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

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.