大劣按蚊丝氨酸蛋白酶cDNA克隆和表达

Cloning and expression of serine proteases from Anopheles dirus haemolymph

目的 克隆、表达大劣按蚊丝氨酸蛋白酶。方法 根据已报道昆虫的前酚氧化酶激活酶 (prophenoloxidase -acti vatingenzyme,PPAE)的保守氨基酸序列设计简并引物 ,以大劣按蚊血细胞mRNA为模板 ,进行RT -PCR扩增 ,克隆和测定插入片段 ;所得序列进行BLAST查询 ,并原核表达其中的两种PPAE样丝氨酸蛋白酶cDNA ,通过SDS -PAGE和Westernblotting检测表达产物。结果和结论 获得了 3种大劣按蚊血细胞丝氨酸蛋白酶cDNA部分序列 ,即AdsP1(5 12bp) ,AdsP2(488bp)和AdsP3(5 12bp)。AdsP1和AdsP3与冈比亚按蚊sP14D1、sP14D2、3种昆虫PPAE和黑腹果蝇Easter很相似 ,推测AdsP1和AdsP3可能是大劣按蚊的PPAE ,起调节大劣按蚊黑化包裹约氏疟原虫反应的作用。另外 ,成功表达了AdsP1和Ad sP3部分cDNA序列。

Objective To clone serine proteases from Anopheles dirus haemolymph and express in JM109.Methods Serine proteases were amplified by RT-PCR with degenerated primers designed based on conserved amino acid sequence of insect PPAEs,then target PCR product was purified and cloned into Pinpoint-Xa-1 vector,the inserted fragments contained in the positive recombinant plasmids verified by PCR and digestion with restriction enzyme were sequenced.After that,obtained sequences were blasted with Genebank sequence database;two PPAE-like serine proteases were expressed in JM109 and detected by SDS-PAGE and Western blotting.Result and conclusion Three kinds of serine proteases(AdsP1,AdsP2 and AdsP3)obtained contain the conserved amino acid residues of serine protease catalytic domain.BLAST indicated that both AdsP3 and AdsP1 share high similarities with Anopheles gambiae sP14D1 and sP14D2,insect PPAEs and Drosophila Easter.Therefore,we have succeeded in cloning three serine proteases from Anopheles dirus haemolymph,AdsP3 and AdsP1 maybe the PPAE of Anopheles dirus involved in regulation of melanotic encapsulation of Plasmodium.In addition,we have succeeded in expressing the AdsP3 and AdsP1 partial cDNA in JM109.