蚯蚓纤溶酶新基因PV_(242)的克隆与表达  

Cloning and Expression of The Novel Gene--PV_(242) of Earthworm Fibrinolytic Enzyme

作　　者：徐义辉[1] 梁国栋[1] 孙兆军[1] 陈飞[1] 付士红[1] 柴玉波[1] 侯云德[1] 

机构地区：[1]中国预防医学科学院病毒学研究所

出　　处：《生物化学与生物物理进展》2002年第4期610-614,共5页Progress In Biochemistry and Biophysics

基　　金：国家高技术研究发展计划 (863 );生物技术资助项目 (10 2 0 8 0 2 0 3 )

摘　　要：从我国特有的双胸蚓属 (Lumbricusbimastus)蚯蚓体内提取到一种有纤维蛋白平板溶解活性的蛋白质 ,采用聚偏二氟乙烯膜 (PVDF)微量测序法测得蛋白质的N端氨基酸序列 ,依此设计简并引物 ,通过RT PCR获得其对应cDNA .该cDNA全长 888bp ,1～ 72 6位核苷酸对应读码框架 (ORF) ,编码含 2 4 2个氨基酸的成熟肽 .终止子 (TAG)位于cDNA的 72 7～ 72 9位 ,其余核苷酸序列为 3′端非编码区 .成熟肽命名为蚯蚓纤溶酶PV2 42 .蛋白质预测得知蛋白质等电点为 4 33,含组氨酸 (His4 4 )及丝氨酸 (Ser191)两个活性位点 ;蛋白质由两个结构域组成 ,表面有活性裂隙 ;该蛋白质属丝氨酸蛋白酶超家族胰蛋白酶类 .经国际基因库等多种查询比较未见PV2 42 基因的报道 ,为首次发现的新基因 ,在国际基因库的输入号为GenBankAF10 96 4 8.随后构建了pTrxFUS PV2 42 重组质粒 ,并在大肠杆菌GI72 4中获得融合蛋白TrxA/PV2 42 的可溶性表达 ,采用离子交换层析法纯化表达蛋白 ,融合蛋白有纤维平板溶解活性 .One kind of protein from earthworm( Lumbricus bimastus )with the molecular mass about 30 ku was extracted by SDS PAGE. Then the N terminal of the protein was sequenced. The PCR primer was designed according to the N terminal sequence, and its cDNA fragment was obtained by RT PCR. The cDNA was cloned into pGEMT vector and sequenced. The sequence showed that the fragment was 888 bp,with the ORF of 726 bp and the 3′ untranslation terminal area. The ORF encoded a protein of 242 amino acids, so the protein was named as PV 242 .The prediction of protein structure shows that the PV 242 has two domains,between which is the active sites His44 and Ser191.At the same time, the p I 4 33 of PV 242 was detected, and it is a protease in trypsin family of serine protease superfamily. PV 242 was expressed in pTrxFUS expression system as the fusion protein TrxA PV 242 in soluble form, the TrxA was a molecular chaperon which made the expressed protein soluble. The fusion protein was purified by ion exchange chromatography. The purified fusion protein TrxA PV 242 has fibrinolytic activity.

关 键 词：蚯蚓纤溶酶 新基因 PV242 克隆 表达 双胸蚓属 蛋白质测序 

分 类 号：R973.2[医药卫生—药品] R394.8[医药卫生—药学]