Cloning and prokaryotic expression of a glutathione peroxidase from tick Hemaphysalis longicornis
10.3969/cjz.j.issn.1002-2694.2012.09.001
- VernacularTitle:长角血蜱谷胱甘肽过氧化物酶的克隆与原核表达
- Author:
Chaoying ZHANG
;
Guowei GUO
;
Zhengchao WANG
;
Xiaohong HUANG
- Publication Type:Journal Article
- Keywords:
Haemaphysalis longicornis;
glutathione peroxidase;
cloning;
site-directed mutagenesis;
prokaryotic expression
- From:
Chinese Journal of Zoonoses
2012;(9):861-868
- CountryChina
- Language:Chinese
-
Abstract:
Ticks transmit various diseases to livestock and human beings. The researches on ticks are of great importance in medical and veterinary sciences. To express a glutathione peroxidase gene (HlGPx) from Hemaphysalis longicornis in Escherichia coli (E. coli) so as to provide basis for further studies, the gene was amplified from cDNAs of H. longicornis adult ticks by PCR prompted by information from EST library. The TGA codon encoding selenocysteine in the gene was mutated into the universal genetic code for cysteine,TGC, by site-directed mutagenesis. E. coli was transformed with the recombinant expression vector pGEX-4T-1/HlGPx'(the mutated HlGPx) and induced to express recombinant HlGPx', which was then analyzed by sodium dodecylsulphate-polyacrylamide gel electrophoresis and Western blotting. The result showed that the complete coding sequence of HlGPx was obtained successfully, the deduced polypeptide was 223 aa, with a calculated molecular mass of 25.0 kDa; the recombinant fusion protein was approximately 51 kDa, correspondent to the calculated. The antibody against glutathione S-transferase (GST) recognized the recombinant protein fused with GST in a Western blotting assay, which confirmed the result above-mentioned. In conclusion, the mutated HlGPx was expressed in E. coli successfully and it could be used for further preparation of immune serum and functional analysis.
