中国人兽共患病学报2012，Vol.28Issue(9)：861-868,8.DOI:10.3969/cjz.j.issn.1002-2694.2012.09.001
长角血蜱谷胱甘肽过氧化物酶的克隆与原核表达
Cloning and prokaryotic expression of a glutathione peroxidase from tick Hemaphysalis longicornis
摘要
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.关键词
长角血蜱/谷胱甘肽过氧化物酶/生物信息学分析/克隆/定点突变/原核表达/Western印迹Key words
Haemaphysalis longicornis/ glutathione peroxidase/ cloning/ site-directed mutagenesis/ prokaryotic expression分类
农业科技引用本文复制引用
张超颖,郭果为,王正朝,黄晓红..长角血蜱谷胱甘肽过氧化物酶的克隆与原核表达[J].中国人兽共患病学报,2012,28(9):861-868,8.基金项目
国家级生物学实验教学示范中心本科生创新性研究计划项目(No.02011520),国家自然科学基金项目(No.31101032,31040084)和福建省自然科学基金项目(No.2010J01145,2008J0004)联合资助 Supported by the Innovative Research Project for Undergraduates from College of Life Science,Fujian Normal University (No.02011520),the Natural Science Foundation of Fujian Province (No.2010J01145 and No.2008J0004),and the Natural Science Fundation of China(No.31101032 and No.31040084). （No.02011520）
