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Recombinant bovine tuberculosis specific antigen protein with three fused genes and preparation method thereof

A technology of fusion antigen and bovine tuberculosis, applied in the field of microbial genetic engineering

Active Publication Date: 2009-09-23
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Brusasca et al. (2001) used ESAT6 antigen to detect specific antibodies in the serum of tuberculosis patients, and the positive rate was 25%, while no corresponding antibodies were detected in the healthy control group

Method used

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  • Recombinant bovine tuberculosis specific antigen protein with three fused genes and preparation method thereof
  • Recombinant bovine tuberculosis specific antigen protein with three fused genes and preparation method thereof
  • Recombinant bovine tuberculosis specific antigen protein with three fused genes and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Cloning of target genes CFP10, ESAT6 and Rv3872

[0036] 1. Source of plasmid and host bacteria

[0037] The original plasmid vector pET-28a (+) used in the present invention and examples is purchased from Novagen, and its physical map is shown in figure 1 shown. Escherichia coli BL21 (DE3) competent cells were purchased from Hubei Wuhan Life Technology Co., Ltd.

[0038] 2. Primer design and synthesis

[0039] Specific primers targeting full-length genes such as CFP10, ESAT6 and Rv3872 were designed according to the published sequence of Mycobacterium bovis AF2122 / 97 (GenBank accession number: NC002945). Primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd. The DNA sequence of the primer pair is as follows:

[0040] CFP10 upstream primer (HindIII): 5′-TCTGAAGCTTGCAGAGATGAAGACCGAT-3′

[0041] CFP10 downstream primer (Linker):

[0042] 5′-AGATCCGCCTCCACCTGAACCGCCACCTCCGAAGCCCATTTGCGAGGACAGCGCCT-3′

[0043] ESAT6 upstream primer (Linker): ...

Embodiment 2

[0059] Embodiment 2, the construction of fusion gene Rv3872-linker-CFP10-linker-ESAT6 (see Figure 4 )

[0060] 1. Construction of intermediate plasmid pET-28a-CFP10-ESAT6

[0061] The fusion gene CFP10- ESAT6. Using the above-mentioned purified PCR products CFP10 and ESAT6 as templates, cfp10 upstream (HindIII) and esat6 downstream (NotI) as primers, PCR was performed with Probest DNA polymerase. Reaction system (50 μL): upstream and downstream primers 300 nmol / L each, CFP10 and ESAT6 PCR products 0.5 μL each, Probest DNA polymerase 1.25 U. The annealing temperature is 63°C (1min), and other conditions are the same as above.

[0062] Recover the PCR product of CFP10-ESAT6 (see Figure 7 ), the pET-28a empty vector and the recovered PCR product were double-digested with HindIII and NotI (purchased from Treasure Bioengineering (Dalian) Co., Ltd.), the digested product was detected by 0.8% agarose gel electrophoresis, and the gel was recovered The kit purifies the digested...

Embodiment 3

[0082] Embodiment 3, expression and purification of target fusion gene in Escherichia coli

[0083] 1. Induced expression of the target gene

[0084] Inoculate the Escherichia coli strain DH5α containing the recombinant expression vector in 3 mL LB liquid medium containing 25 μg / mL kanamycin, and culture it on a shaker at 37°C until OD 600 Reach 0.6-0.8. Take 100 μL of the cultured Escherichia coli bacteria solution and inoculate it into 10 mL of fresh LB liquid medium containing 25 μg / mL kanamycin, culture it with shaking at 37°C for about 3 hours, until OD 600 Reach 0.6-0.8, add isopropylthio-β-D-galactoside (IPTG, purchased from Invitrogen Company) to a final concentration of 0.8mmol / L, continue to cultivate for 3h and collect the thalline ( Figure 8 ).

[0085] 2. SDS-PAGE electrophoresis analysis of expression products

[0086] Preparation of samples for SDS-PAGE electrophoresis

[0087] The induced recombinant Escherichia coli was centrifuged at 8000r / min for 15min...

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Abstract

The invention belongs to the technical field of microbial genetic engineering, specifically relates to a recombinant bovine tuberculosis specific antigen protein with three fused genes and a preparation method thereof. The gene of the bovine tuberculosis specific fusion antigen protein has a nucleotide sequence and an amino acid sequence shown as in a sequence list SEQ ID NO:1. The invention discloses a recombinant Escherichia coli BL21 / pET28a-RCE capable of expressing the bovine tuberculosis specific fusion antigen protein. The Escherichia coli is conserved in China Center for Type Culture Collection (CCTCC) with a conservation number CCTCC NO:M208244. The invention further discloses a preparation method of the three gene fused antigen protein.

Description

technical field [0001] The invention relates to the technical field of microbial genetic engineering. In particular, it relates to a recombinant bovine tuberculosis specific antigen protein and a preparation method of three genes fused. Background technique [0002] Bovine Tuberculosis (bTB) is a chronic zoonotic disease mainly caused by Mycobacterium bovis. my country lists it as a second-class animal disease. The etiological surveys of human tuberculosis at home and abroad have found that Mycobacterium bovis is one of the important causes of human tuberculosis. The report of the seventh meeting of the World Health Organization Expert Committee pointed out: Unless bovine TB is eradicated, the control of human TB will not be successful. Since no effective drugs and vaccines have been found so far for the prevention and treatment of bovine tuberculosis, some countries generally adopt the "quarantine-kill" policy, that is, all positive cattle from quarantine are killed to a...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N1/21C12P21/02C07K19/00C12R1/19C12R1/32
Inventor 郭爱珍刘冬光曹莎陈颖钰廖娟红于清龙凌洁玉陈焕春
Owner HUAZHONG AGRI UNIV
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