中华按蚊防御素基因的克隆、原核表达及其重组产物生物活性的初步评价

目的 克隆、原核表达中华按蚊的防御素基因，并对其表达产物的生物学活性进行评价。方法 根据已发表的埃及伊蚊和冈比亚按蚊防御素基因序列设计合成两对引物．以中华按蚊成蚊cDNA为模板进行PCR扩增，将预期片段克隆测序并进行分析；根据表达质粒pET32a( ）上的克隆位点及测序结果设计PCR引物．将截短的基因片段重组入质粒pET32a( )中进行表达．纯化表达产物并进行体外抑菌试验。结果 PCR扩增产物大小约270bp．目的基因的序列与冈比亚按蚊防御素基因同源性为85％；截取其保守功能区域序列(162bp)．构建成功重组表达质粒pET32a( )-tDEF，含重组质粒的转化菌用IPTG诱导表达后，于Mf26000处可见一预期的特异表达带，该蛋白与抗6-His抗体有特异性的反应；琼脂扩散法显示，纯化的重组防御素融合蛋白未出现抑菌环。结论首次克隆了中华按蚊防御素基冈．其截短片段在大肠杆菌中得到了高效可溶性表达，但重组融合蛋白不具备抑菌活性。

Cloning and prokaryotic expression of Anopheles sinensis defensin gene and preliminary bioactivity evaluation of the recombinant product]

OBJECTIVE: To clone the defensin gene of Anopheles sinensis for its prokaryotic expression and preliminary bioactivity evaluation of the recombinant protein. METHODS: Two pairs of primer were designed and synthesized based on the published defensin sequences, and PCR amplification was performed using the cDNA of An.sinensis as the template. The PCR products were cloned and sequenced. A truncated defensin fragment was cloned into pET-32(a) plasmid and expressed with the induction by IPTG. The expressed product was purified and its bioactivity evaluated with agarose diffusion assay. RESULTS: The PCR product 270 bp in size was identified as the coding sequence for defensin after sequence analysis, which had 85%; homology with the defensin sequence from An.gambiae. The truncated defensin (162 bp) cloned was subcloned into the expression plasmid pET-32(a) and the molecular weight of the target fusion protein was 26 000 after IPTG induction. Preliminary experiment, however, failed to demonstrate bacteriostatic effect of the purified recombinant protein. CONCLUSION: The coding sequence for defensin gene of An.sinensis has been successfully cloned and the truncated fraction can be highly expressed as a soluble fusion protein, which, however, does not possess bacteriostatic activity.