CN107904247A - One kind restructuring Eimeria species Alphavirus particle vaccines and preparation method - Google Patents

Abstract

The present invention provides a kind of recombinant chicken coccidioides A virus particle vaccine and its preparation method, select the A virus replicon carrier pSMART2b based on SFV and Eimeria tenella protective antigen gene Repair_PSII to connect, construct recombinant plasmid pSMART2b-Repair_PSII and use auxiliary The plasmid pSHCAR is packaged into an alphavirus particle vaccine containing the Repair_PSII gene, which has the advantages of good protective effect, high level of induced immunity and high safety.

Description

A kind of recombinant chicken coccidiosis A virus particle vaccine and its preparation method

technical field

The invention provides a recombinant chicken coccidiosis A virus particle vaccine, which can be used to prevent chicken coccidiosis, and also discloses a preparation method of the recombinant chicken coccidiosis A virus particle, which belongs to the technical field of genetic engineering.

Background technique

Chicken coccidiosis is a parasitic protozoan disease caused by coccidia of Sporozoa, Eimeriidae and Eimeria . The disease is distributed worldwide, with a high infection rate and great harm. Coccidiosis-infected chicks are stunted in growth and development, listless, and their feathers stand upright. In severe cases, the chicks will die, and the mortality rate can reach 20%-80%. Adult chickens are mostly worm carriers, and symptoms such as growth retardation, inverted feathers, and decreased egg production rate occur. According to incomplete statistics, the economic loss caused by chicken coccidiosis alone is as high as more than 3 billion US dollars every year.

At present, the prevention and treatment of chicken coccidiosis mainly relies on drugs, but long-term dependence on drug control is not a long-term solution. The first is the problem of drug resistance, such as classic diclazuril, salinomycin, sulfaquinoxaline, sulfachlorpyrazine, sulfadimethoxine and other drugs have been highly resistant to drugs; secondly, the problem of drug residues, Chicken farms have violated the Ministry of Agriculture’s drug withdrawal period regulations for long-term abuse of anticoccidiostats. Insecticides and the increased cost of research and development of new anticoccidiostats by pharmaceutical companies. Affected by the above factors, more and more veterinary pharmaceutical companies at home and abroad have withdrawn from the field of coccidiosis control drugs. At this stage, no new chemically synthesized anticoccidial drugs have been released for many years.

The many disadvantages of drug prevention and control have led researchers to turn their attention to the field of vaccines, hoping to achieve the purpose of preventing coccidiosis in chickens through vaccine immunization.

Vaccines studied at this stage can be roughly divided into three categories: live vaccines, subunit vaccines and DNA vaccines. The production process of live vaccines is complicated, transportation is inconvenient, and there are disadvantages such as loose poison and strong virulence, which are not suitable for the multi-layer cage culture mode under large-scale intensive farming conditions, so it is very difficult to promote live vaccines on a large scale; subunit vaccines There are defects such as low expression of protective protein, difficulty in modification, and low immunogenicity, and it cannot provide strong protection against coccidiosis; DNA vaccines are safer than traditional vaccines, and they will not disperse or produce toxins It can cause other diseases, but DNA vaccines also have many problems: such as low expression of conventional eukaryotic expression vectors, weak antigen immunogenicity, and mutations caused by the integration of foreign genes and host genes, so DNA vaccines are currently only in laboratory research stage, not commercialized. The emergence of alpha virus vectors provides a good idea for solving the above problems.

Alphavirus (Alphavirus) belongs to the Togavirus family (Togavirus family), the genetic material is single-stranded positive-strand RNA, the size of its genome is about 12kb, there are 2 open reading frames, 5 ^{, the} end is the self-replication and transcription of alphavirus The non-structural protein region of the required enzyme and signal, and this end has a cap structure and a strong CMV promoter, this region contains nsP1, nsP2, nsP3 and nsP4, this part has the packaging signal of the virus; 3 ^{, the} end is the coding alphavirus Structural protein sequence, RNA (-) synthesizes whole genome RNA and subgenomic RNA under the action of replicase, and finally translates into viral capsid protein (Capsid), E1, E2, 6K, etc. under the action of subgenome promoter Structural protein, and this end has a termination signal (A69) and a strong transcription termination signal SV40poly(A), the termination signal enables normal transcription and the polyA(A) tail structure enhances its stability and completes its expression. Alphavirus replicon vectors can replicate quickly and at a high level after being introduced into cells, and studies have confirmed that the replication amount of SFV in cells can reach as high as 2×10 ⁵ cells/cell, far exceeding conventional eukaryotic expression vectors. The alphavirus replicon vector can also induce cell apoptosis. After the foreign gene is expressed in the cytoplasm, it will undergo apoptosis and be cleaned up by the host cell without being integrated into the host cell, which greatly improves the safety of the vaccine.

Contents of the invention

The object of the present invention is to provide a recombinant chicken coccidiosis alpha virus particle vaccine and a preparation method, which is a recombinant chicken coccidiosis alpha virus particle vaccine comprising the chicken coccidia Repair_PSII gene.

A kind of recombinant chicken coccidiosis A virus particle vaccine of the present invention and preparation method, its technical solution is as follows:

To be packaged into a recombinant alphavirus particle containing the chicken coccidia Repair_PSII gene, first the recombinant plasmid pSMART2b-Repair_PSII capable of delivering and expressing the gene must be obtained, and its correct construction and expression must be confirmed. Then, the recombinant plasmid pSMART2b-Repair_PSII and the auxiliary plasmid pSHCAR are transfected into alphavirus host cells (such as 293 cells), then the virus stock solution is collected and infected with BHK-21 cells, and finally the recombinant alphavirus particle vaccine containing chicken coccidia Repair_PSII gene is obtained .

1. Construction and expression of recombinant plasmid pSMART2b-Repair_PSII

First extract the total RNA of Eimeria tenella and reverse transcribe it into cDNA, amplify the Repair_PSII gene by PCR; As well as biological sequencing to identify the connection, the sticky end Repair_PSII gene was connected to the alphavirus replicon vector pSMART2b in the same way; then the recombinant plasmid pSMART2b-Repair_PSII was transfected into 293 cells, and Western blot was used to identify the Repair_PSII gene expression;

2. Preparation of Recombinant Chicken Coccidian A Virus Particle Vaccine

The correctly identified and expressed recombinant plasmid pSMART2b-Repair_PSII and the helper plasmid pSHCAR were transfected into 293 cells by liposome method, the medium was changed in 6 hours, and the virus stock solution was collected in 48 hours. Add 1/20 volume of α-chymotrypsin solution (10 g/L) to the virus stock solution, incubate at room temperature for 40 minutes to activate the virus (crack the p62 glycoprotein into E2 and E3 proteins to activate the virus), then add 1/15 volume Aprotinin (10 g/L) was incubated at room temperature for 5 min; an appropriate amount of activated virus stock solution was taken to infect BHK-21 cells, and recombinant chicken coccidia virus particles were collected 48 hours later for transmission electron microscope observation and Western blot Identify the expression of the Repair_PSII gene;

3. Optimization of preparation method of recombinant chicken coccidian A virus particle vaccine

Referring to Invitrogen’s transfection reagent Lipofectamine ^TM 3000 instructions, the recombinant plasmid pSMART2b-Repair_PSII and the helper plasmid pSHCAR were co-transfected into 293 cells at a molar ratio of 1:1.5 (total 2.5ug), the medium was changed for 6 hours, and the virus was collected for 48 hours Stock solution; at the same time, first transfect the auxiliary plasmid pSHCAR 2.5ug into 293 cells, change the medium for 6 hours, then take the recombinant plasmid pSMART2b-Repair_PSII 2.5ug single transfection into 293 cells at 24 hours, change the medium for 6 hours, and collect in 48 hours Virus stock solution; finally, the optimized virus stock solution was used to infect BHK-21 cells respectively, and a small amount of recombinant chicken coccidia virus particles was taken for transmission electron microscopy.

The positive effects of the present invention are: a recombinant alphavirus particle vaccine comprising chicken coccidia Repair_PSII gene is provided, and the SFV-based alphavirus replicon vector pSMART2b is selected to connect with the Eimeria tenella protective antigen gene Repair_PSII to construct The recombinant plasmid pSMART2b-Repair_PSII is packaged with the helper plasmid pSHCAR into an alphavirus particle vaccine containing the Repair_PSII gene, which has the advantages of good protective effect, high level of induced immunity and high safety.

Description of drawings

Fig. 1 Electropherogram of Repair_PSII gene;

Fig. 2 Electropherogram of restriction endonuclease identification pMD18-T-Repair_PSII;

Figure 3 Restriction endonuclease identification recombinant plasmid pSMART2b-Repair_PSII electrophoresis;

Figure 4 Western blot method to identify the expression of recombinant plasmids in 293 cells;

Figure 5 Western blot method for identification of recombinant chicken coccidioides A virus particle diagram;

Fig. 6 transmission electron micrograph of recombination chicken coccidioides A virus particle;

Fig. 7 is the X-gal analysis diagram of recombinant alphavirus particles infecting BHK-21 cells;

Fig. 8 The changes of cytokines in chicken serum after immunization determined by double-antibody sandwich ELISA.

Detailed ways

The present invention is further illustrated by the following examples, which do not limit the present invention in any way. The substantive content of the present invention is further described below with embodiment, but content of the present invention is not limited thereto.

Example 1

Construction, Identification and Expression of Recombinant Plasmid pSMART2b-Repair_PSII

1, Primer 5.0 software design primer PCR method to amplify chicken coccidia Repair_PSII gene, the sequence is shown in SEQ no.1;

Upstream: 5'-CGC GGATCC GATGGACTGGCTTTCGAGCGAG-3'(BamH I)

Downstream: 5'-CCC ATCGAT ACGATACTTGGCAATCTCG-3' (ClaI)

2. Using chicken coccidia cDNA as a template, amplify chicken coccidia Repair_PSII gene by PCR method, purify and connect to cloning vector pMD18-T, transform DH5α competent cells and clone, and cut with restriction enzymes BamHI and ClaI Under the Repair_PSII gene fragment containing cohesive ends, the same method was used to obtain the pSMART2b vector containing BamHI and ClaI cohesive end cuts (see Figure 1-4);

3. Prepare the transfection complex according to the conditions of the endotoxin-free plasmid pSMART2b-Repair_PSII 2.5ug and the transfection reagent Lipofectamine ^TM 3000 7.5ul, and transfer it to 293 cells cultured to 60%-80% confluence. After 6 hours of transfection The medium was changed, and the culture was continued for 48 hours in DMEM medium with a concentration of 2.0% fetal bovine serum and 1.0% double antibody. Collect the 293 cells cultured for 48 hours, lyse the cells and obtain the whole protein, and use rabbit anti-chicken Coccidioides Repair_PSII protein multiple antibody serum for Western blot identification (see Figure 5).

Example 2

Preparation of Recombinant Chicken Coccidian A Virus Particle Vaccine

1. Make a transfection complex with the correctly identified and expressed recombinant plasmid pSMART2b-Repair_PSII, helper plasmid pSHCAR and transfection reagent Lipofectamine ^TM 3000 according to the ratio of 1ug:1.5ug:7.5ul and co-transfect into a confluence of 60-80% The 293 cells were replaced with DMEM medium containing 2.0% fetal bovine serum and 1.0% double antibody for 6 hours and continued to culture for 48 hours, and the virus stock solution was collected after 48 hours.

2. Add 1/20 volume of α-chymotrypsin solution (10g/L) to the virus stock solution, incubate at room temperature for 40 minutes (in order to cleave p62 glycoprotein into E2 and E3 proteins to activate the virus), add 1/15 volume of Aprotinin (10g/L), incubated at room temperature for 5min. Then, the activated virus stock solution was used to infect BHK-21 cells, and after 2 hours, it was replaced with DMEM medium containing 2.0% fetal bovine serum and 1.0% double antibody to continue culturing for 48 hours, and the recombinant chicken coccidia virus particles were collected after 48 hours. Finally, a small amount of alphavirus particles were taken for transmission electron microscopy and Western blot to identify the expression of the Repair_PSII gene (see Figure 6).

Example 3

X-gal Analysis of Infected BHK-21 Cells

The positive cells after X-gal in situ staining are blue cells, and the titer of rSFV-Repair_PSII particles can be estimated initially. Under the magnification of 400× (40× eyepiece + 10× objective lens), randomly find 3 fields of view to calculate the average number of blue cells. Then determine the titer. The number of blue cells in each field of view × plate area / area of a field of view, the result obtained × lml / the actual volume of virus suspension used (ml), that is, the virus titer (IU/ml) is obtained (see Figure 7).

Example 4

Protective Effect of Recombinant Chicken Coccidia A Virus Particle Vaccine Against Eimeria tenella

160 13-day-old healthy chicks were randomly divided into 4 groups, 40 in each group. In the immunization group, according to the immunization program of 14-day-old first immunization, 21-day-old second immunization, and 28-day-old third immunization, each group was intramuscularly injected with a mixture of 100 μl recombinant alphavirus particles and 100 μl adjuvant; the adjuvant control group was injected with only 100 μl adjuvant; set up negative control group (white control) and positive control group (red control) at the same time. Before immunization and before challenge, 5 chickens were randomly selected from each group for heart blood collection and spleen lymphocyte separation, and the serum antibody level, IL-2, IL-4, IL-10 and IFN-γ expression levels were determined by MTT method Chicken spleen lymphocyte proliferation and other indicators. Seven days after the third immunization, 1× ¹⁰ sporulated oocysts of Eimeria tenella were orally inoculated, and the challenge test was carried out, and the survival rate, relative weight gain rate, cecal lesion score, OPG, ACI and other indicators were carried out statistical determination. Finally, the comprehensive anti-coccidial index (ACI) was used to evaluate the immune protection effect of alphavirus particles on chickens (see Figure 8).

The ACI comprehensive evaluation of each group of experimental chickens is shown in table 1:

Table 1. Anti-coccidial index of recombinant plasmid against E. tenella

group Survival rate (%) Relative weight gain (%) lesion value Oocyst value Anti-coccidial index (ACI) rSFV-Repair_PSII group 100 92.98 6 1 185.98 Adjuvant group 90 64.91 twenty four 10 120.91 positive control 90 56.14 27 10 109.14 negative control 100 100 0 0 200

Conclusion: As shown in Table 1, the comprehensive anticoccidial index of the immunized rSFV-Repair_PSII group was 185.98, which was significantly higher than that of the positive control group, which was 109.14, showing a good protective effect.

sequence listing

<110> Jilin University

<120> A Recombinant Chicken Coccidian A Virus Particle Vaccine and Its Preparation Method

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 762

<212>DNA

<213> Eimeria tenella

<400> 1

atggactggc tttcgagcga gaaccaggcg ctgaagttgc cagtatggcc cactgcagtt 60

gagaaggtgc tgctttccga ggaacccact cctgatgatt tcaccgagga gctccaaggg 120

ggtgcaccca acatgaacat gttgcctggc ttcaccacac tggagaacta ccccaaccca 180

atggtcaact cgatggcctg cagaagatgg ggcatggaag tttccttcgt ctgcgaccca 240

gatcatgtgttttctagcgg cacgttggac agaatagagg aacgcctttc caatatccga 300

cacaactcgt cccaccgctg cgtcgacgaa tacgtctcct acccctttgg agtggcgatt 360

gttcaagaac tgccatacgg tgttgatgcg aacacgtttg cgaccgagct catagatcgc 420

tggggacttg gacacagcaa gtgccacgat gggcttctgc tgttgtatgt tgcgtccgac 480

ggtgacgttt cgctcaagtg gaagaaagga gtcgagcctg agatcaactt caggacatat 540

agcgggctcc tgcgcgaatt cagaaagaat gggggaaaac tgtccgctgg aagggctctt 600

gaggccgatg tcattgcagt tggacagcat ctgacagggg aaattcttcc acctcgtcag 660

accccgcatc tcgccgtgtt gctcacaatc gccagtattg tcgcccttgc gtatcttgca 720

tgcgccgcca ctgtcatttc tgacgagatt gccaagtatc gt 762

Claims (2)

1. a kind of Repair_PSII genes, its sequence is as shown in SEQ no.1.

2. one kind restructuring Eimeria species Alphavirus particle vaccines preparation method, comprises the following steps：

1）The structure of recombinant plasmid pSMART2b-Repair_PSII and expression

The total serum IgE and reverse transcription for extracting Eimeria tenella are cDNA, go out Repair_PSII genes by PCR amplification；Even Connect, convert and clone into plasmid pMD18-T-Repair_PSII, and pass through restriction enzyme BamHI and ClaI and biology Sticky end Repair_PSII genes, are connected on Alphavirus vector pSMART2b by sequencing identification connection with method； Then recombinant plasmid pSMART2b-Repair_PSII is transfected into 293 cells, Western blot identification Repair_PSII bases Because of expression；

2）Eimeria species Alphavirus particle vaccines are recombinated to prepare

It will identify and express correct recombinant plasmid pSMART2b-Repair_PSII and helper plasmid pSHCAR liposome methods 293 cells are transfected into, 6 change liquid when small, 48 collect virus stock solution used when small；α-gruel albumen of 1/20 volume is added into virus stock solution used Enzyme solutions（10 g/L）, it is incubated at room temperature 40min and p62 glycoprotein is cracked into E2 and E3 albumen with activated virus；Add 1/15 body Long-pending Aprotinin aprotinin（10 g/L）, it is incubated at room temperature 5 min；Take the virus stock solution used infection BHK-21 after appropriate activation thin Born of the same parents, 48 it is small when after collect restructuring Eimeria species Alphavirus particle and be used to take the photograph transmission electron microscope observing and Western blot identifications The expression of Repair_PSII genes；

3）Recombinate Eimeria species Alphavirus particle vaccines Optimization of preparation

By recombinant plasmid pSMART2b-Repair_PSII and helper plasmid pSHCAR according to molar ratio 1:1.5 common 2.5ug is total to 293 cells are transfected into, 6 change liquid when small, 48 collect virus stock solution used when small；Contaminated using helper plasmid pSHCAR 2.5ug single-turns are first taken Enter 293 cells, 6 change liquid when small, 24 hours whens take recombinant plasmid pSMART2b-Repair_PSII 2.5ug are mono- to be transfected into 293 again Cell, 6 change liquid when small, 48 collect virus stock solution used when small；The virus stock solution used of optimization is infected into BHK-21 cells respectively, up to recombinating Eimeria species Alphavirus particle.