类风湿关节炎患者Ⅱ型胶原蛋白cDNA基因的克隆及序列分析

Cloning and sequence analysis of type Ⅱ collagen protein gene cDNA from patients with rheumatoid arthritis

目的克隆人Ⅱ型胶原蛋白cDNA(hCⅡcDNA)基因。方法以类风湿关节炎患者关节软骨组织的RNA为模板,采用RT-PCR技术扩增hCⅡcDNA基因,将扩增产物与pMD18-T载体连接后转化大肠杆菌感受态细胞DH5α,在LB培养基中培养12～16 h,挑取菌落并接种于LB培养液中,取2μL菌液为模板进行PCR鉴定,PCR结果呈现特异性条带为hCⅡcDNA阳性。对hCⅡcDNA阳性的克隆菌株进行测序。结果克隆基因经测序并通过NCBI BLAST分析后发现,其与Genebank中公布的编码人Ⅱ型胶原蛋白的基因序列(NM_001844)同源性达99%。结论成功克隆出了hCⅡcDNA基因。

Objective To clone the gene of type Ⅱ collagen （ C Ⅱ ） from human ankle. Methods The hC Ⅱ cDNA gene was amplified by RT-PCR. The products was connected with pMD18-T and was inserted into eukaryotic expressing vector PcDNA3.1 （ ＋ ） which was cloned and sequenced. Results A homology search performed by NCBI BLAST re- vealed that the cloned gene from human ankle shared 99% similarity with the nucleotide sequence of C Ⅱ antifungal protein gene （Genbank accession no： NM_001844）. Condution It is success to clone the hC Ⅱ cDNA gene.