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Mycobacterium tuberculosis TB10.4-F1 fusion protein vaccine and preparation method thereof

A TB10.4-F1, TB10.4 technology, applied in chemical instruments and methods, medical preparations containing active ingredients, hybrid peptides, etc., can solve hospitalization and increased mortality, unacceptable virulence, enhanced To achieve the effects of easy cultivation and fermentation, high yield of target protein, and improved expression level

Inactive Publication Date: 2011-10-12
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the 1960s, when children were immunized with inactivated RSV vaccines, they could induce the production of neutralizing antibodies, but they were not protective. Moreover, the use of vaccines enhanced the incidence of children's subsequent infection with wild strains of RSV, and significantly increased hospitalization and mortality.
Live attenuated RSV vaccines, when given to RSV-uninfected infants 1 to 2 months of age, resulted in mild to moderate congestion of the upper respiratory tract, suggesting that further attenuation is needed
Although clinical trials of live attenuated vaccines did not cause vaccine-enhanced disease, doubts remain about their use due to poor immunogenicity, unacceptable virulence, and genetic instability of live attenuated vaccines

Method used

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  • Mycobacterium tuberculosis TB10.4-F1 fusion protein vaccine and preparation method thereof
  • Mycobacterium tuberculosis TB10.4-F1 fusion protein vaccine and preparation method thereof
  • Mycobacterium tuberculosis TB10.4-F1 fusion protein vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Obtaining of TB10.4(N / X), TB10.4(N / B), F1(B / X) genes.

[0037] Using the whole genome of Mycobacterium tuberculosis standard strain H37Rv as a template, TaqPlus DNA polymerase (from TIANGEN, J8422) was used to amplify the TB10.4 gene. The primers used were as follows: THP1: 5'-CATATGTCGCAAATCATGTACAACTACCC -3', THP2 : 5'-GCGCCGGATCCGGCGCCGCCGCCCCATTTGGCGGCTTCGGCCGTGTC-3', TBP2: 5'-CTCGAGTTAGTGGTGGTGGTGGTGGTGGCCGCCCCATTTGGCGGCTTC-3'. Among them, THP1 and THP2 are used to amplify the TB10.4(N / B) gene, and introduce Nde Ⅰ (from TAKALA company, CK8852A), Bam H Ⅰ (from TAKALA company, CK8701A) two restriction sites and linker sequence are used to construct the fusion gene TB10.4-F1; THP1 and TBP2 are used to amplify the TB10.4(N / X) gene and introduce Nde I and xho Ⅰ (from TAKALA company, CK1701A) two restriction sites and 6×His tag, used to express TB10.4 protein alone; PCR amplification conditions are: 95°C 10min; 95°C 30s, 52°C 30s, 72°C 20s (30 cycles);...

Embodiment 2

[0039] Example 2: Construction of recombinant subcloning vectors TB10.4(N / X) / pMD18T, TB10.4(N / B) / pMD18T and F1(B / X) / pMD18T.

[0040] After purifying TB10.4(N / X), TB10.4(N / B) and F1(B / X) genes using the gel recovery kit from Biotech Corporation (the following gel recovery and purification are all performed according to the instructions), each 50ng were ligated with 50ng of pMD18T (from TAKALA, CK5001BA) with 1 μl of T4 DNA ligase (from TAKALA, CK5021B) overnight at 16°C, and the ligated products were transformed by heat shock method with CaCl 2 Prepared E. coli DH5α, Coated Amp + -LB plate, cultured at 37°C for 16 hours, picked several single colonies to inoculate Amp + - LB culture medium, cultured with shaking at 37°C for 12 hours, and then extracting the plasmid. use Nde I and xho Ⅰ After double digestion of recombinant vector TB10.4(N / X) / pMD18T (recombinant plasmid: 700ng; enzyme: 1μl each; 20μl total system, placed at 37℃ for 3h), the target fragment with a size o...

Embodiment 3

[0041] Example 3: Construction of recombinant expression vectors TB10.4 / pET28a, TB10.4(N / B) / pET30a, TB10.4-F1 / pET30a.

[0042]Construction of the recombinant expression vector TB10.4 / pET28a: use the correct TB10.4(N / X) / pMD18T and pET28a plasmids Nde I and xho Ⅰ Digest at 37°C for 3 hours, take the gel to recover 50ng of the purified TB10.4(N / X) fragment and 150ng of the purified expression vector pET28a double digestion product, and use 1μl of T4 DNA ligase to ligate overnight at 16°C. CaCl for transformation by shock method 2 Prepared E. coli DH5α, coated Kan + -LB plate, cultivated at 37°C for 16 hours, picked several single colonies to inoculate Kan + - LB culture medium, cultured with shaking at 37°C for 12 hours, and then extracting the plasmid. use Nde I and xho Ⅰ After double enzyme digestion (2 μg of recombinant plasmid; 2 μl of each enzyme; 50 μl of the total system at 37°C for 5 hours), the target fragment with a size of 310 bp and its corresponding vect...

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Abstract

The invention discloses a mycobacterium tuberculosis TB10.4-F1 fusion protein vaccine and a preparation method thereof. The preparation method comprises the following steps of: connecting the mycobacterium tuberculosis TB10.4 protein gene with the F1 protein gene of respiratory syncytial virus protein, transferring into a colibacillus expressing strain, inducing by IPTG (isopropyl beta-D-1-thiogalactopyranoside) to express the TB10.4-F1 fusion protein, and acquiring the protein with higher purity by metal ion affinity chromatography. The fusion protein has stronger immunogenicity, can excite more balanced Th1 / Th2 immune response, and can prevent infections of the mycobacterium tuberculosis and the respiratory syncytial virus.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method for expressing mycobacterium tuberculosis fusion protein TB10.4-F1 by Escherichia coli cells. Background technique [0002] Mycobacterium tuberculosis( Mycobacterium tuberculosis , MTB) and human respiratory syncytial virus (Human Respiratory Syncytial virus, RSV) are pathogens that infect the body through the respiratory tract and cause a series of respiratory diseases. [0003] MTB can cause human tuberculosis, the most common of which is pulmonary tuberculosis, which causes 2 to 3 million deaths each year. In view of the threat posed by tuberculosis to public health, research on new methods to fight and control tuberculosis infection has become an international hot research issue. The World Health Organization has promoted the fully supervised short-course chemotherapy (DOTS) program in many regions, but it still failed to control the global epidemic of tube...

Claims

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Application Information

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IPC IPC(8): A61K39/295C07K19/00C12N15/62C12N15/70C12P21/02A61P31/06A61K39/04A61K39/155
Inventor 井申荣王瑞博曾韦锟黄芬
Owner KUNMING UNIV OF SCI & TECH
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