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Unlabeled tuberculosis fusion protein ESAT6-Ag85B

A fusion protein, esat6-ag85b technology, applied in the direction of DNA / RNA fragments, hybrid peptides, recombinant DNA technology, etc., can solve unconsidered problems

Inactive Publication Date: 2012-05-16
LANZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in previous studies, fusion proteins with various tags (such as His-Tag, GST-Tag, S-Tag, etc.) were often constructed for the convenience of later purification methods, without considering the potential of such fusion proteins. Whether the label will affect the approval of animal experiments and further clinical trials

Method used

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  • Unlabeled tuberculosis fusion protein ESAT6-Ag85B
  • Unlabeled tuberculosis fusion protein ESAT6-Ag85B
  • Unlabeled tuberculosis fusion protein ESAT6-Ag85B

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Cloning construction of recombinant plasmid pET30a-ESAT6-Ag85B;

[0031] 1. Main materials: Mycobacterium tuberculosis strain H37Rv was obtained from the Gansu Provincial Center for Disease Control; plasmids pET30a, E.coli DH5α and BL21(DE3) were donated by Professor Wang Honghai of Fudan University; primers were synthesized by Shanghai Biotechnology Co., Ltd.; gene sequencing Completed by Beijing Huada Gene Technology; PCR kits, product purification kits, and gel recovery kits were purchased from Dalian Bao Biological Co., Ltd.; genome extraction kits, DNA Marker Ⅲ were purchased from Lanzhou Pengcheng Biological Co., Ltd.; plasmid extraction kits , T4 DNA ligase, restriction endonuclease Eco RI, Hin dⅢ, Nde Ⅰ was purchased from Shanghai Biological Engineering Co., Ltd.

[0032] 2. Main instruments: high-pressure steam sterilizer (Shanghai Shen'an Medical Instrument Factory); electric blast drying oven (Shanghai Yiheng Scientific Instrument Co., Ltd.); ultra-low ...

Embodiment 2

[0038] Induced Expression of Fusion Gene ESAT6-Ag85B

[0039] 1. Main materials: Low molecular weight protein markers were purchased from Dalian Bao Biological Co., Ltd.; kanamycin, LB medium components (tryptone, sodium chloride, yeast extract) and protein electrophoresis related reagents (acrylamide, methylene bismuth Reagents such as acrylamide, Tris, ammonium persulfate, glycine, SDS, TEMED, β-mercaptoethanol, G250, etc.), disodium hydrogen phosphate and sodium dihydrogen phosphate were purchased from Lanzhou Pengcheng Biological Co., Ltd.; IPTG was purchased from Shanghai Biological Co., Ltd. Shenggong Engineering Co., Ltd.

[0040] 2. Main instruments: high-pressure steam sterilizer (Shanghai Shen'an Medical Instrument Factory); electric blast drying oven (Shanghai Yiheng Scientific Instrument Co., Ltd.); ultra-low temperature refrigerator -80°C (USA 813283-1048); Pure water machine AFZ0502U (Chongqing Yiyang Technology Co., Ltd.); ultra-clean bench (Suzhou Purification...

Embodiment 3

[0047] Purification steps of fusion protein ESAT6-Ag85B

[0048] 1. Main materials: Low molecular weight protein markers were purchased from Dalian Bao Biological Co., Ltd.; protein electrophoresis related reagents (acrylamide, methylene bisacrylamide, Tris, ammonium persulfate, glycine, SDS, TEMED, β-mercaptoethanol, G250, etc.) , urea, disodium hydrogen phosphate and sodium dihydrogen phosphate, MW8000-14000 dialysis bags, etc. were purchased from Lanzhou Pengcheng Biological Co., Ltd.; IPTG was purchased from Shanghai Biological Engineering Co., Ltd.

[0049] 2. Main instruments: purification instrument (AKTA purifierTM UPC100 Sweden); DYY12 electrophoresis instrument and vertical electrophoresis tank (Beijing Liuyi Instrument Factory); decolorization shaker TS-1 (Haimen Qilin Medical Instrument Factory); IF258 automatic gel imaging Analysis system (Shanghai Jiapeng Technology Co., Ltd.); pH instrument (PB-16); filter and 0.45 μm filter membrane; microplate reader; ion exch...

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Abstract

The invention relates to an unlabeled tuberculosis fusion protein ESAT6-Ag85B. The genetic engineering technique is used to recombine and fuse genes ESAT6 and Ag85B of tuberculosis mycobacteria to build recombinant plasmids; the induced expression is performed to obtain expression products of recombined genes; finally, the expression products of the recombined genes are purified and are cloned to build a tuberculosis mycobacteria fusion protein ESAT6-Ag85B without any labels. The invention has advantages of solving the subsequent problems that the labels brought by the fusion proteins can affect animal experiments and further affect clinical tests, and effectively purifying the fusion proteins by different chromatographic analysis methods. The unlabeled tuberculosis fusion protein can hopefully be candidate vaccine for preventing tuberculosis.

Description

technical field [0001] The invention relates to a gene engineering recombinant protein, in particular to an unlabeled tuberculosis fusion protein ESAT6-Ag85B, which belongs to the technical field of vaccine manufacture. Background technique [0002] Tuberculosis is one of the serious diseases that endanger human health for a long time. Nearly 1 / 3 of the people in the world are still infected with Mycobacterium tuberculosis. About 5% of tuberculosis infected people will develop into pulmonary tuberculosis patients within 2-5 years, and the rest A latent infection who may develop tuberculosis. my country is one of the countries with a high burden of tuberculosis, and about 130,000 people die of tuberculosis every year. [0003] At present, Bacillus Calmette-Guerin (BCG) vaccination is an important measure to effectively prevent tuberculosis. However, different studies have shown that the protective effect of BCG in different populations is not stable, especially for adults w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K1/20C07K1/18C12N15/62C12N15/70A61K39/04A61P31/06
Inventor 祝秉东章国平王秉翔胡丽娜达泽蛟张颖李小娟唐克峰万艳
Owner LANZHOU UNIVERSITY
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