长角血蜱丝氨酸蛋白酶抑制剂基因的原核表达  

Prokaryotic expression of serpin-2 gene from Haemaphysalis longicornis

作　　者：王路[1,2] 刘光远[2] 谢俊仁[2] 龚真莉[2] 田占成[2] 柴慧萍[2] 贾宁[1] 

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院兰州兽医研究所家畜疫原病原生物学国家重点实验室甘肃省兽医寄生虫学重点实验室,甘肃兰州730046

出　　处：《中国兽医学报》2009年第8期998-1002,共5页Chinese Journal of Veterinary Science

基　　金：国家科技基础条件平台项目(2005DKA21205-3);国家"863"计划资助项目(2006AA10A207);甘肃省自然科学基金资助项目(32S041-A25-035)

摘　　要：根据GenBank中的长角血蜱丝氨酸蛋白酶抑制剂(Haemaphysalis longicornisserine proteinase inhibitor-2,HLS2)基因序列设计特异性引物,以长角血蜱饥饿成蜱总RNA为模板,经RT-PCR扩增出1 164 bp的HLS2DNA片段。将其与pET-30a载体连接,构建pET-30a-HLS2表达载体,转化J M109大肠杆菌,筛选阳性克隆,经双酶切鉴定及测序分析后转化到E.coliBL21(DE3)表达菌株中,经IPTG诱导后收集菌体进行SDS-PAGE电泳分析。优化表达条件后纯化融合蛋白,用Western blotting鉴定其抗原性。结果表明,获得的HLS2 DNA片段与GenBank中的HLS2(序列号AB162827)序列的同源性为98%。构建的pET-30a-HLS2表达载体在大肠杆菌中表达了约为47000的HLS2融合蛋白,主要以包涵体形式存在,IPTG终浓度为1.0 mmol/L、28℃诱导7 h后融合蛋白的表达量最高。Western blotting显示该融合蛋白可与兔抗长角血蜱阳性血清反应。A pair of specific primers were designed according to the （Haemaphysalis longicornis serine proteinase inhibitor-2, HLS2） gene sequence published in GenBank. The HLS2 DNA fragment of 1 164 bp was amplified by RT-PCR from unfed adult tick total RNA and cloned into pET-30a and transformed into E. coli JM109. The positive colonies were identified by restriction endonuclease digestion and sequencing. The pET-30a-HLS2 expression vector was transformed into E. coli BL21 （DE3） and induced by IPTG. The expression product was analyzed by SDS-PAGE and the expression condition was optimized. The recombinant protein was purified and identified by Western blotting. The results indicated that the cloned HLS2 DNA fragment had 98% identity to HLS2 sequence in GenBank （Accession no. AB162827）. A 47 000 HLS2 fusion protein was expressed in E. coli mainly as the inclusion bodies form. The optimum expression of HLS2 fusion protein was obtained with 1.0 mmol/L IPTG for 7 h at 28℃. Western blotting analysis showed the HLS2 fusion protein was recognized by the anti-H, longicornis serum of rabbit.

关 键 词：长角血蜱 丝氨酸蛋白酶抑制剂-2 原核表达 

分 类 号：S852.23[农业科学—基础兽医学] S852.7[农业科学—兽医学]