Method for producing classical swine fever virus subunit vaccine by utilizing bombyx mori bioreactor

A technology for subunit vaccine and swine fever virus, which is applied in the field of preparing recombinant subunit vaccine for swine fever, can solve the problems of silkworm body corruption, false positives, difficulty in culturing, etc., so as to improve screening efficiency, simplify production steps, and save production. cost effect

Inactive Publication Date: 2013-09-25
PU LIKE BIO ENG
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the above-mentioned method of using the BmNPV expression system to infect silkworms, the construction steps of the recombinant BmNPV virus are tedious; Long, difficult to cultivate, high technical requirements, and a high rate of false positives
In addition, the use

Method used

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  • Method for producing classical swine fever virus subunit vaccine by utilizing bombyx mori bioreactor
  • Method for producing classical swine fever virus subunit vaccine by utilizing bombyx mori bioreactor
  • Method for producing classical swine fever virus subunit vaccine by utilizing bombyx mori bioreactor

Examples

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Effect test

Embodiment 1

[0042] This embodiment describes the method for obtaining the classical swine fever virus E2 gene provided by the present invention, comprising the following steps:

[0043] (1) extraction of classical swine fever C strain attenuated vaccine strain virus genome RNA:

[0044] Harbin Veken Biotechnology Development Co., Ltd.'s live vaccine for swine fever (cell source) (the vaccine contains attenuated strain of swine fever lathe, batch number 080011004), diluted with normal saline, and treated according to the TIANamp virus of Tiangen Biochemical Technology (Beijing) Co., Ltd. The RNA extraction kit instructions were used to extract CSFV genomic RNA.

[0045] (2) Cloning of classical swine fever C strain attenuated vaccine strain gene:

[0046] 1. According to the sequence of the classical swine fever virus strain C-ZJ-2008 registered in GenBank (Classical swine fever virus strain C-ZJ-2008, GenBank accession number is HM175885), a pair of primers were designed to amplify the E...

Embodiment 2

[0050] This embodiment describes the method for producing recombinant CSFV E2 protein, expressing recombinant CSFV E2 protein with silkworm or chrysalis, and includes the following steps

[0051] (1) Transform the recombinant vector pFastBac-E2 obtained above into the Escherichia coli competent cell DH10Bac (Invitrogen, Catalog: 10361012) containing the full-length baculovirus genome shuttle vector, recombine in the Escherichia coli cell, and pass the cyanobacteria Bacmid was screened for the recombinant shuttle vector, and the recombinant shuttle vector was named Bacmid-E2.

[0052] (2) Transfect insect sf9 with the recombinant shuttle vector Bacmid-E2 described in step (1), and produce recombinant baculovirus in the cells, named as vBac-E2: prepare 5 × 10 5 cells / ml of sf9 cell suspension, insert 2ml / well of sf9 cells into a 6-well cell culture plate, and let stand at 27°C for more than 1 hour to allow the cells to adhere to the wall. Take a sterile 1.5ml centrifuge tube, a...

Embodiment 3

[0060] This example describes the CSFV genetically engineered subunit vaccine provided by the present invention and its efficacy evaluation.

[0061] The CSFV subunit vaccine is an injection vaccine: including the above-mentioned dilution of the hemolymph of silkworm larvae or silkworm chrysalis infected with the recombinant virus, immune adjuvants, preservatives and stabilizers. The recombinant CSFV E2 protein obtained in Example 3 and the ISA206 adjuvant (SEPPIC company) were prepared in a ratio of 46:54, maintained at 30° C., and stirred at 130 rpm / min for 1.5 h to prepare the recombinant CSFV E2 protein in the final prepared vaccine. The protein content is 50 μg / mL, and each dose is 1 ml, that is, each dose of vaccine contains 50 μg of CSFV E2 antigen.

[0062] Screen 5 piglets with 4-week-old CSFV seronegative piglets, inject 1 dose (1ml / dose) per pig intramuscularly with the vaccine prepared above, and inject 1 dose after 2 weeks. After the first injection, the blood of...

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Abstract

The invention relates to a method for expressing a classical swine fever virus (CSFV) recombinant E2 protein by utilizing a bombyx mori bioreactor of a bombyx mori larvae and/or a bombyx mori chrysalis. With the use of a gene engineering technology, a CSFV E2 gene and upstream 90 nucleotide sequences are recombined into an autographa californica nuclear polyhedrosis virus (AcNPV) genome, a recombinant AcNPV is obtained, and the CSFV recombinant E2 protein is expressed by injecting the recombinant virus into the bombyx mori or the chrysalis. A CSFV subunit vaccine is produced by the recombinant CSFV recombinant E2 protein, and includes a CSFV subunit E2 protein antigen and an immune adjuvant.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a method and a product for expressing a large amount of recombinant E2 protein of classical swine fever virus in silkworms by means of genetic engineering and using the recombinant protein to prepare a recombinant subunit vaccine of classical swine fever. Background technique [0002] Classical Swine Fever (CSF) is caused by Classical Swine Fever Virus (CSFV), which is acute or chronic and has a high fatality rate. Its pathological features are degeneration of small blood vessel walls, multiple hemorrhages in internal organs, Infarction and necrosis are extremely harmful swine infectious diseases. [0003] Chinese patent CN101736017A discloses a method of using recombinant silkworm baculovirus (BmNPV) to produce CSFV E2 protein, using BmNPV to infect silkworm larvae or silkworm chrysalis to express foreign protein (CSFV E2 protein), that is, by constructing recombinant BmNPV, and...

Claims

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Application Information

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IPC IPC(8): C07K14/18C12N15/866A01K67/04A61K39/187A61P31/14G01N33/68
Inventor 张许科孙进忠白朝勇
Owner PU LIKE BIO ENG
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