title: [Prokaryotic expression and polyclonal antibody preparation of antitoxin higA of Mycobacterium tuberculosis].
abstract: OBJECTIVE
  To clone higA gene from genome of Mycobacterium tuberculosis, express higA protein in E.coli and prepare polyclonal antibodies against higA protein of Mycobacterium tuberculosis.
  
  
  METHODS
  HigA gene was obtained by PCR from the genome of Mycobacterium tuberculosis H37Rv strain and inserted into E.coli plasmid pET-32a⁺. The recombinant expression vector pET-32a⁺-higA was then transformed into prokaryotic expression bacterium E.coli BL21, and the recombinant protein higA was expressed in E.coli BL21 under IPTG induction. The protein was identified by SDS-PAGE and Western blotting. Purified protein higA was used to immunize New Zealand rabbits to prepare anti-higA polyclonal antibody. Quality and quantity of the antibody were examined.
  
  
  RESULTS
  HigA gene was successfully amplified and inserted into pET-32a.⁺ DNA sequencing confirmed the positive recombinant plasmid pET-32a⁺-higA screened by PCR and restriction enzyme digestion analysis. After induction with IPTG, the recombinant protein higA with 36.63 kDa was successfully expressed in E.coli BL21. Rabbit anti-higA polyclonal antibody with a good specificity was obtained, and the titer was above 1:3200.
  
  
  CONCLUSIONS
  The higA protein was successfully expressed in E.coli BL21. Rabbit anti-higA antibody with high titer and specificity was prepared.
authors.affiliation: Department of Clinical Laboratory Diagnostics, Weifang Medical University, Weifang 261053, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Liu
authors.affiliation: Department of Laboratory Medicine, Shandong Provincial Key Laboratory of Clinical Laboratory Diagnostics, Hospita1Affiliated to Weifang Medical University, Weifang 261031, China.
authors.forename: Zhengjun
authors.initials: Z
authors.lastname: Yi
authors.affiliation: Department of Medical Microbiology, Weifang Medical University, Weifang 261053, China.
authors.forename: Yurong
authors.initials: Y
authors.lastname: Fu
authors.affiliation: Department of Laboratory Medicine, Third People's Hospital of Heze, Heze 274031, China.
authors.forename: Shuqing
authors.initials: S
authors.lastname: Li
authors.affiliation: Department of Laboratory Medicine, PLA 401 Hospital, Qingdao 266000, China.
authors.forename: Shanshan
authors.initials: S
authors.lastname: Yang
mesh_terms: D000818:Animals; D000906:Antibodies / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D000992:Antitoxins / Q000276:immunology*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D009169:Mycobacterium tuberculosis; D010957:Plasmids / Q000235:genetics
chemical_list: D000906:Antibodies; D000992:Antitoxins