长角血蜱酸性磷酸酶全长cDNA的克隆与原核表达及其表达产物的酶活性分析

Cloning and prokaryotic expression of full-length cDNA encoding acid phosphatase from Haemaphysalis longicornisand analysis of enzyme activity of the expressed protein

通过对长角血蜱饥饿雌性成蜱cDNA表达文库中筛选到的未知基因进行5′RACE扩增,得到了长角血蜱酸性磷酸酶全长cDNA序列。利用生物信息学方法对该基因的核苷酸序列和推导的氨基酸序列进行了分析,同时构建了系统发育树。采用PCR方法扩增目的基因完整的开放阅读框,将其克隆到表达载体pGEX-4T-1中,构建重组质粒,转入BL21(DE3)中,经IPTG进行诱导表达,分别用SDS-PAGE和Wes-tern-blot分析目的蛋白的表达情况和反应原性,同时进行体外酶活性分析。结果表明,成功获得了长角血蜱酸性磷酸酶全长序列,体外高效诱导表达了约67.0 ku的重组蛋白。Western-blot表明该蛋白具有很强的反应原性,且在pH5.0时,该酶水解对硝基苯磷酸二钠的能力最强。

The full-length cDNA encoding was screened from a cDNA library constructed tion of the cDNA ends（5＇ RACE）. The nucleoti were analyzed by the bioinformatics software, acid phosphatase from Haema from unfed female ticks,was ob de sequence of acid phosphatase and the phylogenetic tree was ph ysalis longicornis , which tained by 5＇ rapid amplifica- and the deduced amino acid constructed. The complete open reading frame was amplified by PCR,the PCR product was inserted into expression vector pGEX 4T-1 to construct recombinant plasmid,and the recombinant plasmid was transformed into Escherichia coli BL21 （DE3） for expression. The expression and reacto PAGE and Western-blot, respectively. These result phatase from H. longicornis was obtained successf 67.0 ku in molecular weight. Western-blot showed binant acid phosphatase,once was activated, could at an optimal pH of 5.0. of r dth ecombinant prote at the full-length expressed fusion in were analyzed by SDS- cDNA encoding acid phos protein was approximately that the protein possessed reactogenicity, and the recomhydrolyze para-nitrophenyl phosphate（pNPP） substrate at an optimal pH of 5.0.