中国畜牧兽医 ›› 2020, Vol. 47 ›› Issue (2): 590-596.doi: 10.16431/j.cnki.1671-7236.2020.02.031

• 预防兽医 • 上一篇    下一篇

表达Ag85基因重组卡介苗的构建

黄淑芩1, 黄雯晶2, 李文锋3, 洪锐3, 黄淑坚3, 陈勇3   

  1. 1. 广东药科大学, 广州 510000;
    2. 韶关市植物保护站, 韶关 512000;
    2. 佛山科学技术学院生命科学与工程学院, 佛山 528200
  • 收稿日期:2019-05-08 发布日期:2020-02-28
  • 通讯作者: 黄淑坚, 陈勇 E-mail:sjhuang.foshan@163.com;chenyongtl@hotmail.com
  • 作者简介:黄淑芩(1997-),女,广东韶关人,本科,研究方向:食品化学与微生物,E-mail:1652061588@qq.com;黄雯晶(1992-),女,广东韶关人,硕士,研究方向:家禽传染病诊断与防控,E-mail:757586187@qq.com
  • 基金资助:
    高层次人才科研启动费(gg07042)

A Construction of Recombinant BCG Expressing Ag85 Gene

HUANG Shuqin1, HUANG Wenjing2, LI Wenfeng3, HONG Rui3, HUANG Shujian3, CHEN Yong3   

  1. 1. Guangdong Pharmaceutical University, Guangzhou 510000, China;
    2. Shaoguan Plant Protection Station, Shaoguan 512000, China;
    3. School of Life Science and Engineering, Foshan University, Foshan 528200, China
  • Received:2019-05-08 Published:2020-02-28

摘要: 为研发一种更安全、有效的结核病疫苗,用于预防并清除结核病,本试验构建4株分别表达Ag85AAg85BAg85CAg85D基因的重组卡介苗,参考pMV261载体序列和Tuberculist上登录的H37Rv序列设计引物,通过PCR扩增和无缝克隆技术构建含Ag85基因片段的重组pMV261质粒。分别对重组质粒进行PCR、双酶切鉴定后,通过电击转化转染到卡介苗感受态细胞中,利用Kan耐药基因筛选获得重组卡介苗菌株。采用PCR及Western blotting鉴定Ag85AAg85BAg85CAg85D基因在卡介苗中的表达,再对构建成功的重组卡介苗进行培养,用于后续试验。PCR扩增结果表明,构建的重组卡介苗rBCG/Ag85A、rBCG/Ag85B、rBCG/Ag85C和rBCG/Ag85D目的基因片段大小分别为1 447、1 402、1 453和1 330 bp,与预期大小一致,表明目的基因已成功整合到卡介苗中;Western blotting结果表明,Ag85AAg85BAg85CAg85D基因均可在卡介苗细胞内成功表达。本试验利用基因工程技术成功获得4株分别表达Ag85AAg85BAg85CAg85D基因的重组卡介苗,将其分别命名为rBCG/Ag85A、rBCG/Ag85B、rBCG/Ag85C、rBCG/Ag85D。本研究可为进一步将其他抗原基因插入卡介苗细胞构建重组疫苗及疫苗研究提供材料。

关键词: 结核病; Ag85蛋白; 无缝克隆; 重组卡介苗

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

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