2) After incubating the primary antibody overnight, the membranes were washed three times with TBST on a shaker for 5min each. (3) The secondary antibody (goat anti-rabbit, code A0277, from Biyunyan) was diluted to the appropriate concentration with 1 XTSST and incubated with the membrane for 2h at room temperature, and the membrane was washed three times with 1 XTSST 5min each time on a shaker.

Chemiluminescence, development, fixation: (1) first, the liquid on the membrane was blotted dry with filter paper (2) two luminescent reagents A and B were mixed in equal volumes in an EP tube, the luminescent reagents were applied to a glass plate, the membrane was leveled face down with a gun and the time was taken for 2 min. (3) And uniformly dispensing the prepared ECL chemiluminescence liquid on an NC film, and exposing by using a chemiluminescence gel imaging system after 10-30 s. The results are shown in FIG. 1.

Preparation of Et-VKVQ protein

3.1 primer design

Designing primer sequences aiming at the upstream and the downstream of a target gene:

the upstream primer Et-VKVQ-PaF:5 'GGCGGATCCGTCAAAGTTCAGCAGCA GCAGCAGC 3' (SEQ ID NO: 7);

the downstream primer Et-VKVQ-PaR:5 'GGCAAGCTTCGGGAGGGCCTCCTCCGCC AAAGAA 3' (SEQ ID NO: 8).

3.2 Gene amplification

(1) PCR reaction system and amplification conditions: using pMD18-T-Et-VKVQ plasmid as template, PrimeSTAR high fidelity enzyme is used to amplify gene target fragment, and the specific reaction system and conditions are as follows (Table 7):

TABLE 7 PCR reaction System (unit: μ L) of Et-VKVQ

PCR reaction procedure:

pre-denaturation at 94 ℃ for 5min → denaturation at 94 ℃ for 30sec → annealing at 60 ℃ for 30sec → extension at 72 ℃ for 2min → extension at 72 ℃ for 10min → 4 ℃; for a total of 30 cycles.

(2) Electrophoresis: and (3) carrying out 1% agarose gel electrophoresis on the PCR product, and observing the result under an ultraviolet detector. The size of the product was approximately 1200bp, consistent with the expected product size.

(3) And (3) recovering a PCR product: the target fragment was recovered with a DNA gel recovery kit and subjected to an enzymatic cleavage reaction (Table 8):

TABLE 8 Et-VKVQ cleavage reaction System (unit: μ L)

After incubation for 3h at 37 ℃, 1% agarose gel electrophoresis is carried out, and the target fragment after enzyme digestion is recovered by a DNA gel recovery kit.

3.3 treatment of the expression vector

mu.L (1. mu.g/. mu.L) of pET30a plasmid was digested simultaneously with BamHI and Hind III to create the following (Table 9) digestion reactions which cleaved the plasmid:

TABLE 9 digestion reaction System of plasmid (unit: μ L)

After incubation at 37 ℃ for 3h, electrophoresis was performed on a 1% agarose gel. Recovering the plasmid after enzyme digestion by using a DNA gel recovery kit;

3.4 ligation of the digestion vector to the digestion fragment

The enzyme-digested Et-VKVQ and pET30a form a ligation reaction according to the composition of Table 10 (Table 10), and pET30a-Et-VKVQ recombinant prokaryotic expression plasmids are constructed;

TABLE 10 ligation reaction System of digestion vector and fragment (Unit:. mu.L)

3.5 ligation product transformation E.coli DH 5. alpha

(1) Add 10 μ L of ligation product to 100 μ L e.coli DH5 α competent cells in an ice bath. Mix by gentle rotation and ice-bath for 30 min.

(2) Placing the centrifuge tube into a water bath preheated to 42 ℃, and standing for 90 s.

(3) The tube was quickly transferred to an ice bath to cool the cells for 1-2 min.

(4) Add 800. mu.L SOC medium to each tube, incubate 45min at 37 ℃ with slow shaking.

(5) The culture was spread on LB agar plates (containing 50. mu.g/mL kanamycin sulfate), the plates were left at room temperature until the liquid was absorbed, the plates were inverted, and cultured overnight at 37 ℃ (about 12-16 h).

3.6 colony PCR method for identifying transformed clones

Several colonies were picked and inoculated into 10mL of LB medium (containing 50. mu.g/mL kanamycin sulfate), and after overnight culture at 37 ℃ with shaking, the cultures were used to screen for positive clones by the following PCR reaction (Table 11):

TABLE 11 PCR reaction System (unit: μ L) for identifying transformed clones

PCR reaction procedure: as above.

Taking 10 mu L of PCR reaction product, carrying out 1% agarose gel electrophoresis, selecting positive bacterial colony, shaking bacteria, and sequencing.

3.7 plasmid petiole

The positive clone is verified by sequencing, and the plasmid is arranged to be extracted in small scale.

3.8 inducible expression of pET30a-Et-VKVQ in expression bacteria

The constructed pET30a-Et-VKVQ plasmid was transformed into BL21(DE3) competent cells, and then uniformly spread on LB plates (containing 50. mu.g/mL kanamycin sulfate), followed by being placed upside down in an incubator at 37 ℃ overnight.

From the transformed plate, a single clone was selected, inoculated into 4L of LB medium (containing 50. mu.g/mL kanamycin sulfate), cultured to OD600 of 0.5-0.8, and added to the culture solution to a final concentration of 0.1mM IPTG, followed by induction of expression at 15 ℃ and 37 ℃ respectively.

And (3) analyzing and identifying an induced expression result by SDS-PAGE, centrifuging the culture solution after induction at 12000rpm for 5min, removing the supernatant, adding PBS (phosphate buffer solution) to resuspend and precipitate, adding SDS-PAGE loading buffer solution, heating the sample at 100 ℃ for 10min, and centrifuging to obtain the supernatant for electrophoresis. And (3) performing 100V stabilized voltage electrophoresis 10min before electrophoresis, after the bromophenol blue indicator enters the separation gel, performing 200V stabilized voltage electrophoresis until the bromophenol blue band moves to 1cm away from the bottom of the gel, taking out the gel, dyeing the gel with Coomassie brilliant blue dyeing solution, and then transferring the gel into a decoloring solution, and decoloring until the background is clear. The results are shown in FIG. 2.

Protein purification, inclusion body with 20mM PBS (pH7.2), 150mM NaCl containing 1% Triton X-100, 2mM EDTA, 2mM DTT washing, 20mM PB (pH7.2), 150mM NaCl, 8M Urea, 20mM Imidazole buffer solution dissolved inclusion body and balanced Ni-IDA column, finally with different concentrations of Imidazole balanced buffer elution target protein, and collecting each elution component for SDS-PAGE analysis detection. The results are shown in FIG. 2.

Purifying by Ni-IDA affinity chromatography, collecting high purity Lane 5-11, adding into treated dialysis bag, dialyzing at 4 deg.C into buffer solution 1 × PBS (pH7.4), 4mM GSH, 0.4mM GSSG, 2mM EDTA, 0.4 mM-Arginine for renaturation, and dialyzing Et-VKVQ protein into storage solution 1 × PBS (pH7.4) and 10% Glycerol solution for about 6-8 h. After the renaturation by dialysis, the supernatant was filtered through a 0.22 μm filter and dispensed, and was frozen to-80 ℃.

3.9 immunoblot (Western blot) analysis of recombinant proteins

And (3) carrying out immune activity identification on the recombinant Et-VKVQ protein by using an immunoblotting (Western blot) method. The primary antibody was murine his monoclonal antibody (Sigma) and the secondary antibody was goat anti-murine IgG-HRP (Sigma). The results are shown in FIG. 3.

Immunoprotection assay for 4 Et-VKVQ

4.1 materials

Coccidian oocysts: the eimeria tenella Guangdong strain sporulated oocysts were preserved by the zooecism research institute of animal health institute of Guangdong academy of agricultural sciences, Guangdong province, and rejuvenated in coccidiless chicks before use.

Chicks: the green south yellow chicks are provided by animal science research institute of agriculture academy of sciences of Guangdong province and are raised in a sterilized special animal house; the chicken coop and the utensils are strictly disinfected, and the chicken coop can freely eat and drink purified water; before the experiment, the chicks are observed to have clinical symptoms and whether coccidian oocysts exist in the excrement is continuously checked for 3 days for later use.

Feed: the chick breeding material is customized by the animal science research institute of the Guangdong province academy of agricultural sciences, and does not contain any anticoccidial drugs.

4.2 test methods

Grouping: weighing 180 test chicks of 1 day old one by one, removing lean or overweight chicks, selecting healthy chickens with individual weight difference within 10g, and randomly dividing into 6 groups of 30 chicks each.

And (3) treatment:

emulsification of Et-VKVQ recombinant protein: uniformly mixing the Et-VKVQ recombinant protein obtained after 3.9 purification with Freund's adjuvant (FCA) according to the ratio of 1: 1; repeatedly sucking with No. 7 needle syringe until no diffusion occurs within 5 min.

Test chickens were immunized with pCDNA3.1-Et-VKVQ eukaryotic plasmid (intramuscular injection in the leg) or pET30a-Et-VKVQ recombinant protein (subcutaneous injection) at 1, 7, and 14 days old, respectively, and a non-immune infected group and a non-immune non-infected group were used as controls. Each 21-day-old was orally infected with 5 × 104 fresh e.tenella sporulated oocysts. Observing and recording the mental state, feed intake, excrement condition and the like of the chicken flocks every day; weighing dead chicks, performing a autopsy, and if the chicks die due to Eimeria tenella infection, the lesion score is + 4; all chicks were weighed one by one on day 7 post infection, necropsied, and scored for cecal lesions. Specific test groupings are detailed in table 12:

table 12 experimental group design

Anticoccidial index evaluation criteria:

relative rate of weight gain: the weight of the chickens is weighed at the beginning and the end of the test respectively, and the average weight gain and the relative weight gain rate are calculated. Relative weight gain rate (weight gain rate in each group/weight gain rate in non-immune non-infected group) × 100%.

Survival rate: the number of dead chickens in each group is recorded, and the death cause is determined by autopsy and the survival rate is calculated. Survival rate (number of surviving chickens/number of chickens in test group at end of test) × 100%.

The lesion value is: slaughtering the chickens 7 days after infection, scoring intestinal lesions of each chicken according to a lesion scoring method designed by Johnson and Reid (1970), and converting the lesion scores into lesion values;

and (3) lesion scoring: (when the bilateral cecal lesions are inconsistent, the serious side is taken as the standard):

0 point, no macroscopic lesion;

1, the cecum wall has little scattered petechia, the cecum wall is not thickened, and the content is normal;

2, the number of lesions is large, the content of the caecum is obviously bloody, the wall of the caecum is slightly thickened, and the content is normal;

3 minutes, a large amount of blood or a caecum core (blood clots or a banana-shaped block like grey cheese) exists in the caecum, the caecum wall is obviously thickened, and the content of excrement in the caecum is low;

4 points, because of the large amount of blood or intestinal core full of cecal enlargement, the intestinal core contains or does not contain fecal dregs, and the dead chicken due to coccidiosis is recorded in 4 points.

The lesion value (0-40) is the average lesion score (0-4). times.10 for each test group.

Oocyst value: fecal oocysts were counted by the Macmester counting method, and the number of fecal Oocysts (OPG) per group was determined, and the number of oocysts was calculated and converted from Table 13 to obtain an oocyst value.

TABLE 13 conversion of oocyst count to oocyst value

Anticoccidial index (ACI): ACI is calculated as (relative rate of weight gain + survival) × 100- (lesion value + oocyst value).

Judging the immune effect standard: ACI is more than 180, which is high-efficiency; ACI of more than 160 and less than 180 belong to middle-effect; ACI 120 < 160 is inefficient; ACI <120 was not effective against coccidia.

4.3 test results

Observation of clinical symptoms:

the non-immune infected control group test chickens gradually showed reactions such as decreased feed intake, poor spirit and the like after being infected with sporulated oocysts. On the 4 th day after infection, blood dung is discharged from the pCDNA3.1 group, the pET30a group and the non-immune infection control group, the water intake is reduced, the disease is more serious on the 5 th day and the 6 th day, cecal examination shows caecum lesion, bleeding or blood clot with different degrees, and no lesion is observed in other organs; the plasmid + recombinant protein immune group, the recombinant protein + plasmid immune group and the non-immune non-infection control group have no blood dung, and are fed with normal drinking water.

The test results show that the coccidian resistance indexes of the pCDNA3.1 group and the pET30a group are both lower than 120, and the coccidian resistance indexes all present ineffective coccidian resistance effects; the plasmid + recombinant protein immune anticoccidial index is 188.68, and the recombinant protein + plasmid immune group anticoccidial index is 189.86, and has high-efficacy anticoccidial effect. The results are detailed in Table 14.

TABLE 14 evaluation of the immunoprotective Effect of Et-VKVQ

The amino acid and nucleotide sequences in this application are as follows:

SEQ ID NO: 1(Et-VKVQ nucleotide sequence)

GTCAAAGTTCAGCAGCAGCAGCAGCAGCAGCAAAGCAGCCCTTCCCCCTTGCAGAGAGCGCCTGCAGAGAGCTTTTCCGCGATGGATCAAACAGCAGCAGCAGCGCTAGAGATGCTGCAGCTGCAGCAGCAGCAGCAGCAGCAGCAGCAGGAGCAGCAGGAAGCTGAAAAGTTGAATTCAAACAACTTCCTGGCCACTGCATCGCCACATGCTGTCTCTACACTGCAAACAGAAAGTCAAAATCCTTGGCTGTCGGGAGCTCTGAAGACATTCATAAGCAAGTTCAATATCCCGGTTGTTCACGGTTCTGGGGTGTACGTGGACCTCGGGAAAACGAGTTCCGGATACCGCCAGCCGGGAGGACTCTGTCCAGTTTTTGGGAAATATATTAAAATTGAAAAGCAAAATTCAAATTACAAAACTTTTCTCGAAGACTTTCCAGTTTCGGGATCTTCCGAAAGGCCTTTGCCCGGAGGATTCAACTTGGCGTATGTCACCAGCAGCGGACGCGCCTTTTCTCCGGTTGCCGATTCTTTCCTCGTGGATGCTTTTGGCTTGACGGGGCCATCGGACCCGATATCTCGCTGCGCGAAGTACAGCTACTTGATGGAGCCCCGGAAGCCAGGAACAAATGGCACTTTTGATTACAAGTTGCCTTTTGTTTACGACGCAGCAGCAAAAAGCTGCTTCGTGCTGCACGTCAGCATGCAGAAGCTCACTGGCAGCAGATATTGCTCCTCGAACGGGACCCCCGCTGGCCTCTTTTGGCCCTGCTTCCGCCCCGCCAAAAGCGCCTCAGCTTCGACCCATCTTGTCTATGGCTCTGGGCAAATGGGCGCAGACCCAGAGGCTTGGAAAAAGAAGTGCCCCTCGAGAGCCGTGAAGGACGCGGTGTTCGGACAGTGGGGGGTGGGGGGCTGCACGGCGTTGGCGGCGAACTCTTCGGGTGTACGTACACTGCAAGTTGCATACAAAAGCAAATGTTGGGAAACTCTTTTTGACTTCTCTGCTTCTGACGAAGCTGTTGCTTCCAACCCCGACAACTGGGACTCCATTTGGCCTACGCTCGACAGCAGCGCCTCCGTCTCAGCGGGAGTTGGGCAAAACTACGCCAACTTCTTCCCCAGCAGCTCCGGGCCAGGGGGGACTTGTGTTATTTTTGCTGCTGTACCCTCTTGCTTCCTGAGGGCCCCCGGGCACACAGCCTACACCTCAGTGGGTTCTTTGGCGGAGGAGGCCCTCCCG

SEQ ID NO 2(Et-VKVQ amino acid sequence)

VKVQQQQQQQQSSPSPLQRAPAESFSAMDQTAAAALEMLQLQQQQQQQQQEQQEAEKLNSNNFLATASPHAVSTLQTESQNPWLSGALKTFISKFNIPVVHGSGVYVDLGKTSSGYRQPGGLCPVFGKYIKIEKQNSNYKTFLEDFPVSGSSERPLPGGFNLAYVTSSGRAFSPVADSFLVDAFGLTGPSDPISRCAKYSYLMEPRKPGTNGTFDYKLPFVYDAAAKSCFVLHVSMQKLTGSRYCSSNGTPAGLFWPCFRPAKSASASTHLVYGSGQMGADPEAWKKKCPSRAVKDAVFGQWGVGGCTALAANSSGVRTLQVAYKSKCWETLFDFSASDEAVASNPDNWDSIWPTLDSSASVSAGVGQNYANFFPSSSGPGGTCVIFAAVPSCFLRAPGHTAYTSVGSLAEEALP

SEQ ID NO:3(Et-VKVQ-F)

GTCAAAGTTCAGCAGCAGCAGCAGC

SEQ ID NO:4(Et-VKVQ-R)

CGGGAGGGCCTCCTCCGCCAAAGAA

SEQ ID NO:5(Et-VKVQ-EuF)

CTTGGTACCGAGCTCGGATCCGCCACCGTCAAAGTTCAGCAGCAGCAGCAGC

SEQ ID NO:6(Et-VKVQ-EuR)

GGTTTAAACGGGCCCTCTAGACGGGAGGGCCTCCTCCGCCAAAGAA

SEQ ID NO:7(Et-VKVQ-PaF)

GGCGGATCCGTCAAAGTTCAGCAGCAGCAGCAGC

SEQ ID NO:8(Et-VKVQ-PaR)

GGCAAGCTTCGGGAGGGCCTCCTCCGCCAAAGAA

The foregoing shows and describes the general principles and features of the present invention, together with the advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

<110> institute of animal health of academy of agricultural sciences of Guangdong province

<120> preparation method of Eimeria tenella recombinant polypeptide vaccine VKVQ and application method thereof in chicken coccidiosis resistance

<130>20200730

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<212>DNA

<213> Eimeria tenella (Eimeria tenella)

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cctgcagaga gcttttccgc gatggatcaa acagcagcag cagcgctaga gatgctgcag 120

ctgcagcagc agcagcagca gcagcagcag gagcagcagg aagctgaaaa gttgaattca 180

aacaacttcc tggccactgc atcgccacat gctgtctcta cactgcaaac agaaagtcaa 240

aatccttggc tgtcgggagc tctgaagaca ttcataagca agttcaatat cccggttgtt 300

cacggttctg gggtgtacgt ggacctcggg aaaacgagtt ccggataccg ccagccggga 360

ggactctgtc cagtttttgg gaaatatatt aaaattgaaa agcaaaattc aaattacaaa 420

acttttctcg aagactttcc agtttcggga tcttccgaaa ggcctttgcc cggaggattc 480

aacttggcgt atgtcaccag cagcggacgc gccttttctc cggttgccga ttctttcctc 540

gtggatgctt ttggcttgac ggggccatcg gacccgatat ctcgctgcgc gaagtacagc 600

tacttgatgg agccccggaa gccaggaaca aatggcactt ttgattacaa gttgcctttt 660

gtttacgacg cagcagcaaa aagctgcttc gtgctgcacg tcagcatgca gaagctcact 720

ggcagcagat attgctcctc gaacgggacc cccgctggcc tcttttggcc ctgcttccgc 780

cccgccaaaa gcgcctcagc ttcgacccat cttgtctatg gctctgggca aatgggcgca 840

gacccagagg cttggaaaaa gaagtgcccc tcgagagccg tgaaggacgc ggtgttcgga 900

cagtgggggg tggggggctg cacggcgttg gcggcgaact cttcgggtgt acgtacactg 960

caagttgcat acaaaagcaa atgttgggaa actctttttg acttctctgc ttctgacgaa 1020

gctgttgctt ccaaccccga caactgggac tccatttggc ctacgctcga cagcagcgcc 1080

tccgtctcag cgggagttgg gcaaaactac gccaacttct tccccagcag ctccgggcca 1140

ggggggactt gtgttatttt tgctgctgta ccctcttgct tcctgagggc ccccgggcac 1200

acagcctaca cctcagtggg ttctttggcg gaggaggccc tcccg 1245

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<213> Eimeria tenella (Eimeria tenella)

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Val Lys Val Gln Gln Gln Gln Gln Gln Gln Gln Ser Ser Pro Ser Pro

1 5 10 15

Leu Gln Arg Ala Pro Ala Glu Ser Phe Ser Ala Met Asp Gln Thr Ala

20 25 30

Ala Ala Ala Leu Glu Met Leu Gln Leu Gln Gln Gln Gln Gln Gln Gln

35 40 45

Gln Gln Glu Gln Gln Glu Ala Glu Lys Leu Asn Ser Asn Asn Phe Leu

50 55 60

Ala Thr Ala Ser Pro His Ala Val Ser Thr Leu Gln Thr Glu Ser Gln

65 70 75 80

Asn Pro Trp Leu Ser Gly Ala Leu Lys Thr Phe Ile Ser Lys Phe Asn

85 90 95

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

100 105 110

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

115 120 125

Tyr Ile Lys Ile Glu Lys Gln Asn Ser Asn Tyr Lys Thr Phe Leu Glu

130 135 140

Asp Phe Pro Val Ser Gly Ser Ser Glu Arg Pro Leu Pro Gly Gly Phe

145 150 155 160

Asn Leu Ala Tyr Val Thr Ser Ser Gly Arg Ala Phe Ser Pro Val Ala

165 170 175

Asp Ser Phe Leu Val Asp Ala Phe Gly Leu Thr Gly Pro Ser Asp Pro

180 185 190

Ile Ser Arg Cys Ala Lys Tyr Ser Tyr Leu Met Glu Pro Arg Lys Pro

195 200 205

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

210 215 220

Ala Ala Lys Ser Cys Phe Val Leu His Val Ser Met Gln Lys Leu Thr

225 230 235 240

Gly Ser Arg Tyr Cys Ser Ser Asn Gly Thr Pro Ala Gly Leu Phe Trp

245 250 255

Pro Cys Phe Arg Pro Ala Lys Ser Ala Ser Ala Ser Thr His Leu Val

260 265 270

Tyr Gly Ser Gly Gln Met Gly Ala Asp Pro Glu Ala Trp Lys Lys Lys

275 280 285

Cys Pro Ser Arg Ala Val Lys Asp Ala Val Phe Gly Gln Trp Gly Val

290 295 300

Gly Gly Cys Thr Ala Leu Ala Ala Asn Ser Ser Gly Val Arg Thr Leu

305 310 315 320

Gln Val Ala Tyr Lys Ser Lys Cys Trp Glu Thr Leu Phe Asp Phe Ser

325 330 335

Ala Ser Asp Glu Ala Val Ala Ser Asn Pro Asp Asn Trp Asp Ser Ile

340 345 350

Trp Pro Thr Leu Asp Ser Ser Ala Ser Val Ser Ala Gly Val Gly Gln

355 360 365

Asn Tyr Ala Asn Phe Phe Pro Ser Ser Ser Gly Pro Gly Gly Thr Cys

370 375 380

Val Ile Phe Ala Ala Val Pro Ser Cys Phe Leu Arg Ala Pro Gly His

385 390 395 400

Thr Ala Tyr Thr Ser Val Gly Ser Leu Ala Glu Glu Ala Leu Pro

405 410 415

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<212>DNA

<213> Eimeria tenella (Eimeria tenella)

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gtcaaagttc agcagcagca gcagc 25

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<212>DNA

<213> Eimeria tenella (Eimeria tenella)

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<212>DNA

<213> Eimeria tenella (Eimeria tenella)

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<212>DNA

<213> Eimeria tenella (Eimeria tenella)

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ggcggatccg tcaaagttca gcagcagcag cagc 34

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<212>DNA

<213> Eimeria tenella (Eimeria tenella)

<400>8

ggcaagcttc gggagggcct cctccgccaa agaa 34