Construction method of polycistron expression vector

An expression vector and polycistronic technology, which is applied in the field of construction of polycistronic expression vectors, can solve problems such as hindering protein transportation and affecting protein functions, and achieve the effect of wide range, simplified operation steps and few operation steps

Inactive Publication Date: 2009-12-23
HENAN UNIVERSITY OF TECHNOLOGY
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Problems solved by technology

If the expression product is a pharmaceutical protein, it may hinder the transportation of the protein in the body, or the multimeric form m

Method used

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  • Construction method of polycistron expression vector
  • Construction method of polycistron expression vector
  • Construction method of polycistron expression vector

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specific Embodiment 1

[0043] Chromogranin A (CGA) antifungal-derived peptide CGA-N46 was secreted and expressed in Bacillus subtilis using a tricistronic expression vector.

[0044] The restriction endonucleases used in the following steps were all purchased from NEB Company, the primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd., the T-vector pMD18-T was a product of TAKARA Company, and the DNA sequencing was provided by Shanghai Yingjun Biotechnology Co., Ltd. The company is done.

[0045] (1) Synthesis of two pairs of primers:

[0046] Because the T-vector pMD18-T that implementation example uses has multiple cloning sites (MCS), the expression vector pSBPTQ (Li Ruifang, etc. Acta Microbiology, 2006, 46 (5): 714-719.) is the subtilis subtilis constructed by the inventor Bacillus secretion expression vector, the carrier has the Bacillus subtilis levansucrase gene (sacB) promoter and signal peptide sequence, the restriction endonuclease site at the MCS place downstream of the pr...

specific Embodiment 2

[0060] Expression of Calreticulin (CRT)-derived Anticancer Polypeptide CRT-N58 in Escherichia coli Using Tricistronic Expression Vector

[0061] The restriction endonucleases used in the following steps were all purchased from NEB Company, the primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd., the T-vector pMD18-T was a product of TAKARA Company, and the DNA sequencing was provided by Shanghai Yingjun Biotechnology Co., Ltd. The company is done.

[0062] (1) Synthesis of two pairs of primers:

[0063] The T-vector pMD18-T used in the implementation example has multiple cloning sites (MCS), the expression vector is commercial pET-3c, and pET-3c is an E. source protein. The first pair of primers were designed according to the CRT cDNA. The 5′ end of the upstream primer introduced the start codon atg, and the downstream primer introduced an additional base a, large intestine, between the 3′ end of the NheI site and the 5′ end of the complementary sequence of ...

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Abstract

The invention relates to a construction method of a polycistron expression vector, which comprises the following steps: designing two pairs of primers by utilizing a commercial T-vector as an intermediate vector, introducing Nhel and Spel enzyme cutting sites and a ribosome bind site (RBS) in each primer, connecting an amplified product with the T-vector, cutting the obtained recombinant T-vector by using Nhel and Spel enzymes, and connecting an obtained DNA segment by using a DNA joining enzyme to enable an individual heterologous gene to increase progressively in the manner of 2n-1 (n is Gene number of the back recombinant T-vector), finally cutting the DNA segment containing a plurality of heterologous genes through a restriction enzyme corresponding to the enzyme cutting sites at two ends of the DNA segment, subcloning to the downstream of an expression vector promoter, and then obtaining the polycistron expression vector. The invention has the advantages of simple operation and wide application scope, and is applicable to all proteins, in particular to the expression of small peptides. The constructed expression vector can be directly converted into a procaryotic host cell and provide a technical platform for solving the bottleneck problem of low expression in the prokaryotic protein gene engineering expression.

Description

technical field [0001] The invention relates to a method for constructing an expression vector, in particular to a method for constructing a polycistronic expression vector. Background technique [0002] Usually, the protein expression method is to connect the target gene to be cloned with the carrier, transform the recombinant into the recipient bacteria, screen and identify the correct clone for amplification, and then transfer it into the host cell for expression. This is the most widely used method in protein expression research, but low expression level has always been a bottleneck problem in protein expression research. In order to increase the expression level, scientists have adopted different improvement measures, such as tandem expression (Dai, et al.Mar Biotechnol, DOI 10.1007 / s10126-008-9125-6) and multicistronic expression (Yang Lijun, etc. China Biotechnology Journal, 2006, 26 11: 45-47), etc. In the above-mentioned method for constructing an expression vecto...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/74C12N15/75C12R1/19C12R1/125
Inventor 李瑞芳薛雯雯
Owner HENAN UNIVERSITY OF TECHNOLOGY
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