Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Expression purification in colibacillus and activity identification method of recombinant thymulin alpha-1

A technology for expression and purification of Escherichia coli, which is applied in the field of medical bioengineering, can solve problems such as low yield, high production cost, and cumbersome steps, and achieve the effects of reducing production cost, shortening cycle, and short time consumption

Inactive Publication Date: 2005-12-14
NANJING UNIV
View PDF0 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The composition of the chest tissue is complex, so the natural Thyα 1 The purification steps are complicated and the yield is very low
Thyα currently in clinical use 1 It is manufactured by chemical synthesis, and the steps are cumbersome
And even if the efficiency of each step synthesis is higher, the final yield of multi-step synthesis is not ideal, so the production cost is higher

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Expression purification in colibacillus and activity identification method of recombinant thymulin alpha-1
  • Expression purification in colibacillus and activity identification method of recombinant thymulin alpha-1
  • Expression purification in colibacillus and activity identification method of recombinant thymulin alpha-1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0038] 1. Take 1 μL chemically synthesized rhThyα for PCR reaction 1 Template (5nmol / L) was added to 36.75μL sterile water, followed by 50pmol 5' end primer and 3' end primer, 5μL 10×PCR buffer, 1μL 10mmol / L dNTP, 4μL 25mmol / L MgCl 2 and 0.25 μL Taq enzyme, and amplify immediately after mixing. The reaction conditions were denaturation at 94°C for 1 min; then the following 26 cycles were performed: 94°C for 45 s, 54°C for 1 min, 72°C for 45 s; and extension at 72°C for 8 min.

[0039] 2. rhThyα 1 Cloning plasmid (Thyα 1 Construction of / pMD18-T Vector) Take 4 μL of PCR amplification product, mix with 5 μL of ligation buffer and 1 μL of ligation vector (containing ligase), and incubate at 16° C. for 30 min. Take 5 μL of the ligation product to transform into DH5α competent cells, use KpnI / SacI double enzyme digestion to screen and identify positive clones, and perform sequence determination.

[0040] 3. rhThyα 1 Expression plasmid (Thyα1 / pET32b) construction Thyα 1 The / ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The present invention relates to medicine biological engineering technology, and is expression and purification in colibacillus and activity identification method of recombinant human thymulin alpha-1. Through PCR, chemically synthesized recombinant human thymulin alpha-1 gene is amplified and cloned to pMD18-T vector, and through KpnI / SacI restriction and connection, the thymulin gene is inserted to the downstream of thioredoxin in expression vector pET32b. Recombinant plasmid is converted to expression host BL21(DE3), and through IPTG induction, Zn-Sepharose affinity chromatographic purification and renaturation, thioredoxin-thymulin alpha-1 fusion protein in the amount of 35 mg / L may be obtained from the bacteria liquid. Through further blood coagulation factor Xa restriction, Zn-Sepharose affinity chromatography and reverse efficient liquid chromatographic purification, recombinant human thymulin is obtained.

Description

technical field [0001] The invention relates to the technical field of medical bioengineering. The present invention further relates to a recombinant human thymosin alpha 1 Expression purification and activity identification method in Escherichia coli. Background technique [0002] The thymus plays a vital role in the immune system of animals. It regulates the maturation, differentiation and function of T cells by producing a series of polypeptides. In 1972, Goldstein et al. purified and identified thymus tissue, and discovered one of the active ingredients with a molecular weight of 12600Da (1) . Subsequent studies have shown that the protein is composed of three polypeptides with a molecular weight below 4000Da (2) . Thymosin component 5 is one of them. It has a powerful immune-stimulating effect, and can even replace the thymus to rebuild immune function in thymus-removed or immune-deficient animals (3) . Thymosin fraction 5 is composed of 10 to 15 major component...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12
Inventor 刘建宁孙自勇陈均勇张菁吴盛
Owner NANJING UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com