一种新的螯虾丝氨酸蛋白酶抑制物基因的克隆表达及其活性分析

Cloning and expressing of a new serine proteinase inhibitor gene of crayfish and its activity analysis

根据本课题组从克氏原螯虾中新发现的丝氨酸蛋白酶抑制物的基因序列(GenBank登录号CD644775)设计一对引物,应用逆转录-聚合酶链式反应(RT-PCR)技术,从螯虾血淋巴细胞中扩增出丝氨酸蛋白酶抑制物基因PCI188,将其连入原核表达载体pET-32a,转化至大肠杆菌Rosetta株和BL21株中进行蛋白表达,结果该蛋白只在前者表达。表达产物用免疫转印检测,出现50kD的特异性条带,与螯虾PCI188基因编码的蛋白大小相符。将融合蛋白纯化后免疫新西兰兔,用免疫血清与螯虾血淋巴作用后测定酚氧化酶活力,结果显示,酚氧化酶活力有所升高,从而首次证实螯虾PCI188编码的蛋白对丝氨酸蛋白酶有抑制作用。

The specific primers were designed referring to the reported gene sequences （GenBank accession numberCD644775） of serine proteinase inhibitor first found from crayfish Procambarus clarkii. Serine proteinase inhibitor gene （PCI188） was amplified by reverse transcription polymerase chain reaction （RT-PCR） method using total RNA isolated from crayfish hemolymph as template. The amplified eDNA fragments were purified and inserted into pET-32a vector, then transformed into E, coli BL21 （DE3） and Rosetta. They induced by IPTG for the expression of recombinant protein. The protein was only expressed in Rosetta other than in BL21.The expressed fusion protein was identified by SDS-PAGE and Western-blotting. The results showed that 50 kD fusion protein was identical with the known protein coded by gene PCI188 of crayfish. Recombinant protein was purified and used to immunize the rabbit. Then phenoloxidase （PO） activities were measured by speetrophotometer after the immunized serum incubated with crayfish haemolymph. The results showed that immunized serum increased the PO aetivity of crayfish. This paper is the first to prove that the protein coded by gene PCI188 of crayfish can inhibit serine proteinase [ Acta Zoologcica Sinica 51 （6） ： 1109-1114, 2005].