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A construction and expression method for co-expressing foreign proteins of baculovirus by using multi-copy genes

A baculovirus and exogenous protein technology, applied in the field of genetic engineering, can solve the problems of decreased expression efficiency of exogenous protein, no baculovirus expression system, etc., and achieve the effect of high-efficiency expression and increase of expression amount

Active Publication Date: 2021-04-27
NANYANG NORMAL UNIV
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Problems solved by technology

However, there are also reports in the literature that when multiple copies of exogenous genes are transcribed and translated, each gene and the promoter will interact with each other, resulting in a decrease in the expression efficiency of the exogenous protein.
However, there is no systematic study on the relationship between the copy number of the foreign gene and its protein expression in the baculovirus expression system

Method used

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  • A construction and expression method for co-expressing foreign proteins of baculovirus by using multi-copy genes
  • A construction and expression method for co-expressing foreign proteins of baculovirus by using multi-copy genes
  • A construction and expression method for co-expressing foreign proteins of baculovirus by using multi-copy genes

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Embodiment 1

[0041] A method for constructing and expressing a foreign protein of baculovirus co-expressed by using multiple copies of genes, comprising the following steps:

[0042] 1. PCR amplification of firefly luciferase gene:

[0043] Using the plasmid Pet28a-Fluc as a template, a pair of specific primers BSFlucF and XhoFlucR were designed, wherein the BSFlucF primer was added with BamHI and SmaI restriction sites, and the XhoFlucR primer was added with an XhoI restriction site. Utilize PCR method to amplify then and obtain Fluc gene fragment, gene sequence length is 1671bp (result see figure 1 ), after PCR amplification, the PCR product was sent to Beijing Huada Gene for sequencing verification.

[0044] 2. Construction of multi-copy exogenous gene transfer vector driven by p10 promoter:

[0045] The correctly sequenced Fluc gene fragment was digested by SmaI and XhoI and then ligated into the same restriction site of pFBDM-IG to obtain the vector pFBDM-p10F-IG; then pFBDM-p10F-IG...

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Abstract

The invention discloses a method for constructing and expressing foreign proteins of baculovirus co-expressed by using multi-copy genes. The firefly luciferase Fluc gene is used as the target gene to construct polyhedron polh promoter and p10 promoter controlled containing Recombinant baculoviruses with 1-3 gene copies were used to infect the SF9 cell line, and the cells were collected after infection to detect the activity of firefly luciferase. The beneficial effect of the present invention is: the method provided by the present invention, after detection, it is found that compared with the unmodified baculovirus, the expression of the firefly luciferase gene is significantly increased by 2-5 times, effectively improving the expression level of the exogenous gene in the baculovirus. The expression level in the virus system is suitable for the production of proteins with natural activity, which is of great significance. It provides a new strategy for constructing recombinant viruses for production using the baculovirus multi-gene expression system, realizing the realization of exogenous genes. High-efficiency expression in the baculovirus multi-gene expression system.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for constructing and expressing a baculovirus exogenous protein co-expressed by using multi-copy genes. Background technique [0002] At present, the baculovirus expression system is widely used in drug research and development, vaccine production, gene therapy, recombinant baculovirus insecticides and other fields. Because the baculovirus has a powerful polyhedron (polh) promoter and p10 promoter, the expressed foreign protein has good biological activity after modification and processing, and its genome can accommodate the insertion of large foreign gene fragments, etc. Therefore, the insect-baculovirus expression system has become a recognized excellent eukaryotic expression system. In recent years, drugs and vaccines produced by recombinant baculoviruses have been successfully marketed, such as the human papillomavirus vaccine produced by the baculovirus...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/67C12N15/66C12N15/866
CPCC12N9/0069C12N15/66C12N15/67C12N15/86C12N2710/14143C12Y113/12007
Inventor 刘阳坤姚伦广李娜胡小敏王铁军谷娟娟尹延震
Owner NANYANG NORMAL UNIV
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