Pronuclei fusion expression vector and application thereof

A technology of fusion expression and expression vector, applied in the field of genetic engineering, to achieve the effect of good solubility and easy purification

Inactive Publication Date: 2014-04-09
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The fusion expression system has obvious advantages, but there are still some areas to be improved

Method used

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  • Pronuclei fusion expression vector and application thereof
  • Pronuclei fusion expression vector and application thereof
  • Pronuclei fusion expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1, the construction of fusion expression vector pPDI

[0043] 1. PCR amplification of PDI gene

[0044] PDI is obtained from the Conus snail cDNA library, therefore, the PDI gene was amplified using the Conus snail cDNA as a template by PCR method. According to the analysis of RACE results by computer software, a pair of primers were designed and synthesized:

[0045] The upstream primer is 5’–ATGGCTAGCATGAAGTTTTCATCTTGTT-3’,

[0046] The downstream primer is 5'-GATCCCAGTTCATCTCTTGGCAGATTCTCA-3'.

[0047] For the convenience of cloning, NheI and BamHI restriction enzyme recognition sequences were added to the upstream and downstream primers respectively.

[0048] The PCR reaction was carried out according to the Takara system method.

[0049] 2. Construction of transfer vector pGEM-PDI containing PDI gene

[0050] The PDI gene amplified by PCR was detected and recovered by agarose gel electrophoresis, and then double-cut by Nhe I and BamH I and inserted ...

Embodiment 2

[0057] Example 2. Application of Fusion Expression Vector pPDI

[0058] The conus snail PPI gene and lt14a gene were inserted into the SacI and HindIII restriction restriction windows to construct fusion expression vectors pPDI-PPI and pPDI-lt14a.

[0059] Transform pPDI-PPI, pPDI-lt14a into E. coli BL21C DE3). Inoculate a single colony in 50ml kanamycin-resistant LB medium, culture overnight at 37°C, 250rpm, inoculate 20mL of the overnight culture into 2L ammonium-resistant LB medium, cultivate at 37°C, 250rpm until OD600=0.6 , adding 100mM IPTG and 20% glucose to a final concentration of 1mM and 0.2%, respectively, 18°C, 250rpm induction culture for 14h, and centrifuged to harvest the cells, sonicate the cells, centrifuge to collect the supernatant, and use Ni 2+ Chelating Sepharose affinity chromatography purification. For the expression and purification of pPDI-lt14a fusion protein, see Figure 5 .

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Abstract

The invention provides a pronuclei fusion expression vector which comprises a nucleotide fragment. The nucleotide fragment is composed of a promoter, a gene for coding a PDI (Protein Disulfide Isomerase) parasporal protein, and a joining region which are serially connected, wherein the joining region contains an enterokinase cleavage site and a multiple cloning site. The pronuclei fusion expression vector is capable of efficiently expressing an exogenous gene in a form of a fusion protein, is good in solubility of an expression product, simple in purification, and has natural bioactivity of the exogenous gene. The pronuclei fusion expression vector is suitable for mass production of a recombinant protein, is suitable for research and production of a genetic engineering product and a biological agent, and can also be used for researching a protein function and conformation relationship.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a prokaryotic fusion expression vector and its application. Background technique [0002] There are two types of expression systems for conventional genetic engineering: prokaryotic expression systems and eukaryotic expression systems. The expression of exogenous gene in prokaryotic cells is to make the cloned exogenous gene to synthesize exogenous gene expression products rapidly and efficiently in prokaryotic cells by fermentation. Prokaryotic expression system is currently the most well-understood and most widely used expression system. [0003] When expressing foreign genes in prokaryotic cells, fusion and non-fusion recombinant proteins can generally be produced due to different experimental designs. When expressing a foreign gene in a non-fusion manner (that is, the amino acid sequence of the obtained recombinant protein is consistent with the target product...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/66C12N15/10
Inventor 徐安龙王磊任政华王晓敏唐炜邹帆陈尚武
Owner SUN YAT SEN UNIV
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