生物技术通报 ›› 2022, Vol. 38 ›› Issue (7): 269-277.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1299

• 研究报告 • 上一篇    下一篇

霍乱弧菌溶血素HlyA的原核表达、纯化及多克隆抗体制备与鉴定

王光丽1,2(), 范婵1,2, 王辉1,2, 卢惠芳1,2, 夏灵尹1,2, 黄健1,2, 闵迅1,2()   

  1. 1.遵义医科大学附属医院医学检验科,遵义 563003
    2. 遵义医科大学检验医学院,遵义 563003
  • 收稿日期:2021-10-14 出版日期:2022-07-26 发布日期:2022-08-09
  • 作者简介:王光丽,女,硕士研究生,研究方向:细菌致病机制;E-mail: 1947952722@qq.com
  • 基金资助:
    国家自然科学基金项目(31800130)

Prokaryotic Expression,Purification,Identification,and Polyclonal Antibody Preparation of Vibrio cholerae Hemolysin HlyA

WANG Guang-li1,2(), FAN Chan1,2, WANG Hui1,2, LU Hui-fang1,2, XIA Ling-yin1,2, HUANG Jian1,2, MIN Xun1,2()   

  1. 1. Department of Clincal Laboratory,Affiliated Hospital of Zunyi Medical University,Zunyi 563003
    2. School of Laboratory Medicine,Zunyi Medical University,Zunyi 563003
  • Received:2021-10-14 Published:2022-07-26 Online:2022-08-09

摘要:

原核表达、纯化霍乱弧菌HlyA蛋白,制备并鉴定其多克隆抗体。PCR扩增霍乱弧菌hlyA基因并克隆入pET28a、pET32a和 pCold TF载体中构建重组表达载体;将重组载体pET28a-hlyA、pET32a-hlyA和pCold TF-hlyA转化E. coil BL21(DE3)中,进行表达条件优化及表达形式鉴定。获取可溶性形式的HlyA蛋白行Ni-NTA柱纯化,纯化的HlyA蛋白免疫BALB /c小鼠以制备多克隆抗体,并用间接ELISA法检测抗体效价,以评估其免疫原性。再以Western blot法分析抗体对霍乱弧菌中HlyA蛋白的特异性识别,并行质谱验证。分析纯化的HlyA蛋白的溶血活性及其抗体的中和活性。pET28a-hlyA、pET32a-hlyA载体只能诱导出包涵体表达的HlyA蛋白,pCold TF-hlyA载体诱导出可溶性表达的HlyA蛋白。经Ni-NTA柱纯化后获得较纯的HlyA蛋白,该蛋白不能裂解兔红细胞,但免疫小鼠可获得较高效价的多克隆抗体;Western blot和质谱鉴定均显示HlyA多克隆抗体能特异性识别霍乱弧菌中的HlyA蛋白,且该抗体可有效抑制霍乱弧菌分泌上清液的溶血活性。成功获得可溶表达的HlyA蛋白,免疫小鼠后获得高效价的抗HlyA多克隆抗体,为后续研究HlyA蛋白在霍乱弧菌致病过程中的作用奠定了基础。

关键词: 霍乱弧菌, HlyA蛋白, 原核表达, 多克隆抗体

Abstract:

It is aimed to express and purify hemolysin HlyA protein of Vibrio cholerae in prokaryotic cells,and to prepare and identify the polyclonal antibody of mouse anti-HlyA. The hlyAgene was amplified from the genome Vibrio cholerae via PCR and cloned into the expression plasmid pET28a,pET32a and pCold TF to construct the recombinant plasmid pET28a-hlyA,pET32a-hlyA and pCold TF-hlyA, and they were transformed into E. coli BL21(DE3). The conditions used to induce expression were optimized and the expressive forms were identified. The soluble form of hlyA protein was purified by Ni-NTA column. The purified HlyA protein was used as antigen for immunization of BALB/c mice to prepare polyclonal antibody. The antibody titer was determined to test the immunogenicity by indirect ELISA. The antibody specific recognition of HlyA protein in Vibrio cholerae was analyzed by Western blot and verified by mass spectrometry. The hemolytic activity of the purified hlyA protein and the neutralizing activity of the antibody were analyzed. The HlyA protein was expressed as inclusion bodies in the pET28a-hlyA and pET32a-hlyA vectors,and as soluble form in the pCold TF-hlyA vector. HlyA was purified by affinity chromatography on an Ni-NTA agarose column to get higher purity. The recombinant HlyA protein could not lyse rabbit red blood cells,but immunized mice can obtain polyclonal antibody with higher titer. The results of Western blot and mass spectrometry showed that the HlyA polyclonal antibody can specifically recognize the HlyA protein in Vibrio cholerae,and the antibody can effectively inhibit the hemolytic activity of Vibrio cholerae. The soluble form of HlyA protein and the high titer anti-HlyA polyclonal antibody were successfully obtained,which laid the foundation for the follow-up study of the role of HlyA protein in the pathogenic process of V. cholerae.

Key words: Vibrio cholerae, HlyA protein, prokaryotic expression, polyclonal antibody