表达Ag85基因重组卡介苗的构建

doi:10.16431/j.cnki.1671-7236.2020.02.031

Abstract

Abstract: In order to develop a safer and more effective tuberculosis vaccine for the prevention and elimination of tuberculosis,this study constructed four recombinant BCG vaccines expressing Ag85A,Ag85B,Ag85C and Ag85D genes,respectively.According to the pMV261 vector sequence and the H37Rv sequence registered on Tuberculist,primers were designed,and the recombinant pMV261 plasmid containing Ag85 gene fragment was constructed by PCR amplification and infusion cloning.The recombinant plasmid was identified by PCR,double enzyme digestion and transformed into BCG receptive cells by electroporation.Kan resistance was used to screen the recombinant BCG strain.The expression of Ag85A,Ag85B,Ag85C and Ag85D genes in BCG vaccine were identified by PCR and Western blotting,and then the successfully constructed recombinant BCG vaccines were cultured for follow-up experiment.The results of PCR amplification showed that the target gene fragment of recombinant BCG vaccines rBCG/Ag85A,rBCG/Ag85B,rBCG/Ag85C and rBCG/Ag85D were 1 447,1 402,1 453 and 1 330 bp,indicating that the target gene had been successfully integrated into BCG vaccine.Western blotting results showed that Ag85A,Ag85B,Ag85C and Ag85D genes could be successfully expressed in BCG cells.In this experiment,four recombinant BCG vaccines expressing Ag85A,Ag85B,Ag85C and Ag85D genes were successfully obtained by genetic engineering and named rBCG/Ag85A,rBCG/Ag85B,rBCG/Ag85C and rBCG/Ag85D,respectively.It could provide materials for further inserting other antigen genes into BCG cells to construct recombinant vaccine and vaccine research.

Key words: tuberculosis; Ag85 protein; in-fusion cloning; recombinant BCG vaccine

CLC Number:

Q782

HUANG Shuqin, HUANG Wenjing, LI Wenfeng, HONG Rui, HUANG Shujian, CHEN Yong. A Construction of Recombinant BCG Expressing Ag85 Gene[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(2): 590-596.

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A Construction of Recombinant BCG Expressing Ag85 Gene

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