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Gene knockout type baculovirus expression vector

A technology of baculovirus and expression vector, which is applied in genetic engineering, plant gene improvement, biochemical equipment and methods, etc., can solve the problem of low expression level, and achieve high protein expression level, excellent high-yield characteristics, and excellent production traits Effect

Active Publication Date: 2022-04-12
陕西杆粒生物科技有限公司
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Problems solved by technology

[0005] The purpose of the present invention is to provide a gene knockout type high-yield recombinant baculovirus vector, aiming to solve the problem of low expression level mentioned in the background technology

Method used

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  • Gene knockout type baculovirus expression vector
  • Gene knockout type baculovirus expression vector
  • Gene knockout type baculovirus expression vector

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Embodiment 1

[0024] 1. Knockout of Ac29-33 gene.

[0025] Using primers (SEQ ID NO:1 and SEQ ID NO:2) with 50bp upstream and downstream homology arms of Ac29-33, using the pTriEx1.1 plasmid as a template, the ampicillin resistance gene fragment was amplified, and a 1023bp PCR was obtained Product (SEQ ID NO:3).

[0026] The obtained PCR product was transformed into the E. coli strain HS996 with RedET plasmid and Bacmid by electroporation, induced by arabinose to produce recombinase, and the recombined E. coli was screened on the ampicillin resistance plate and verified by colony PCR. Positive clones were obtained. At this point the Ac29-33 DNA fragment has been replaced by the ampicillin resistance gene fragment. Bacmid was extracted from Escherichia coli HS996 and named BacmidΔAc29-33 after sequencing and identification (see appendix for knockout strategy figure 1 ).

[0027] After linearization of BacmidΔAc29-33 by Bsu36I restriction endonuclease, it was used in subsequent experiment...

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Abstract

The invention discloses a gene knockout type baculovirus expression vector, and relates to the technical field of biology. According to the gene knockout type baculovirus expression vector, five continuous non-essential genes Ac29, Ac30, Ac31, Ac32 and Ac33 on the baculovirus expression vector are knocked out at the same time, and then the gene knockout type baculovirus expression vector is obtained. According to the gene knockout type baculovirus expression vector disclosed by the invention, the expression level of foreign proteins is improved by 80%. Meanwhile, the proliferation characteristic of the virus is not influenced. And the production cost of enterprises can be obviously reduced by improving the expression yield. The baculovirus expression vector can be used in the industrial field of biological products, especially in the industrial field of subunit vaccines.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a gene knockout type baculovirus expression vector. Background technique [0002] Baculovirus is a double-stranded DNA virus that specifically infects arthropods. Autographa californica nucleopolyhedrovirus (AcMNPV) is the type species of baculovirus. Since Smith GE and others first expressed human β-interferon gene in insect cells with baculovirus in 1983 (Mol Cell Biol. 1983; 3:2156-65.), due to its low cost, high yield, and various translation Post-modification system and other advantages, the baculovirus expression vector system is widely used in scientific research and production. [0003] However, compared with the prokaryotic expression systems (Escherichia coli, Bacillus subtilis) and yeast expression systems commonly used in industry, the yield of the baculovirus expression vector system is not satisfactory, which leads to the high production cost of the baculovirus express...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/866C12N15/54C12N15/34
CPCY02A50/30
Inventor 不公告发明人
Owner 陕西杆粒生物科技有限公司
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