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Mycobacterium tuberculosis fusion protein AR2, construction, expression and purification method and application thereof

A Mycobacterium tuberculosis fusion protein technology, applied in the field of Mycobacterium tuberculosis fusion protein AR2 and its construction, expression and purification, can solve the problems of ineffective prevention of adult tuberculosis and the failure to develop new anti-tuberculosis vaccines, and achieve long-lasting The effects of immune protection, good specificity and biological activity, and broad application prospects

Active Publication Date: 2022-02-01
ANHUI UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have confirmed that it can effectively prevent severe meningeal tuberculosis and systemic miliary tuberculosis in newborns and children, but it cannot effectively prevent the occurrence of pulmonary tuberculosis in adults
Following the application of BCG vaccine for more than 80 years, no effective new anti-TB vaccine has been developed in the world

Method used

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  • Mycobacterium tuberculosis fusion protein AR2, construction, expression and purification method and application thereof
  • Mycobacterium tuberculosis fusion protein AR2, construction, expression and purification method and application thereof
  • Mycobacterium tuberculosis fusion protein AR2, construction, expression and purification method and application thereof

Examples

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Embodiment 1

[0090] Example 1: Construction, expression and purification of Mycobacterium tuberculosis fusion protein AR2

[0091] The construction, expression and purification method of mycobacterium tuberculosis fusion protein AR2 comprises the following steps,

[0092] S1: Extract the gene sequences encoded by Rv1988 and Rv1886c from the whole genome sequence of M.tb H37Rv provided by Genbank database;

[0093] S2: Using pET28a and pET30b as vectors, construct pET30b-Rv1988 recombinant plasmid and pET28a-Rv1886c recombinant plasmid;

[0094] Specifically, S201: Design primers corresponding to Rv1988 and Rv1886c with reference to the multiple cloning sites of prokaryotic expression vectors pET28a and pET30b, as shown in Table 1 below, and perform PCR gene amplification using the H37Rv genome as a template; reaction conditions for gene amplification and PCR The reaction systems are shown in Table 2 and Table 3 below, respectively.

[0095] Table 1 Gene and primer information

[0096] ...

Embodiment 2

[0244] Embodiment 2 provides the application of Mycobacterium tuberculosis fusion protein AR2 in the preparation of tuberculosis subunit vaccine, the specific application process includes the following steps,

[0245] 1) preparing cationic liposome DDA;

[0246] Cationic liposome DDA was prepared by film method: CHCl 3 and CH 3 OH is mixed as a solvent in a volume ratio of 9:1, and 20 mg of DDA is added and dissolved, and N is slowly introduced 2 Blow dry the above solvents and leave overnight at room temperature to allow them to dry completely. Prepare a 5 mg / ml stock solution with sterilized 1×PBS; bathe in water at 60°C for 1 hour (vortex regularly to fully dissolve it), and cool down.

[0247] 2) using cationic liposome DDA to prepare DC adjuvant, DM adjuvant and DMC adjuvant respectively;

[0248] Specifically, DC adjuvant:

[0249] 5'-T*C*C*A*T*G*A*C*G*T*T*C*C*T*G*A*C*G*T*T-3', oligonucleotide The sequence was synthesized by Suzhou Synbio Biotechnology Co., Ltd., a...

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Abstract

The invention discloses a mycobacterium tuberculosis fusion protein AR2, a construction, expression and purification method and application thereof. The construction, expression and purification method comprises the following steps: S1, extracting gene sequences coded by Rv1988 and Rv1886c from a whole genome sequence of mycobacterium tuberculosis H37Rv; S2, constructing a pET30b-Rv1988 recombinant plasmid and a pET28a-Rv1886c recombinant plasmid by taking pET28a and pET30b as carriers; S3, synthesizing Rv1988-Rv1886c, namely, a fusion protein AR2, according to the extracted gene sequences of the Rv1988 and the Rv1886c; S4, by taking the plasmid pET28a as a carrier, constructing a recombinant plasmid pET28a-AR2; S5, transferring the recombinant plasmids constructed in the step S2 and the step S4 into an expression vector E.coli BL21 strain; and S6, expressing and purifying the fusion protein AR2 and subcomponent proteins rRv1988 and rRv1886c. According to the invention, the fusion protein AR2 and an adjuvant are combined to construct a tuberculosis subunit vaccine, the vaccine can induce strong cellular immune response in a mouse body and promote generation of memory T cells, and the tuberculosis subunit vaccine is expected to become a more effective novel candidate vaccine for tuberculosis in the later period.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to mycobacterium tuberculosis fusion protein AR2 and its construction, expression and purification method and application. Background technique [0002] Mycobacterium tuberculosis (MTB), the pathogen of tuberculosis, is one of the most adaptable human pathogens to the human environment. It can survive in human macrophages in a latent state for a long time. It is estimated that one-third of the world's population has latent infection with Mycobacterium tuberculosis, and 10% of this population progresses from latent TB to active TB when they are immunocompromised. Control strategies for TB rely heavily on diagnosis of the disease and long-term treatment with at least three different anti-tuberculosis drugs. But this has led to the continued development of multidrug-resistant TB. In addition, due to the prevalence of HIV, concurrent Mycobacterium tuberculosis infection has gra...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/31C07K14/35C07K1/22C12N1/21A61K39/04A61K39/39A61P31/06C12R1/32
CPCC12N15/70C07K14/35A61K39/04A61K39/39A61P31/06A61K2039/55555Y02A50/30
Inventor 王晓春施自伦李辉蔡霞程龙强
Owner ANHUI UNIV OF SCI & TECH
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