草地螟羧肽酶A基因的克隆、表达及序列分析

Cloning,expression and sequence analysis of carboxypeptidase A gene from Loxostege sticticalis(Lepidoptera:Pyralidae)

羧肽酶是昆虫体内重要的消化酶系之一。利用甜菜夜蛾Spodoptera exigua(Hübner)围食膜多克隆抗体免疫筛选草地螟Loxostege sticticalis L.中肠cDNA表达文库,得到编码羧肽酶A的全长cDNA克隆。该cDNA克隆全长1 380 bp(GenBank登录号EU924506),开放阅读框长1 302 bp,编码434个氨基酸,预测分子量和等电点分别为49.1 kDa和9.56。序列含有胰蛋白酶切割位点和催化特征,具有典型的羧肽酶A特性。将该基因与pET30载体重组后,经IPTG诱导,蛋白在大肠杆菌中获得了表达。

Carboxypeptidase A （CPA） plays an important role in the digestion of dietary proteins in insects. LstiCPA, a full-length cDNA clone encoding earboxylesterase was obtained by screening cDNA expression library of the midgut of Loxostege sticticalis L. using the polyclonal antiserum against Spodoptera exigua （Htibner） invertebrate intestinal mucin （IIM）. LstiCPA is 1 380 bp in length （GenBank accession no. EU924506） , and the open reading frame （ORF） encodes 434 amino acids, with the predicted MW 49.1 kDa and pI 9.56. LstiCPA possesses a trypsin cleavage site and a catalytic zinc site characterized by CPA. LstiCPA was recombined into pET30 vector. The Western blotting analysis demonstrated that LstiCPA protein was expressed at a low level after IPTG induction. The enzymatic activity of LstiCPA was 0. 0005 U/mL with the FAPP as substrate.