Three-antigen fusion gene vaccine of mycobacterium tuberculosis as well as preparation method and application of three-antigen fusion gene vaccine

An antigen gene and fusion gene technology, applied in the field of genetic engineering, can solve the problems of poor tuberculosis effect and low level of vaccine-induced cellular immune response.

Active Publication Date: 2013-08-28
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The object of the present invention is to provide a kind of Mycobacterium tuberculosis three antigen fusion gene vaccine and its preparation method and application, described this Mycobacterium tuberculosis t

Method used

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  • Three-antigen fusion gene vaccine of mycobacterium tuberculosis as well as preparation method and application of three-antigen fusion gene vaccine
  • Three-antigen fusion gene vaccine of mycobacterium tuberculosis as well as preparation method and application of three-antigen fusion gene vaccine
  • Three-antigen fusion gene vaccine of mycobacterium tuberculosis as well as preparation method and application of three-antigen fusion gene vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: Prediction of the target antigen gene of p846 gene vaccine

[0063] Through the analysis of BLAST network database, DNAstar biological software, and network database (http: / / www.syfpeithi.com / scripts / MHCServer.dll / home.htm), comprehensive hydrophilicity and hydrophobicity, softness, antigen index, surface accessibility, and HLA class I, II molecular binding, and other parameters, combined with previous studies, selected three dominant antigens derived from Mycobacterium tuberculosis H37Rv strain: Rv3615c, Mtb10.4 and Rv2660c, of which Rv3615c and Mtb10.4 also exist in cattle type Mycobacterium tuberculosis BCG, while Rv2660c is missing in BCG.

[0064] Such as figure 1 , 2 As shown, using pcDNA3.1 as a plasmid vector, a fusion gene of Rv3615c, Mtb10.4 and Rv2660c three antigens arranged in tandem was inserted through the NheI / XhoI restriction site, and a Flag tag was fused at the 3' end of the fusion gene for in vitro detection.

Embodiment 2p846

[0065] Construction of embodiment 2p846 tuberculosis gene vaccine

[0066] The present invention utilizes DNA primer direct synthesis and PCR methods to sequentially amplify three segments of antigen-encoding genes from the 5' to 3' end, and then mix the obtained three individual antigen gene segments as templates, and after denaturation, overlap the complementary The sequences were ligated, and finally the p846 full-length gene fused with three tuberculosis antigen genes was amplified by PCR using the upper and lower primers at the 5' and 3' ends. At the same time, Nhe I and Xho I restriction sites are respectively placed on both ends of the gene, and after double digestion, it can be connected into the eukaryotic expression vector pcDNA3.1(+) or the prokaryotic expression vector pET28a that has undergone the same double digestion.

[0067] In order to construct the fusion gene of the three antigens Rv3615c, Mtb10.4 and Rv2660c, we arranged the base sequences of the three ant...

Embodiment 3

[0080] Example 3 pET28a-846, pGEX-Rv3615c, pGEX-Mtb10.4,

[0081] Construction of pGEX-Rv2660c prokaryotic expression plasmid and protein expression and purification

[0082] In order to obtain a large amount of pET-28a-846 protein antigen, referred to as TFP846 protein, and the protein encoded by a single antigen gene, we used Nhe I and Xho I to connect the sequenced and identified correct three-antigen fusion gene 846 connected to pcDNA3.1 Double enzyme digestion, and connection with the prokaryotic expression vector pET28a after corresponding enzyme digestion, constructed the pET28a-846 prokaryotic expression plasmid. Since the molecular weights of individual antigens Rv3615c, Mtb104 and Rv2660c are relatively small, and they all encode proteins with a size of about 10KDa, it is not easy to purify when connected to the prokaryotic expression vector pET28a. Therefore, we redesigned three pairs of primers and constructed pGEX-Rv3615c , pGEX-Mtb10.4, the prokaryotic expressio...

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Abstract

The invention discloses a fusion protein gene which is formed by sequentially connecting an antigen gene Rv3615c, an antigen gene Mtb10.4 and an antigen gene Rv2660c in series from a 5' end to a 3' end. The invention also provides a three-antigen fusion gene vaccine of mycobacterium tuberculosis as well as a preparation method and application of the three-antigen fusion gene vaccine of the mycobacterium tuberculosis, wherein the three-antigen fusion gene vaccine is composed of a carrier, and a segment of the fusion protein gene is inserted to the carrier. Rat immunological experiments show that the three-antigen fusion gene vaccine of the mycobacterium tuberculosis can be used for inducing a better cellular immunological response. The three-antigen fusion gene vaccine of the mycobacterium tuberculosis, disclosed by the invention, can be used for inducing a specific killing response and a stronger CD4<+> and CD8<+>T cellular immune protection reaction having an important protection effect on mycobacterium tuberculosis infection, can be used for effectively resisting the mycobacterium tuberculosis infection, and plays a better protection effect on tuberculosis infected rats.

Description

Technical field: [0001] The invention belongs to the field of genetic engineering and relates to a recombinant gene vaccine, in particular to a vaccine for treating and preventing tuberculosis, specifically a tuberculosis three-antigen fusion gene vaccine and its preparation method and application. Background technique: [0002] Tuberculosis is a traditional infectious disease that poses a major threat to human health. About 1 / 3 of the world's population is infected with tuberculosis, of which about 10% of the population will be acutely ill, and the other 90% of the population is mainly latent infection. In recent years, due to the prevalence of multidrug-resistant tuberculosis, adverse reactions to chemotherapy drugs, human immunodeficiency virus (HIV) infection, the use of immunosuppressants, and senile tuberculosis, the body's immune function is low, making refractory tuberculosis Anti-tuberculosis treatment is facing a huge challenge. In particular, the emergence of mul...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K19/00C12N15/66A61K48/00A61P31/06
Inventor 熊思东徐薇董春升岳艳孔红梅
Owner SUZHOU UNIV
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