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New high-efficiency secretion and expression system of colibacillus and application thereof

A technology of expression cassettes and vectors, applied in the direction of using vectors to introduce foreign genetic material, from leech inhibitors, specific peptides, etc., which can solve problems such as complicated operations

Inactive Publication Date: 2011-08-17
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Existing secretory expression vectors, such as PIN-III-ompA, adopt the Escherichia coli outer membrane protein (ompA) signal peptide sequence, followed by a multiple cloning site for inserting foreign genes. Finally, there is often a redundant linking sequence between the signal peptide gene and the target gene. In this way, in order to obtain the correct target product at the N-terminal, site-directed mutagenesis must be used to remove the redundant sequence between the signal peptide gene and the target gene. very complicated

Method used

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  • New high-efficiency secretion and expression system of colibacillus and application thereof
  • New high-efficiency secretion and expression system of colibacillus and application thereof
  • New high-efficiency secretion and expression system of colibacillus and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Amplification of expression cassette gene in embodiment 1 expression vector pTASH

[0024] Extract the template plasmid pTASH from the original Escherichia coli strain containing the pTASH plasmid (Tan Shuhua et al., Chinese invention patent 01113526.3), and design and synthesize the following primers:

[0025] Forward primer pkk223-3TAC-f: 5' GGA TCC AAG CTG TGG TAT GGC TGT GCA GGT CGTAAATC 3' (underline indicates BamH I restriction site)

[0026] Reverse primer pkk223-3rrnBT2-r: 5' GGA TCC AGC GTT TCT GGG TGA GCA AAA ACA GGAAG 3' (underline indicates BamH I restriction site)

[0027] Using the plasmid pTASH as a template, the polymerase chain reaction was carried out under the action of Pfu DNA polymerase. The composition of the 50 μl reaction system is as follows: 20 pmol each of forward primer and reverse primer; 5 units of Pfu DNA polymerase; 5 μl of 10× reaction buffer; 1 μl of dNTP (10 mM each); 2(25mM) 3μl; plasmid DNA 1μl; make up to 50μl with sterile wat...

Embodiment 2

[0029] Example 2 Construction of Double Expression Cassette Recombinant Hirudin Efficient Secretion Expression Plasmid

[0030] The plasmid pMD-19T-TASH was extracted by alkaline lysis, digested with BamH I, and DL2,000 TM DNA Marker was used as a control, separated by 1.5% agarose gel electrophoresis, and the digested fragment was recovered to obtain an expression cassette fragment with a size of about 1.1 kb.

[0031] Plasmid pTASH was extracted from the original strain and digested with BamH I to obtain linearized pTASH with the same cohesive ends as the expression cassette fragment. To prevent linearized pTASH from self-cyclization during the ligation reaction, its 5' phosphate group needs to be converted to a hydroxyl group using the dephosphatase CIAP. The 40 μl reaction system is as follows: 4 μl of 10× reaction buffer, 2 μl of CIAP, 34 μl of linearized pTASH, reacted at 37°C for 4 hours; inactivated at 65°C for 30 minutes to terminate the reaction, and then precipitat...

Embodiment 3

[0035] Example 3 High-efficiency secretory expression of recombinant hirudin III

[0036] The selected expression cassette was inserted into the recombinant hirudin III double expression cassette clone p(TASH) inserted in the forward direction 2 , insert 30mlLBA (tryptone 1%, yeast powder 0.5%, NaCl 1%, Amp100μg / ml, pH7.0) liquid culture medium and carry out seed liquid culture, culture condition is 37 ℃ 220rpm shaking culture 14 hours, then with 2 % inoculum size was transferred to fermentation medium (tryptone 1%, yeast powder 0.5%, sodium glutamate 4%, malt powder 1.0%, Amp100μg / ml, KH 2 PO 4 0.374%, Na 2 HPO 4 12H 2 O 1.75%, pH 7.0), cultured at 37° C. and 220 rpm for 24 hours, and measured the antithrombin activity in the supernatant of the fermentation broth.

[0037] The bioactivity of hirudin was measured with reference to Markwardt's thrombin titration method (F.Markwardt, Hirudin as an inhibitor of thrombin, Methods Enzymol.19 (1970) 924-932.): add 200 μl of 0....

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Abstract

The invention discloses a novel high-efficiency secretion and expression vector jof colibacillus, a preparation method thereof and application thereof. The expression vector is a cyclic plasmid deoxyribonucleic acid (DNA) vector, and comprises a procaryotic copy initial point, a selection marker gene and two series-wound exogenous gene expression box units. Each series-wound exogenous objective (target) gene expression box unit respectively comprises a tac strong promoter, an L-Asparaginasum signal peptide degenerate gene, an exogenous objective (target) gene and a high-transcription terminator rrnBT1T2 which are sequentially arranged with one another. The novel high-efficiency secretion and expression vector disclosed by the invention can secrete and express a polypeptide / protein objective product at a high level in the host cell of the colibacillus, and the expression product can be automatically formed into a correct intramolecular disulfide bond and secreted into cell periplasm / and culture solution, so that the invention is very good for the downstream separation, extraction and purification of the expression product, and has an important application value.

Description

technical field [0001] The invention relates to a high-efficiency secretion expression vector containing two series-connected gene expression cassette units, which is used in the field of genetic engineering and uses Escherichia coli as a host, as well as its preparation method and application. Background technique [0002] There is no general rule for the expression of polypeptide proteins in genetic engineering technology. In order to obtain effective expression, different expression systems and expression methods are adopted for different target products due to their different structural properties. Escherichia coli is the earliest prokaryotic expression system, and its expression methods mainly have three forms: non-fusion expression, fusion expression and secretory expression. Misfolding of peptide chains and mismatching of disulfide bonds during the renaturation process of non-fusion expression methods often lead to a greatly reduced yield of active products. In addit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/815C12N15/70C12N15/11C12N15/66
Inventor 谭树华吴梧桐黄翠翠吕静
Owner CHINA PHARM UNIV
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