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Mycobacterium tuberculosis ag85ab chimeric gene vaccine, its preparation method and application

A technology of Mycobacterium tuberculosis and genetic vaccine, applied in the field of biomedicine, can solve the problems of unsatisfactory pathological damage, etc., and achieve the effect of strong anti-TB cellular immune response and better therapeutic effect.

Active Publication Date: 2011-12-07
GUAN DINGTAI HAIGUI BIOLOGICAL TECH CO LTD
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Problems solved by technology

[0008] These two chimeric gene vaccines have shown good effects in animal experiments in the prevention of Mycobacterium tuberculosis infection, but later we found that when they were used for experimental treatment of animals infected by Mycobacterium tuberculosis (especially for the treatment of drug-resistant Mycobacterium tuberculosis), Although this chimeric gene vaccine can effectively reduce the number of Mycobacterium tuberculosis in infected tissues, it is not satisfactory for reducing the pathological damage caused by Mycobacterium tuberculosis (such as pathological damage such as tuberculosis nodules produced by experimental animal tuberculosis)

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  • Mycobacterium tuberculosis ag85ab chimeric gene vaccine, its preparation method and application
  • Mycobacterium tuberculosis ag85ab chimeric gene vaccine, its preparation method and application
  • Mycobacterium tuberculosis ag85ab chimeric gene vaccine, its preparation method and application

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preparation example Construction

[0056] In a preferred embodiment, the preparation method of the chimeric Mycobacterium tuberculosis gene vaccine of the present invention comprises the following steps:

[0057] (1) Select the 245-250 KpnI restriction site or the 430-435 Acc I restriction site that can be inserted into the exogenous DNA fragment in the Ag85a gene, and use endonuclease Kpn I or endonuclease, respectively Acc I digests the previously constructed eukaryotic expression vector pVAX1 containing the Ag85a gene, linearizes it, and dephosphorylates it with alkaline phosphatase;

[0058] (2) Use a pair of recognition sequences with endonuclease Kpn I respectively GGTACC primers, or a pair of endonuclease Acc I recognition sequences GTCTAC The primers were used to amplify the DNA fragment encoding the amino acid sequence of Ag85b protein 125-282 by polymerase chain reaction;

[0059] (3) Connect the dephosphorylated linear Ag85a gene vector of step (1) and the DNA amplification fragment encoding the a...

Embodiment 1

[0071] Example 1: Preparation of nucleic acid vaccine containing Ag85ab chimeric gene pVAX1 plasmid (HG85abA plasmid)

[0072] Main experimental materials :

[0073] The pVAX 1 vector plasmid was purchased from Invitrogen

[0074] The pVAX1-85a containing the Mycobacterium tuberculosis Ag85a gene and the PET28A-85B plasmid containing the Mycobacterium tuberculosis Ag85b gene derived from the pVAX1 plasmid vector were prepared by Shanghai Haigui Biotechnology Co., Ltd. (Li Z, Song D, Zhang H, et al., Improved humorol immunity against Tuberculosis ESAT-6 antigen by chimeric DNA prime and protein boost strategy. DNA Cell Biol, 2006, 25(1):25-29).

[0075] Alkaline phosphatase and Taq DNA polymerase are products of NEB Company.

[0076]Restriction endonucleases BamH I, Hind III and dNTP Mix (mixture) are products of Takara Company. Restriction endonucleases Acc I, Kpn I and T4 DNA ligase are products of MBI Company.

[0077] AXYGEN plasmid extraction kit and DNA gel recovery...

Embodiment 2

[0124] Example 2: Preparation of nucleic acid vaccine containing Ag85ab chimeric gene PVAX1 plasmid (HG85abK plasmid)

[0125] Main experimental materials: Same as Example 1

[0126] Preparation

[0127] (1) Primer Design and Synthesis : Designed for PCR amplification of Ag85b gene (coding amino acids 125-282) fragment containing Kpn I restriction site The following upstream primer P3 and downstream primer P4 oligonucleotide sequences:

[0128] P3: 5'-CACATCACGATACCG TCGATGGCCGGCTCGTC-3' (SEQ ID NO: 6)

[0129] P4: 5'-CACATGCGAATACCG TAACGAACTCTGCAGGTC-3' (sequence 7)

[0130] The two sequences were sent to Invitrogen for synthesis.

[0131] (2) PCR amplification of the fragment of the Ag85b gene (encoding amino acids 125-282) : The method is the same as in Example 1, but the above-mentioned P3 / P4 primers are used.

[0132] (3) Preparation of dephosphorylated linear pVAX1-Ag85A plasmid : The method is the same as in Example 1, but Kpn I enzyme is used.

[...

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Abstract

The invention provides a mosaic gene, which comprises a gene for coding tubercle bacillus protein Ag85a and a gene which is mosaiced at a Kpn I recognition sequence at sites 245-250 and / or an Acc I recognition sequence at sites 430-435 of Ag85a gene for coding the tubercle bacillus protein Ag85b. The invention provides a mosaic gene-containing mosaic tubercle bacillus gene vaccine, wherein the Ag85a gene is connected to a eukaryotic expression vector. The invention also provides a preparation method of the mosaic tubercle bacillus gene vaccine. The method comprises the following steps of: performing PCR (Polymerase Chain Reaction) amplification on an Ag85b gene segment; inserting the amplified Ag85b gene segment into the Ag85b gene-containing eukaryotic expression vector; and connecting by using ligase. The mosaic tubercle bacillus gene vaccine can be used for treating drug-resistant tubercle bacillus infection and can also be used in combination with levamisole serving as an adjuvantto induce stronger anti-tubercular cell immunological response.

Description

technical field [0001] The invention relates to novel vaccine technology in the field of biomedicine, in particular to a tuberculosis chimeric gene vaccine developed by chimeric gene technology that can be used to prevent or treat tuberculosis, especially a chimeric gene vaccine for treating drug-resistant tuberculosis-infected tuberculosis patients . technical background [0002] So far, although it is known that BCG can prevent tuberculosis in children, including severe cerebral tuberculosis and miliary tuberculosis, it cannot effectively prevent pulmonary tuberculosis in adults. Therefore, the United States, Canada and other countries have not routinely vaccinated BCG. Since the 1980s, due to the growing problem of drug resistance of Mycobacterium tuberculosis, as well as social problems such as AIDS, immigration and poverty, the incidence of tuberculosis has rebounded worldwide. The World Health Organization (WHO) reported in 2004 that there are more than 9 million new ...

Claims

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Application Information

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IPC IPC(8): C12N15/62A61K48/00A61K39/39A61P31/06
CPCA61K39/04C07K2319/00A61K2039/53C07K14/35A61P31/06
Inventor 李忠明吴雪琼刘庆良梁艳张平静张俊仙阳幼荣
Owner GUAN DINGTAI HAIGUI BIOLOGICAL TECH CO LTD
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