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

A kind of preparation method of mycobacterium tuberculosis immunogenic protein esat6

A technology of Mycobacterium tuberculosis and immunogen, which is applied in the field of biomedicine, can solve the problems of small molecular weight of ESAT6 protein, poor antigenicity of ESAT6 protein, and low soluble protein expression, and achieve high antigenicity, high purity and high expression. Effect

Active Publication Date: 2022-04-05
上海晶诺生物科技有限公司
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the method for preparing ESAT6 protein, there is still an increase in the fusion protein component, and the antigenicity of the obtained ESAT6 protein is poor; or the antigenicity of the obtained ESAT6 protein is good, but most of the prokaryotic expressed ESAT6 protein exists in the form of inclusion bodies In the precipitation, a small amount exists in the lysate supernatant in a soluble form, and the obtained ESAT6 protein has a small molecular weight, which leads to the problems of low expression level and low recovery rate of the soluble protein

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
  • A kind of preparation method of mycobacterium tuberculosis immunogenic protein esat6
  • A kind of preparation method of mycobacterium tuberculosis immunogenic protein esat6
  • A kind of preparation method of mycobacterium tuberculosis immunogenic protein esat6

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Construction and identification of pET-28a-CFP10-ESAT6 recombinant plasmid

[0050] 1. Primer design, synthesis and amplification of target genes CFP10 and ESAT6

[0051] With the Mycobacterium tuberculosis H37Rv genome as a template, primers CFP10-F / R are used to amplify the CFP10 gene (the nucleotide sequences of the primers CFP10-F / R are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively), Utilize primer ESAT6-F / R to amplify ESAT6 gene (the nucleotide sequence of primer ESAT6-F / R is shown in SEQ ID NO.5 and SEQ ID NO.6 respectively), amplified product is separated through nucleic acid gel electrophoresis For DNA fragments, use Omega’s glue back kit to recover the target DNA fragments. The recovered DNA is digested with NcoI / BamHI and BamHI / XhoI restriction enzymes respectively, and recovered by Omega’s Cycle-pure recovery kit. DNA, spare.

[0052] 2. Construction of pET-28a-CFP10-ESAT6 recombinant plasmid

[0053] Use the LB solid plate to culture the E. ...

Embodiment 2

[0054] Example 2 Expression and purification of CFP10-ESAT6 recombinant fusion protein

[0055] 1. Transformation and verification of Escherichia coli BL21(DE3)

[0056] Take BL21(DE3) competent cells and mix them with 5 μL of pET-28a-CFP10-ESAT6 recombinant plasmid constructed in Example 1, put them in an ice bath for 30 min, heat shock at 42°C for 90 s, remove them from the ice bath for 2 min, add 900 μL of SOC medium and place at 37 ℃ shaker, recover at 200rpm for 1h, centrifuge at 5000rpm for 1min, leave 100μL and remove the supernatant, gently pipette and mix the bacteria, spread on LB solid plate (containing 40μg / mL kanamycin sulfate), place in 37 ℃ incubator, after culturing for 12 hours, pick the monoclonal colony grown on the plate to inoculate LB liquid medium, and cultivate overnight in a shaker at 200 rpm at 37 ℃, after the bacterial liquid particles are sequenced by Qingke Company to verify that there is no mutation in the sequence, proceed to the next step Induc...

Embodiment 3

[0076] Example 3 Obtaining of Immunogen Protein ESAT6

[0077] 1. Enzyme digestion of CFP10-ESAT6 recombinant fusion protein

[0078] The CFP10-ESAT6 recombinant fusion protein obtained after purification in Example 2 above was cleaved with protease TEV 4° C. and dialyzed overnight, and the fusion protein washed with 25% elution buffer E on the DEAE column was washed with a macromolecular dialysis bag at pH 8.0, 20mM Tris-HCl dialyzed for 3-4h, 4 ℃ overnight dialyzed digestion.

[0079] 2. Purification of ESAT6 immunogenic protein

[0080] (1) Removal of CFP10-ESAT6 recombinant fusion protein

[0081] Principle: Remove the uncut CFP10-ESAT6 recombinant fusion protein after enzyme digestion, and separate the CFP10-ESAT6 recombinant fusion protein and the cut CFP10 protein and ESAT6 protein with Ni column. Since the CFP10-ESAT6 recombinant fusion protein has a HIS tag, it will bind On the Ni column, the CFP10 protein and ESAT6 protein interact, and the two proteins will exist...

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 invention discloses a preparation method of mycobacterium tuberculosis immunogenic protein ESAT6. The present invention provides a CFP10‑ESAT6 recombinant fusion protein, the amino acid sequence of which is shown in SEQ ID NO.1. The recombinant fusion protein has a high expression level, supernatant expression, and good activity; after the recombinant fusion protein is purified, digested with a nickel column, and eluted, the Mycobacterium tuberculosis immunogen protein ESAT6 is prepared; the method prepares The ESAT6 protein has high expression, good antigenicity, good activity, and high purity, which solves the problems of poor antigenicity, poor solubility, and prokaryotic expression of ESAT6 protein. It is an early diagnosis of tuberculosis and a new vaccine. The development has laid a foundation and has broad application prospects.

Description

technical field [0001] The invention belongs to the technical field of biomedicine. More specifically, it relates to a preparation method of Mycobacterium tuberculosis immunogenic protein ESAT6. Background technique [0002] Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis (MTB) infection. According to the 2019 Global Tuberculosis Report released by WHO, 1 / 4 of the world's population is currently infected with Mycobacterium tuberculosis, and about 10 million new cases occur each year. Tuberculosis is responsible for approximately 1.2 million deaths among HIV-negative people. my country is one of the 30 countries with a high burden of tuberculosis in the world. According to the data of the fourth national tuberculosis epidemiological survey, the number of people infected by Mycobacterium tuberculosis in the country exceeds 400 million. Therefore, how to detect and prevent tuberculosis early has attracted worldwide attention. [0003] ESAT6 protein...

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 Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C07K14/35C07K1/22C07K1/18
CPCC07K14/35C12N15/70C07K2319/00C07K2319/50
Inventor 周亚凤申兆兴黎勇宋淑婷
Owner 上海晶诺生物科技有限公司
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