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A kind of avian reticuloendotheliosis virus subunit vaccine injection compound and its preparation method

A subunit vaccine and poultry reticuloendothelial technology, applied in biochemical equipment and methods, viruses, microorganisms, etc., can solve the problems of lack of control effect, few expression products, complicated development process, etc., and achieve good commercialization To develop prospects, prevent or treat infection effects

Active Publication Date: 2021-06-29
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Cui Zhizhong once reported the use of attenuated strains to infect breeders to induce the production of maternal antibodies to block the early infection of chicks; recently reported the use of baculovirus expression system and Pichia pastoris expression system and eukaryotic expression plasmid pCAGGS on REV envelope gene The expression of gp90 is induced to obtain a recombinant protein with certain immunoprotective properties, but the above methods have certain limitations. The weakened virus immunogen in the former has the risk of returning to strength and rejuvenation, and the antibody titer induced by the latter is still biased. However, it still cannot produce enough protection. In addition, the development process is complicated, technical, and the disadvantages of obtaining less expression products and high cost also limit the clinical application of this method.
[0004] The use of prokaryotic expression systems to prepare recombinant proteins with simple structures is still the main method for producing subunit vaccines or immunogens. It has the advantages of relatively simple operation, large amount of expression products, easy purification, and low cost, but the immunogenicity is relatively low. People often use immune adjuvants to improve the immunogenicity of prokaryotic expression products
Traditional immune adjuvants are oil-emulsion adjuvants, which can improve the immunogenicity and immune protection ability of inactivated vaccines and protein antigens, but compared with attenuated vaccines, there is still a big gap, and often fail to meet the needs of clinical production. prevention effect

Method used

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  • A kind of avian reticuloendotheliosis virus subunit vaccine injection compound and its preparation method
  • A kind of avian reticuloendotheliosis virus subunit vaccine injection compound and its preparation method
  • A kind of avian reticuloendotheliosis virus subunit vaccine injection compound and its preparation method

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Experimental program
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Effect test

Embodiment 1

[0040] The preparation of embodiment 1 REV immunization antigen

[0041] Specific method: using designed primers

[0042] Upstream primer: 5-CCGGAATTCTCCTCCATACCTACCTATTACA-3', the nucleotide sequence of which is shown in Seq ID No:1;

[0043] Downstream primer: 5-AGAGTCGACTCAATTAGGGAGTGCCGTGT-3, the nucleotide sequence of which is shown in Seq ID No:2;

[0044] To amplify the REV-gp90 gene, the steps are: use a DNA extraction kit to extract viral DNA from REV-infected cells, use the DNA as a template, and use the 20 μL PCR reaction system in the Molecular Biology Experiment Manual as a benchmark for the REVgp90 gene Amplification, PCR reaction conditions: 95°C for 3min, make it fully denatured and then enter the cycle system: denature at 95°C for 30s, anneal at 61°C for 30s, extend at 72°C for 70s, cycle 30 times, and finally extend at 72°C for 10min. The amplified product was obtained by nucleic acid electrophoresis test figure 1 The nucleic acid band corresponding to the...

Embodiment 2

[0049] The preparation of embodiment 2 nucleic acid sustained-release adjuvants

[0050] The preparation method of the nucleic acid sustained-release adjuvant is as follows: No. 7 white oil and Siben 80 are mixed at a volume ratio of 94% and 6%, respectively, and then aluminum stearate is added to the above mixture at a mass ratio of 2%, and the mixture is autoclaved at 116°C Bacteria, cooled to 50°C, shake vigorously until transparent;

[0051] Then add all the thioated-modified CpG-ODN at 100 μg / mL, mix well, and prepare an oil emulsion;

[0052] Wherein CpG-ODN is a fully thio-modified 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' nucleic acid molecule, and its nucleotide sequence is shown in Seq ID No:3.

Embodiment 3

[0053] Example 3 Preparation of Avian Reticuloendotheliosis Virus Subunit Vaccine Injection Complex

[0054] Specific steps are as follows:

[0055] The purified gp90 protein antigen was diluted to 1.0 mg / mL with PBS, then mixed according to the volume ratio of protein antigen:immune adjuvant at 1:1, shaken on the shaker for 1 hour, and emulsified by ultrasonic breaker, ultrasonic emulsified The conditions are: the power is 350W, the working time is 3s, the intermittent time is 2s, the temperature is controlled at 25°C, and the ultrasound is stopped after the emulsification is complete.

[0056] The method for judging the complete emulsification is as follows: Dip the immunogen in the centrifuge tube with a syringe needle, shake the needle to drop the liquid into pre-cooled water, and judge that the emulsification is complete if it does not spread within 30 minutes.

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Abstract

The invention relates to the field of biological immunity, and provides an avian reticuloendotheliosis virus (REV) subunit vaccine injection compound and a preparation method thereof, including an avian reticuloendotheliosis virus (REV) subunit vaccine antigen and a corresponding immune adjuvant The above-mentioned antigens are obtained and combined with adjuvants for immunization. The subunit vaccine is prepared by amplifying the primers designed according to the REV envelope protein gp90 gene to obtain the target gene, and the recombinant protein is prepared by expressing Escherichia coli to obtain the vaccine antigen. After the recombinant antigen is emulsified and mixed with the thiolated nucleic acid slow-release adjuvant, the bird is immunized, which can effectively prevent or treat the infection of the bird REV, and has a good prospect for commercial development.

Description

technical field [0001] The invention relates to the field of biological immunity and provides an avian reticuloendotheliosis virus (REV) subunit vaccine injection compound and a preparation method thereof. Background technique [0002] Reticuloendothelial hyperplasia virus (REV) is a single-stranded RNA virus that can cause immunosuppression and reticuloendothelial tissue hyperplasia in infected chickens. Epidemiological surveys in Guangxi, Anhui, Jiangxi and other places in my country in the past three years show that the infection rate of REV is above 30%, second only to Marek's disease, showing that double or multiple infections are the main The main popular trend, brings serious economic loss to my country's poultry industry every year. The virus can be transmitted vertically and horizontally in chicken flocks. In the natural state, the former is the dominant type. Lateral transmission mostly occurs in chickens with low immunity or insect bites and contaminated vaccinatio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/21A61K39/39A61P31/14
CPCA61K39/12A61K39/39A61K2039/54A61K2039/55561C12N2740/13034
Inventor 郭慧君袁霏李宏梅
Owner SHANDONG AGRICULTURAL UNIVERSITY
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