简单异尖线虫D-天冬氨酸蛋白酶基因的克隆和表达  

Cloning and Expression of D-like Aspartic Protease of Anisakis simplex

作　　者：倪芳[1] 徐世三[1] 王逸难[1] 余长茂[1] 罗大民[1] 

机构地区：[1]厦门大学生命科学院,厦门361005

出　　处：《中国寄生虫学与寄生虫病杂志》2012年第4期294-298,共5页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(No.81171595);福建省自然科学基金(No.2010J01229)~~

摘　　要：目的克隆简单异尖线虫Ⅲ期幼虫(L3)的D-天冬氨酸蛋白酶基因(AsAP)全长,研究其表达蛋白的特性。方法根据GenBank中简单异尖线虫D-天冬氨酸蛋白酶基因表达序列标签的部分信息,设计特异引物并用cDNA末端快速扩增技术得到AsAP全长序列,分析推导的蛋白序列特征,并预测其三级结构。用RT-PCR扩增简单异尖线虫L3的AsAP基因编码序列,产物用EcoRⅠ和SalⅠ双酶切,连入表达载体pET32а(+),转化大肠埃希菌(E.coli)BL21(DE3),以异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,表达产物经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测。结果简单异尖线虫L3的AsAP基因全长1 753 bp,编码453个氨基酸,与锡兰钩虫(Ancylostoma ceylanicum)的D-天冬氨酸蛋白酶相似性达65%。该蛋白具有两个保守的催化域,1个活性中心翼环,S2和S3亚位点各1个;具有由20个氨基酸组成的N端信号肽,构成疏水性强的跨膜域。不同浓度的IPTG(0.2～1.6 mmol/L)诱导对AsAP表达的影响较小,1.0 mmol/LIPTG诱导2 h后表达量达到最高水平。结论克隆并表达了简单异尖线虫的D-天冬氨酸蛋白酶。Objective To clone and express the full length of D-like aspartic protease gene （AsAP） of the third stage larvae of Anisakis simplex. Methods According to the partial information of D-like aspartic protease encoding gene of A. simplex from GenBank, specific primers were designed to amplify 3＇end and 5＇ end of AsAP gene using rapid amplification of cDNA ends （RACE）, and the full length of the D-like aspartic protease gene was obtained. Using total RNA of the third-stage larvae of A. simplex, coding sequence of the AsAP gene was amplified by reverse transcription- PCR （RT-PCR）. The PCR product was digested by EcoR Ⅰ and SalⅠ , and cloned into pET32 vector. The recombinant plasmid was checked by double enzyme digestion and sequencing, and the positive recombinant plasmid was transformed into E. eoli BL21 （DE3）. Expression of the protein induced by IPTG under gradient concentration and different time was conducted. Result A 1 753 bp full length of AsAP was obtained, which contained 30 bp 5＇UTR, 361 bp 3＇UTR and a 1 362 bp open reading frame （ORF） encoding 453 amino acids with a predicted molecular mass of Mr 50 726. It showed 65% identity with the D-like aspartic protease of Ancylostoma ceylanicum. The predicted amino acid sequence contains two conserved catalytic motif, an active site flap, an S2 subsite and an S3 subsite. A 20 amino acids signal peptide was found in the N-terminus, with significant hydrophobic property. Different concentration of the IPTG （0.2-1.6 mmol/L） showed little effect on the expression, and the production of the protein was up to maximum after 2 hours induction. Conclusion The AsAP gene has been cloned and expressed.

关 键 词：简单异尖线虫 D-天冬氨酸蛋白酶 克隆 原核表达 

分 类 号：R941.524[医药卫生—药剂学]