一种广州管圆线虫新基因——胶原蛋白全长cDNA的克隆和鉴定

Cloning and Identification of the Full-Length cDNA Sequence of Angiostrongylus cantonensis Collagen

目的克隆和鉴定广州管圆线虫(Angiostrongyluscantonensis)新基因——胶原蛋白(collagen,COL)全长cDNA序列。方法根据表达序列标签(expressionsequencetag,EST)中部分序列和文库载体序列设计引物,采用PCR技术从广州管圆线虫幼虫的cDNA文库中分段扩增cDNA序列,用DNAMAN软件拼接分段扩增的序列以得到全长cDNA序列;利用多种生物信息学软件对cDNA全长序列进行同源性搜索与结构分析。根据cDNA全长序列,设计新的引物从文库中扩出包含胶原蛋白全长开放阅读框(openreadingframe,ORF)的cDNA片段,PCR产物纯化后克隆到pMD18-T载体上并测序。重组T载体经双酶切后切下的新基因AcCOL亚克隆到原核表达质粒pET32a(+)中。结果克隆一个广州管圆线虫新基因全长cDNA序列;序列比对、蛋白结构域搜索及结构分析结果显示,该新基因所编码的是一种胶原蛋白。构建的新基因重组质粒经双酶切后证明含有与目的片段长度相符的插入片段。结论克隆并鉴定了广州管圆线虫新胶原蛋白编码基因全长,成功构建了该基因的原核表达重组质粒pET32a(+)-AcCOL。

Objective To clone and identify a novel collagen （COL） cDNA of Angiostrongylus cantonensis, Methods According to the sequences of a known EST of A. cantonensis and the vector, primers were designed to amplify the cDNA sequence from an A. cantonens/s larvae cDNA hbrary by RACE like PCR. The amplified cDNA sequences were spliced, that is going through a process of homologue identitifcation and underwent structural analysis using diverse bioinformatics programs, by a DNAMAN software to obtain the full-length cDNA sequence. According to the full-length cDNA sequence, a pair of primers were designed to amplify the full-length open reading frame （ORF） sequence of the novel gene from the cDNA library. The PCR product was cloned into the pMD18-T vector and sequenced to confirm the full-length ORF of the AcCOL cDNA. The AcCOL cDNA was further subcloned into the expression plasmid pET32a （＋）. Results A novel full-length cDNA sequence of A. cantonens/s was cloned. Based on the sequence alignment, Motif Scan and structural analysis by softwares, the cDNA sequence is encoding the collagen of A. cantonensis. Conclusion The coding sequence for collagen of A. cantonensis was first cloned and identified, and its recombinant prokaryotic expression plasmid pET32a （＋）-AcCOL was successfully constructed.