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Cattle mycobacterium Mce4E protein, preparation method and uses thereof

A technology of Mycobacterium bovis and protein, which is applied in the biological field, can solve problems such as the research and application report of Mycobacterium bovis Mce4E protein, and achieve rapid detection, good repeatability, and improved specificity and sensitivity. Effect

Inactive Publication Date: 2010-10-20
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are no reports on the research and application of Mce4E protein from Mycobacterium bovis at home and abroad

Method used

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  • Cattle mycobacterium Mce4E protein, preparation method and uses thereof
  • Cattle mycobacterium Mce4E protein, preparation method and uses thereof
  • Cattle mycobacterium Mce4E protein, preparation method and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The construction of embodiment 1 genetically engineered bacteria

[0039] According to the mce4E gene sequence of H37Rv strain and Mycobacterium bovis AF2122 / 97 strain (NO: NC_002945, NC_000962) in GeneBank, a pair of PCR primers (synthesized by Shanghai Sangon Biotechnology Co., Ltd.) for amplifying the mature protein gene were designed and synthesized. mce4E-S: 5′-ATAT GGTACC GCAGTGCTGACTTATCTTTCGT-3' (the KpnI restriction site is underlined). mce4E-AS:5′-TTAA GAGCTC TCAGAAGTCGTCCCGTTCCGCG-3 (the horizontal line is the Sac I restriction site). Genomic DNA of Mycobacterium bovis was extracted by CTAB method. Using Mycobacterium bovis chromosomal DNA as a template, using mce4E-S and mce4E-AS as primers, the mce4E mature protein gene was amplified by PCR according to the instructions of the TaKaRa Ex Taq PCR kit. The total volume of the PCR reaction was 50 μL, and the amount of template DNA added was 5 μg. PCR program: Heat denaturation at 98°C for 8 minutes, addi...

Embodiment 2

[0041] Expression and purification of embodiment 2 recombinant protein

[0042] Inoculate the host bacteria containing the expression vector into LB liquid medium, and wait for the bacteria to grow to A 600 At about 0.8, add a final concentration of 0.2mmol / L IPTG, and after induction at 26°C for 6 hours, take out the bacterial liquid and centrifuge, and the bacterial cell is lysed with lysis buffer (50mmol / L Tris HCl, 0.5mmol / L EDTA, 50mmol / L NaCl, 5 % glycerol, pH=8.0) resuspended, sonicated under ice-bath conditions, 8000-10000rpm / min, centrifuged for 10min, the precipitate was the inclusion body, and the supernatant and precipitate were detected by SDS-PAGE electrophoresis ( Figure 4 ). Results A specific and massively expressed protein band appeared at the expected 45ku, and the expression amount accounted for 53.3% of the total bacterial protein.

[0043] The obtained inclusion body protein was sequentially washed with washing solution I (containing 10mmol / L Tris HCl,...

Embodiment 3

[0044] The dialysis refolding of embodiment 3 protein

[0045] Put the eluted protein into the dialysis bag and add 8mol / L, 6mol / L, 4mol / L, 2mol / L, 0mol / L urea in the base buffer (50mmol / L Tris HCl, 1mmol / LEDTA, 1mmol / L L GSSG, 5mmol / L GSH, 10% glycerol, 10mmol / L PMSF) were dialyzed for renaturation, each concentration was dialyzed at 4°C for 4-8h, and finally dialyzed in pH7.4 PBS buffer overnight. After reconstitution, centrifuge at 10000rpm / min for 10min at 4°C, discard the precipitate, and the supernatant is the reconstituted protein.

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Abstract

The present invention provides a mycobacterium bovis Mce4E protein, and has an amino acid sequence which is shown in a sequence table SEQID NO.2, or an amino acid sequence which has the same function and is formed by replacement, deletion or addition of one or a plurality of amino acids of the sequence. The present invention also provides a preparation method of Mce4E, including the steps of construction of genetic engineering bacteria, in vitro induced expression, separation and purification, renaturation and other steps. The present invention further provides the genetic engineering bacteria PET30 / Mce4E CGMCC No.1865 for the in vitro preparation of Mce4E. The Mce4E protein and mycobacterium bovis positive serum of the present invention can generate a specific serological reaction, so the Mce4E protein can be further used as an antigen for the development and detection of the test kits; the yield of the Mce4E protein can achieve 20mg / L in each culture media by using the preparation method of the present invention.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to mycobacterium bovis Mce4E protein, its in vitro preparation method and application. Background technique [0002] Bovine tuberculosis is a chronic, consumptive zoonotic disease mainly caused by Mycobacterium bovis. At present, there are about 4.5 million active pulmonary tuberculosis patients in the country, and about 1.45 million new active pulmonary tuberculosis patients are diagnosed every year. About 130,000 people die of tuberculosis every year, and about 15% of them are caused by Mycobacterium bovis infection. The incidence rate of bovine tuberculosis in my country presents an upward trend, and some provinces and autonomous regions have reached a positive rate of 10%; it is found that many wild animals (such as badgers, buffaloes, lions, antelopes, white-tailed deer, etc.) can be infected with Mycobacterium bovis. The control and eradication of bovine tuberculosis poses a serious...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/35C12N1/21C12N15/31G01N33/569
Inventor 赵德明徐广贤周向梅杨建民尹晓敏马李颖
Owner CHINA AGRI UNIV