Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (7): 269-277.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1299

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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 Online:2022-07-26 Published:2022-08-09
  • Contact: MIN Xun E-mail:1947952722@qq.com;zmchj2001@163.com

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