Sequence analysis and expression of groE gene encoding heat shock proteins of Brucella abortus isolates

Brucella abortus 국내 분리주의 Heat Shock Protein 암호 groE 유전자의 염기서열 분석과 발현

  • Kim, Tae-Yong (ChoongAng Vaccine Laboratory) ;
  • Kim, Ji-Young (College of Veterinary Medicine, Chungnam National University) ;
  • Chang, Kyung-Soo (College of Medicine, University of Kentucky) ;
  • Kim, Myung-Cheol (College of Veterinary Medicine, Chungnam National University) ;
  • Park, Chang-Sik (Research Center for Transgenic Cloned Pigs, Chungnam National University) ;
  • Han, Hong-Ryul (College of Veterinary Medicine, Seoul National University) ;
  • Jun, Moo-Hyung (College of Veterinary Medicine, Chungnam National University)
  • 김태용 ((주)중앙백신연구소) ;
  • 김지영 (충남대학교 수의과대학) ;
  • 장경수 (켄터키대학교 의과대학) ;
  • 김명철 (충남대학교 수의과대학) ;
  • 박창식 (충남대학교 형질전환복제돼지연구센터) ;
  • 한홍율 (서울대학교 수의과대학) ;
  • 전무형 (충남대학교 수의과대학)
  • Accepted : 2005.01.09
  • Published : 2005.03.25

Abstract

GroE that is a heat shock protein composed of GroEL and GroES is known as an immunodominant target of both the humoral and cellular immune responses in bovine brucellosis. This study was carried out to characterize groE gene encoding heat shock proteins of B. abortus isolated in Korea and to evaluate the immunogenicity of the GroE protein expressed in E. coli system. In PCR the specific signals with the size of 2,077 bp were detected in five strains isolated from the mammary lymphnodes of the dairy cattle that were serologically positive and the reference strains. In comparison of the sequences of nucleotides and amino acids among the strains, GroES showed 100% identity in both sequences. GroEL was evaluated 99.0~99.9% in nucleotides and 98.0~100% homology in amino acids. The groE gene including groES and groEL was inserted into pET29a vector and constructed pET29a-GroE recombinant plasmids. The inserted groE was confirmed by digestion with Nco1 and EcoR1 endonucleases and nucleotide sequencing. E. coli BL (DE3) was transformed with pET29a-GroE, named as E. coli BL (DE3)/pET29a-GroE. In SDS-PAGE, it was evident that the recombinant plasmid effectively expressed the polypeptides for GroES (10 kDa) and GroEL (60 kDa) in 0.5, 1 and 2 hours after IPTG induction. The immuno-reactivity of the expressed proteins were proved in mouse inoculation and Western blot analysis.

Acknowledgement

Supported by : 한국과학재단 우수연구센터

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