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Chicken Infectious Anemia Virus Genetic Engineering Vaccine

A technique for encoding genes and recombining baculoviruses, applied in genetic engineering, viruses, vaccines, etc., can solve problems such as weakened immune response, low expression of VP1, strong virulence, etc., to reduce production costs, improve assembly efficiency, good immune effect

Active Publication Date: 2020-11-20
苏州世诺生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, CN110028558A provides a method for co-expressing the VP1 and VP2 proteins of truncated CIAV to prepare subunit vaccines. Since the N-terminus of VP1 has a large amount of R and has a large number of positive charges, VP1 itself is easily degraded by alkaline protease , so that the expression level of VP1 is low, and the truncated VP1 removes the R-rich part of the N-terminus and cannot be assembled into VLP. Even if it is co-expressed with VP2, the expression level and assembly efficiency of VP1 protein are still low
As another example, CN109136198A provides a method for preparing recombinant fowlpox virus live vector vaccine expressing CIAV VP1 and VP2 genes, but compared with baculovirus, poxvirus vector safety is low, and repeated vaccination of poxvirus vector vaccine may produce Anti-vector immunization leads to a weakened immune response; inactivated vaccines cause unsatisfactory immune effects due to the immunosuppression caused by CIAV; attenuated live vaccines have residual virulence and the possibility of strong virulence

Method used

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  • Chicken Infectious Anemia Virus Genetic Engineering Vaccine
  • Chicken Infectious Anemia Virus Genetic Engineering Vaccine
  • Chicken Infectious Anemia Virus Genetic Engineering Vaccine

Examples

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preparation example Construction

[0069] In some embodiments, a method for preparing a chicken infectious anemia virus genetic engineering vaccine specifically includes:

[0070] 1) Prepare a nucleic acid molecule for encoding a chicken infectious anemia virus fusion protein (which can be defined as CIAV-Fu);

[0071] 2) Constructing a recombinant vector, cloning the nucleic acid molecule in step 1) into a shuttle vector to obtain a recombinant shuttle vector containing the target gene;

[0072] 3) Transform the recombinant shuttle vector into DH10Bac bacteria, select the recombinant bacteria, extract the genome and transfect Sf9 cells to obtain recombinant baculovirus;

[0073] 4) Cultivate the Sf9 cells, automatically assemble into virus-like particles (VLP) in the cells, and then recombinantly express and produce a fusion protein (defined as CIAV-VLP);

[0074] 5) Adding the CIAV-VLP into the adjuvant to obtain the vaccine.

[0075] The terminology used in this specification is for the purpose of describi...

Embodiment 1

[0128] Example 1 Construction and Identification of Transfer Vector pF-CIAV-Fu

[0129] 1. CIAV-Fu gene amplification and purification The codon-optimized CIAV-Fu gene (SEQ ID NO: 1) was synthesized in Shanghai Sunny Biotechnology Co., Ltd. and cloned into the pUC17 vector to obtain the pUC-Fu plasmid vector. Using the pUC-Fu plasmid as a template, CIAV-Fu-F and CIAV-Fu-R are used as upstream and downstream primers for PCR amplification (the sequences of CIAV-Fu-F and CIAV-Fu-R are shown in SEQ ID NO: 3 and 4 Shown), the amplification system is shown in Table 1.

[0130] Table 1 CIAV-Fu gene amplification system

[0131]

[0132] The reaction conditions were: 95°C pre-denaturation for 5 minutes; 94°C denaturation for 45 seconds, 54°C annealing for 45 seconds, 72°C extension for 1 minute, 35 cycles; 72°C extension for 10 minutes.

[0133] Perform gel electrophoresis on the PCR product to verify the size of the target gene, such as figure 1 As shown, the target band appea...

Embodiment 2

[0145] Example 2 Construction of recombinant baculovirus genome Bac-CIAV-Fu

[0146]1. Transformation of DH10Bac bacteria Take 1 μl of pF-CIAV-Fu plasmid in Example 1 and add 100 μl of DH10Bac competent cells to mix, ice bath for 30 minutes, heat shock in 42°C water bath for 90 seconds, ice bath for 2 minutes, add 900 μl without Amp LB liquid medium, cultured at 37°C for 5 hours. After 100 μl of bacterial solution was diluted 81 times, 100 μl of diluted bacterial solution was spread on LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal and IPTG, and cultured at 37°C for 48 hours.

[0147] 2. Pick a single colony Use an inoculation needle to pick up large white colonies, then streak on LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal, and IPTG, culture at 37°C for 48 hours, and then pick A single colony was inoculated into LB liquid medium containing gentamicin, kanamycin, and tetracycline for culture, the strains were preserved, and t...

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Abstract

The invention discloses a chicken infectious anemia virus genetic engineering vaccine. The chicken infectious anemia virus genetic engineering vaccine comprises fusion protein, the coding gene sequence of which is as shown in SEQ ID NO:1. The antigenicity, the immunogenicity and the function of the vaccine are similar to that of natural protein; the expression level is relatively high; the immunogenicity is high; the vaccine does not have pathogenicity to poultries, such as chickens; relatively high humoral immunity can be generated in the body of a poultry; an immunized poultry can resist strong toxicity and counteract toxicity; furthermore, the vaccine can be prepared by large-scale serum-free suspension culture through a bioreactor; and thus, the production cost of the vaccine is greatly reduced.

Description

technical field [0001] The invention relates to a genetic engineering vaccine, in particular to a chicken infectious anemia virus genetic engineering vaccine. The preparation method and application thereof belong to the technical field of animal immunity medicine. Background technique [0002] Chicken Infectious Anemia (CIA) is an immunosuppressive infectious disease caused by Chicken Infectious Anemia Virus (CIAV). Chicken is the main natural host of CIA, and chickens of all ages are susceptible to infection, among which chicken embryos and chicks less than 14 days old are the most susceptible and have the highest incidence rate. The characteristic lesions after CIAV infection are anemia and pale visible mucous membranes. The clinical symptoms of affected chickens are mainly depression, emaciation, beak anus, local skin is blue-purple, and prolongation of coagulation time. The pathological changes are mainly bone marrow atrophy, appearance Aplastic anemia and systemic lymp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/12A61P31/20C07K19/00C12N15/62C12N15/866
CPCA61K39/12A61K2039/552A61P31/20C07K14/005C07K2319/00C12N15/86C12N2710/14043C12N2750/10022C12N2750/10034C12N2800/105
Inventor 曹文龙孔迪滕小锘
Owner 苏州世诺生物技术有限公司
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