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Rabbit hemorrhagic disease virus recombinant subunit vaccine and preparation method thereof

A subunit vaccine and virus technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, antiviral agents, etc., can solve the problem of low expression level of foreign proteins, improve the immune effect, simplify the purification process, The effect of improving protein expression

Active Publication Date: 2014-05-14
QILU ANIMAL HEALTH PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the expression level of the foreign protein is usually still much lower than that of the polyhedrin protein expressed by the wild-type virus

Method used

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  • Rabbit hemorrhagic disease virus recombinant subunit vaccine and preparation method thereof
  • Rabbit hemorrhagic disease virus recombinant subunit vaccine and preparation method thereof
  • Rabbit hemorrhagic disease virus recombinant subunit vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] ——Construction of recombinant insect cell baculovirus

[0101] 1. Amplification of Rabbit Viral Hemorrhagic Disease Virus (RHDV) Structural Protein VP60 Gene

[0102] Extract the genomic RNA of the RHDV Beijing strain owned by Qilu Animal Health Company, according to the genome sequence of a strain of RHDV published on NCBI (the protein sequence of the protein published by GENBANK: (accession number: NP_740333.1), and the corresponding The gene sequence can be found in the rabbit plague virus genome, protein number: NP740333.1.), design and synthesize VP60 gene upstream and downstream primers (synthesized by Shanghai Sangong) VP60-F (sequence 2) / VP60-R (sequence 3) . Then use RT-PCR to amplify the VP60 gene, clone the amplified gene into pMD19-T vector (pMD19-VP60), and store it in E.coli as a plasmid.

[0103] 2. Construction of transfer vectors containing insulator cis-acting elements and melittin signal peptide

[0104] (1) Construction of vector containing melitt...

Embodiment 2

[0134] ——Batch preparation of recombinant baculovirus seed virus

Embodiment 3

[0137] ——Batch preparation of recombinant baculovirus seed virus for Sf9 cell production

[0138] Cells cryopreserved in liquid nitrogen were inoculated into 250ml shaker flasks after recovery, and cultured on a constant temperature shaker at 27°C with a rotation speed of 110r / min. When the cell density reaches 3.0×10 6 When the cells / ml is above, scale up to a 1L shake flask according to the volume ratio of 1:2, and finally scale up to a 3L shake flask. 3L shake flask cells up to 2.0 x 10 6 In cells / ml, inoculate the recombinant baculovirus according to MOI=0.05-0.1, culture in suspension at 27°C for 80-96 hours, harvest the culture medium, take a sample to determine the titer of HA, quantitatively aliquot it, and freeze it to store it, which is the virus species for production. Indicate the date of harvest, generation of poisonous seeds, etc.

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Abstract

The invention relates to a rabbit hemorrhagic disease virus recombinant subunit vaccine and a preparation method thereof. A vaccine production strain is a recombinant autographa californica nuclear polyhedrosis virus CGMCC No.8052 (China General Microbiological Culture Collection Center Number 8052), an expression vector adopted by the strain is a secretory expression vector and is provided with six histidine tags easy for protein detection and analysis; the strain is used as the production strain for producing the rabbit hemorrhagic disease virus recombinant subunit vaccine; the vaccine is free from viral genome, animal safety hazard and potential dispersity, is high in immunity pertinence, and can be used for exciting immunized animals to generate sufficient immune antibodies and long protective efficacy. RHDV-like (rabbit hemorrhagic disease virus-like) particles are produced according to the technology provided by the invention, the hemagglutination titer of virus liquid obtained from a 1000L bioreactor is (1 to 32768) to (1 to 262144), the protein expression quantity of the RHDV particles is about 150mg / L, 1L of semi-finished virus liquid can be prepared into 5,000-100,000 standard vaccines, and the preparation efficiency is higher than the domestic technical level by 8-20 times; the recombinant subunit vaccine is added with an adjuvant, so that the immune effect of the vaccine is improved; and the vaccine is a novel vaccine remarkably superior to an inactivated tissue vaccine produced in the prior art.

Description

technical field [0001] The invention relates to a recombinant subunit vaccine of rabbit viral hemorrhagic disease virus and a preparation method thereof. The vaccine is a recombinant subunit vaccine produced by using an insect baculovirus expression system and has the effect of resisting rabbit viral hemorrhagic disease, and belongs to the field of veterinary biological products and biotechnology. Background technique [0002] Rabbit hemorrhagic disease (RHD) is a highly contagious and fatal disease. Its hosts include wild rabbits and domestic rabbits. It has become a major threat to the survival of wild rabbits and the development of rabbit derivative industries. [0003] RHDV (Rabbit hemorrhagic disease virus, RHDV) is a virus that causes RHD. the virus causing rabbit hemorrhagic disease.Journal of Virology.1990(64)3331-3336; F Parra and M Prieto. Purification and characterization of a calicivirus as the causative agent of a lethal hemorrhagic disease in rabbits..Journal ...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/866A61K39/12A61P31/14C12R1/93
Inventor 闵庆如易小萍范志永冀海明王秀梅张大鹤宋晓飞徐龙涛张连秀王蕾禚宝山鲍海忠
Owner QILU ANIMAL HEALTH PROD
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