长角血蜱谷胱甘肽过氧化物酶的克隆与原核表达(英文)

Cloning and prokaryotic expression of a glutathione peroxidase from tick Hemaphysalis longicornis

目的长角血蜱(Hemaphysalis longicornis)不仅吸食家畜血甚至人血,还是多种疾病的传播媒介,对该蜱的研究具有重要的医学和兽医学意义。在大肠杆菌中表达长角血蜱的谷胱甘肽过氧化物酶(glutathione peroxidase)(HlGPx),为该酶在长角血蜱中的生理作用研究奠定基础,为该病媒的免疫防控提供参考。方法根据长角血蜱EST文库提示的片段序列的信息,设计引物采用PCR方法从长角血蜱成蜱的cDNA中扩增谷胱甘肽过氧化物酶基因。利用定点突变将该基因编码序列中一个编码硒半胱氨酸的密码子TGA突变为通用半胱氨酸密码子TGC,与表达载体pGEX-4T-1重组,转化BL21(DE3)pLysS,IPTG诱导表达,SDS-PAGE及免疫印迹分析。结果成功获得了长角血蜱谷胱甘肽过氧化物酶全长序列,突变后在大肠杆菌中诱导表达了约51kDa的重组蛋白,Western-blot显示表达产物与抗GST抗体有很强的交叉反应。结论突变的HlGPx可以在大肠杆菌中表达并可用于下一步制备免疫血清及功能分析。

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.