Method for preparing cyprini herpesvirus II antigen coated polyhedrosis based on baculovirus expression system

A technology of carp herpes virus and expression system, which is applied in the fields of virus/bacteriophage, botany equipment and methods, biochemical equipment and methods, etc. It can solve the problems of no research reports, many influencing factors, complicated preparation process, etc., and achieve biosafety High performance, high expression level, and low production cost

Active Publication Date: 2017-06-13
苏州培恩特生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the preparation process of sodium alginate antigen microspheres is more complicated, and there are many influencing factors; plasmopolyhedron is a protein polyhedron crystal embedded with virus particles formed by insect plasmopolyhedrosis virus in infected cells
[0005] Therefore, the preparation of polyhedrons encapsulating CMV-II antigens through the rod-shaped expression system may serve as a good vaccine, but so far, there has been no relevant research report

Method used

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  • Method for preparing cyprini herpesvirus II antigen coated polyhedrosis based on baculovirus expression system
  • Method for preparing cyprini herpesvirus II antigen coated polyhedrosis based on baculovirus expression system
  • Method for preparing cyprini herpesvirus II antigen coated polyhedrosis based on baculovirus expression system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Preparation method of polyhedron encapsulating carp herpesvirus type II antigen

[0039] (1) Use QIAGEN Viral RNA Mini Kit (Qiagen Company) to extract Bombyx mori plastiform polyhedrosis genomic RNA, and use RNA PCR KitVer.3.0 (Qiagen Company) to convert viral RNA into cDNA according to the instructions in the product catalog, and use SEQ ID NO: 1 as Forward primer CPV-PH-EI ( Eco RI restriction site), with SEQ ID NO:2 as the reverse primer ( Xba I enzyme cutting site), the 5' end and 3' end bands were synthesized by PCR Eco RI and Xba The coding sequence of the plasmid polyhedrin gene at the I site was cloned into the T-vector to obtain the pMD19T-PH plasmid. After the plasmid was verified by sequencing, use Eco RI and Xba I digest the pMD19T-PH plasmid, recover the plasmid polyhedrin gene fragment, and clone it into pFastBac Dual Eco RI and Xba I site, construct the recombinant transfer plasmid pFastBac Dual-polh.

[0040] (2) Synthesize the fusi...

Embodiment 2

[0046] Example 2 Preparation method of polyhedron encapsulating carp herpesvirus type II antigen

[0047] Compared with Example 1, in step (5), culture at 26°C for 4 days, collect the supernatant of the cultured cells infected with the virus, and obtain the P1 generation recombinant virus BmNPV-VP3-cyHV-polh. Inoculate silkworm cultured cells with the P1 recombinant virus, culture at 26°C until the cells become diseased, collect the cell supernatant to obtain the P2 virus; in step (6), use insect needles to get the P2 recombinant virus to inoculate the silkworm chrysalis, and keep at 25°C for 5 days Around the same time, the silkworm chrysalis becomes ill. The rest of the steps are the same, through repeated differential centrifugation at 1000 rpm and 3000 rpm, the cytoplasmic polyhedron can be purified from the hemolymph of the silkworm chrysalis, which is protein microcrystals of about 2-3 microns. About 1.5×10 8 polygons. Microscopic results and electrophoresis results s...

Embodiment 3

[0048] Example 3 Preparation method of polyhedron encapsulating carp herpesvirus type II antigen

[0049] Compared with Example 1, in the step (6), the P2 generation recombinant virus is inoculated into the 4th instar silkworm larvae, and the rest of the steps are the same, and can be purified to the cytoplasmic polyhedron through repeated differential centrifugation at 1000 rpm and 3000 rpm. Body, protein microcrystals of about 2-3 microns; about 10 8 polygons. Microscopic results and electrophoresis results show that the present invention clearly obtains the polyhedron wrapping the cyprin herpesvirus type II antigen.

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Abstract

The invention relates to the technology of antigen protein expression, and particularly relates to a method for preparing a cyprini herpesvirus II antigen coated polyhedrosis based on a baculovirus expression system. According to the method, by designing and recombining bombyx mori nuclear polyhydrosis virus BmNPV-VP3-cyHV-polh, 1-186, 993-1197, 603-783 and 85-186 regional sequences of ORF72, ORF66, ORF81 and ORF82 and the coding sequence of the 1-279 region of a VP3 gene of bombyx mori cytoplasmic polyhedrosis virus structural protein are connected in series to form a fused sequence which is controlled by baculovirus P10 promoter; and the bombyx mori cytoplasmic polyhedrosis protein gene is controlled by a polyhedrin gene promoter of baculovirus. The virus is used for inoculating bombyx mori or bombyx mori culture cells, the recombinant virus expressed cyprini herpesvirus II antigen protein can be coated in bombyx mori cytoplasmic polyhedrosis; the formed polyhedrosis can be purified by simple differential centrifugation; the purified polyhedrosis is cracked under a basic condition, the polyhedrosis protein can be precipitated by centrifuging, and the cyprini herpesvirus II antigen is reserved in supernatant, so that the cyprini herpesvirus II antigen can be quickly and conveniently obtained.

Description

technical field [0001] The invention relates to the technical field of viral genetic engineering, in particular to a method for preparing a polyhedron encapsulating carp herpesvirus type II antigen based on a baculovirus expression system. Background technique [0002] Hemorrhagic gill disease of heterogeneous gibel carp is caused by Cyprinid herpesvirus II (CyHV-2) infection. At present, the prevention and treatment of this disease mainly focuses on enhancing the immune ability of fish, reducing stress response, polyculture, and reducing stocking density. , strengthening disease detection, timely removal of diseased fish and other aspects of control. It is generally believed that immune prophylaxis is one of the most effective methods for the prevention and treatment of fish viral diseases. At present, fish vaccines mainly include inactivated vaccines (killed vaccines), attenuated vaccines, subunit vaccines, DNA vaccines, and protein subunit / DNA combined vaccines. Inactiv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/866C12N7/04A61K39/245A61P31/22C12N15/62
CPCA61K39/00C07K14/005C07K2319/00C12N7/00C12N15/86C12N2710/14022C12N2710/14043C12N2710/16022C12N2710/16034C12N2710/16051C12N2800/105
Inventor 贡成良曹广力胡小龙薛仁宇刘波李坤
Owner 苏州培恩特生物科技有限公司
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