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Genetic engineering bacterium expressing snake venom kininogenase as well as constructing method and application thereof

A technology of genetically engineered bacteria and kininogenase, which is applied in the field of genetically engineered bacteria that efficiently express human snake venom kininogenase, can solve the problems that drugs cannot maximize their effects, cannot be administered intravenously, and have low purity , to achieve the effect of simplifying the follow-up purification work, low cost and high purification efficiency

Inactive Publication Date: 2012-12-26
CHINA AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the kininogenase used clinically in China is extracted from the pancreas of mammals. The purity is not high and it has certain antigenicity. After entering the human body, it may induce a series of antigen-antibody reactions. It has been reported that the injection of pancreatic hormone Peptidase leads to fixed drug eruption and severe erythema multiforme drug eruption, and intravenous administration cannot be realized, so that the drug cannot exert its maximum effect

Method used

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  • Genetic engineering bacterium expressing snake venom kininogenase as well as constructing method and application thereof
  • Genetic engineering bacterium expressing snake venom kininogenase as well as constructing method and application thereof
  • Genetic engineering bacterium expressing snake venom kininogenase as well as constructing method and application thereof

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Effect test

Embodiment 1

[0036] Embodiment 1 Amplification of snake venom kininogenase gene

[0037] Total RNA was extracted from Agkistrodon halys in Jiangsu and Zhejiang provinces, and the VK gene was amplified by RT-PCR. Use 1% agarose gel electrophoresis to detect and recover the target band, then connect to the pGEM-T vector overnight at 4°C with T4 DNA ligase, and then transform into Escherichia coli DH5α; the transformant is first screened by blue and white, and then amplified by PCR (Amplification program: pre-denaturation at 94°C for 5 min; denaturation at 98°C for 10 s, annealing at 60°C for 15 s, extension at 72°C for 1 min, 30 cycles; extension at 72°C for 10 min, end of the reaction) and sequence identification. ( figure 1 )

Embodiment 2

[0038] The construction of embodiment 2 expression vector and the transformation of escherichia coli

[0039] Design upstream primer 5'- CCATGG TCATTGGAGGTGATGAATGTAACATA-3' (underlined part is Nco I restriction site) and downstream primer 5'- GCGGCCGCTCACGGGGGGCATGTCA-3' (the underlined part is the NotI restriction site), amplified by PCR method to obtain the VK gene without signal peptide. After the PCR product was digested by Nco I and NotI, it was inserted into the NcoI and NotI sites of the expression vector pET30a, and the plasmid was transformed into Escherichia coli DH5α, and the plasmid of the transformant was extracted and digested by NcoI and NotI. After sequencing and identification ( figure 2 ), into Escherichia coli BL21(DE3).

Embodiment 3

[0040] Induced expression of embodiment 3 recombinant escherichia coli

[0041] (1) Pick the positive single clone of recombinant Escherichia coli containing the plasmid pET30a / VK and add it to 5 mL LB liquid medium containing Kana antibiotics, culture with shaking at 37°C to make it OD 600 The value reaches 0.5~0.6;

[0042] (2) Take 0.5 mL of bacterial liquid, and centrifuge at 10,000 rpm to precipitate bacterial cells. Add 100 μL of 1×SDS gel loading buffer to the cells, and let it cool naturally in a boiling water bath for 10 minutes. This is the uninduced negative control;

[0043] (3) Add IPTG to the remaining 4.5 mL of the bacterial liquid to make the final concentration reach 1 mM, continue the induction culture for 4 h, take 0.5 mL of the bacterial liquid every 1 h, and centrifuge at 10,000 rpm to precipitate the bacterial cells. Example 4 Broken cells extract supernatant

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Abstract

The invention provides a genetic engineering bacterium expressing recombinant snake venom kininogenase efficiently. A snake venom kininogenase gene in Agkistrodon halys pallas is cloned through reverse transcription, a pronucleus recombinant expression plasmid pET30a / VK (venom kininogenase) is constructed and then is transferred into escherichia coli, and the strains which can secrete and express efficiently are screened out. A terminal N of foreign protein expressed by the expression system is provided with an His protein tag, purification is carried out by adopting a Ni column eluting method, follow-up purification operation of recombinant protein is greatly simplified, purity of the obtained recombinant protein reaches up to more than 90%, and purification efficiency is high and cost is low, thus the genetic engineering bacterium disclosed by the invention is applicable to large-scale preparation of protein.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a genetically engineered bacterium for highly expressing human snake venom kininogenase, its construction method and application. Background technique [0002] Kinikogenase is a proteolytic enzyme in animals that exists in the tissues of many organs. Pancreatic kininogenase in snake venom is the earliest known and most clearly studied. Pancreatic Kallidinogenase, also known as Pancreatic Kallikrein, is a type of endo-proteolytic enzyme that exists in various tissues in mammals, especially in snake venom. It acts on kininogen in the body to release kinin, thus exerting a series of pharmacological effects: dilating microvessels, improving peripheral blood circulation; promoting prostaglandin secretion, dilating small arteries, increasing renal blood flow; activating fibrinolysis The enzyme system hydrolyzes fibrin, reduces blood viscosity, prevents thrombus formation, and...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N9/64C12R1/19
Inventor 罗云波许文涛黄昆仑马骉张雅楠宋梦薇王云鹏
Owner CHINA AGRI UNIV
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