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Method for constructing and expressing multicopy gene co-expressing baculovirus exogenous protein

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 increased expression

Active Publication Date: 2018-05-11
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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  • Method for constructing and expressing multicopy gene co-expressing baculovirus exogenous protein
  • Method for constructing and expressing multicopy gene co-expressing baculovirus exogenous protein
  • Method for constructing and expressing multicopy gene co-expressing baculovirus exogenous protein

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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 a multicopy gene co-expressing baculovirus exogenous protein. The method comprises constructing recombinant baculovirus having 1-3 genecopy numbers and controlled by polyhedrin polh promoters polh and p10 through a firefly luciferase Fluc gene as a desired gene, infecting a SF9 cell line, then collecting cells and detecting the activity of the firefly luciferase. A detection result shows that compared with the unimproved baculovirus, the baculovirus improved by the method has the firefly luciferase gene expression quantity significantly increased by 2-5 times. The method significantly improves the expression quantity of the exogenous gene in the baculovirus system, is suitable for production of proteins with natural activity, has a great significance, provides a novel recombinant virus construction strategy for the baculovirus multi-gene expressing system and realizes the efficient expression of the exogenous gene in thebaculovirus multi-gene expressing 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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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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