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Recombinant calmette-Guerin bacillus vaccine for secretion of tumour necrosis factor-alpha and its constructing method

A technology of tumor necrosis factor and recombinant BCG, applied to other methods of inserting foreign genetic materials, antineoplastic drugs, recombinant DNA technology, etc., to reduce the risk of tumor recurrence, reduce medical expenses, and improve the efficacy of drugs

Inactive Publication Date: 2008-01-09
SHANGHAI JIAO TONG UNIV
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Problems solved by technology

However, the research on improving the curative effect of bladder tumor by constructing recombinant BCG that secretes TNF-α by means of genetic engineering technology has not been reported yet.

Method used

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  • Recombinant calmette-Guerin bacillus vaccine for secretion of tumour necrosis factor-alpha and its constructing method
  • Recombinant calmette-Guerin bacillus vaccine for secretion of tumour necrosis factor-alpha and its constructing method
  • Recombinant calmette-Guerin bacillus vaccine for secretion of tumour necrosis factor-alpha and its constructing method

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Embodiment 1

[0032] Step 1 Construction and Identification of BCG Shuttle Expression Vector

[0033] 1. Synthesis of amplification primers

[0034] According to the gene sequences of Ag85B signal peptide and TNF-α, the main secretory antigens of BCG, which are already available in GeneBank, the amplification primers for Ag85B signal peptide and TNF-α were designed respectively as

[0035] Ag85B sense: 5′-GGATCCATGACAGACGTGAG-3′

[0036]antisense: 5′-CTGCCGTGCCGATCATCAAT-3′

[0037] TNF-αsense: 5′-AGCGAATTCATGAGCACTGAAAGCATGAT-3′

[0038] Antisense: 5′-AGCGTCGACTCACAGGGCAATGATCCCAA-3′

[0039] Sequencing primer on pMV261 vector: 5′-GACAACTTGAGCCGTCCGTC-3′

[0040] 2. Load the Ag85B signal peptide gene into the pMV261 vector

[0041] Synthesize the signal peptide gene and clone it into pUCm-T vector

[0042] Ligation (TAKARA) system: pUCm-T vector 1μl

[0043] Signal peptide fragment (about 50ng / μl) 4μl

[0044] Solμtion I 5μl

[0045] 16°C,...

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Abstract

The invention relates to a reorganization BCG vaccine for secreting tumor necrosis factor-alpha and its construction, which uses the genetic engineering technology to clone the secretion BCG vaccine Ag85b signal peptide and the TNF-alpha gene piece to pcMv261, therefore obtain the BCG vaccine shuttle express carrier pcMstnf-alpha, then use the electricity transformation technology to induce pMSTNF-alpha to the BCG to reconstruct BCG vaccine rBCGTNF-alpha. Depend upon pcMstnf-alpha duplication in the BCG and the signal peptide secretion, it can secrete TNF-alpha efficiently.

Description

technical field [0001] The present invention relates to a recombinant BCG that secretes tumor necrosis factor-α and its construction method, in particular to a recombinant BCG containing BCG secreted antigen Ag85B signal peptide fragment and tumor necrosis factor-α (TNF-α) and its construction method The obtained recombinant bacillus Calmette-Guerin rBCGTNF-α is used for preventing and treating superficial bladder tumors, and belongs to the technical field of genetic engineering. Background technique [0002] Since Morales first reported the application of Bacillus Calmette-Guerin (BCG) bladder infusion in the treatment of superficial bladder tumors in 1976, BCG as an effective immune adjuvant has achieved good results in the prevention and treatment of bladder tumors. One of the most effective adjuvant treatments for tumors. Although BCG has good efficacy in the prevention and treatment of superficial bladder tumors, there are still many problems to be solved. The first i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09C12N15/63C12N15/28C12N15/87A61K38/17A61K38/19A61K39/00C12Q1/68A61P35/00A61P31/06
Inventor 夏术阶刘海涛孙晓文
Owner SHANGHAI JIAO TONG UNIV
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