Construction technology of recombinant insect cross virus expression vector

A construction method and expression vector technology, which are applied in the construction field of insect recombinant hybrid virus expression vector, can solve the problems of long time virification, small host insects, difficult industrialization level, etc., and achieve a wide host range and realize industrialization. The effect of level development and production level improvement

Inactive Publication Date: 2004-09-22
ZHEJIANG UNIV
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Problems solved by technology

However, the BmNPV expression system also has disadvantages: because BmNPV host is specific and only has the ability to infect silkworms, only silkworm cultured cells can be used in the construction of recombinant viruses, and silkworm cells have the characteristics of slow growth and subculture, so It takes a long time for the construction of recombinant virus, screening and the multiplication of a large number of viruses for expression
But its shortcomings are also obvious, that is, the host insects are small and difficult to raise, and it is difficult to be suitable for the development of industrialization level.

Method used

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  • Construction technology of recombinant insect cross virus expression vector
  • Construction technology of recombinant insect cross virus expression vector
  • Construction technology of recombinant insect cross virus expression vector

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Experimental program
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Embodiment Construction

[0022] 1. Materials: Insect cultured cells Sf21 and silkworm BmN cells, AcNPV and BmNPV viruses, Sf21 cell culture medium TC-100 adopts GibcoBRL products, and 10% fetal bovine serum is added before use. Serum ESF921 medium, the product of Expression system company, the culture temperature is 27 ℃, silkworm larvae 1-3 instars are raised on artificial feed, reared at 29 ℃, 4-5 instars are raised in mulberry leaves, reared at 23-25 ​​℃, genetic manipulation institute The various enzymes and PCR amplification kits used are TA cloning kits, which are products of Takara Shuzo Co., Ltd., and the plasmid pCR2.1 was purchased from Invitrogen Company in the United States. The reagents are products of LIFETECHNOLOGIES, and the DNA sequence was determined on a DNA sequencer according to the kit provided by PE Applied Biosystems.

[0023] 2. Construction of hybrid recombinant virus: the construction process is as follows figure 1 express.

[0024] First, the DNA helicase gene was cloned ...

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Abstract

The present invention relates to construction technique of insect recombination hybridization virus expression carrier, it uses isogenous recombination priciple to construct hybridization reconstruction virus expression carrier HyNPV situated between AcNPV and BmNPV. Said expression carrier solves the problem of narrow host range of BmNPV expression system and degradation of recombination object product by protease. It possesses the advantage of broad host range, not only can infect domestic silkworm, but also can infect commonly used Sf21, Sf9 and Tn-5 culture cell, also can use Sf21, Sf9 culture cell to instead domestic silkworm cell to hasthen the preparation of recombination virus.

Description

technical field [0001] The invention relates to a construction method of an insect recombinant hybrid virus expression vector. Background technique [0002] The production of recombinant proteins by means of genetic engineering such as recombinant DNA is the main driving force behind the rapid development of the biopharmaceutical industry in recent years. At present, there are three different levels of expression systems for the production of recombinant proteins: prokaryotic, insect and mammalian cells. Among them, the Baculovirus Expression Vector System, or BEVS for short, has become the most effective due to its high-efficiency expression, relatively complete protein post-translational processing, large-scale expression and production using insect individuals or their cultured cells, and good safety. one of the eukaryotic expression systems. At present, there are two main types of BEVS: the Autographa californica nuclear polyhedrosis virus A...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09C12N15/63C12N15/64C12N15/86
Inventor 吴小锋
Owner ZHEJIANG UNIV
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