创伤后增生性疤痕中α1(Ⅰ)及(Ⅲ)型前胶原基因片段的克隆及鉴定

Cloning and Identification of the Gene Fragments of α 1(Ⅰ)and(Ⅲ) Procollagen Genes in the Hypertrophic Scar After Trauma

【目的】胶原蛋白是构成细胞外基质的主要组分之一 ,它的过量沉积是创伤后增殖性瘢痕形成的原因之一 ,本实验的目的就是要克隆出疤痕中α 1(Ⅰ )型及 (Ⅲ )型前胶原基因片段并进行鉴定。【方法】本研究根据α 1(Ⅰ )型及 (Ⅲ )型前胶原基因的核苷酸序列分别设计 3条引物 ,从瘢痕组织中经RT PCR分别扩增Ⅰ及Ⅲ型前胶原基因的第 2外显子 (Exon 2 )区域的基因片段 (ⅠF2 ,ⅢF2 ) ,克隆到T载体 (pGEM Tvector)。得到阳性重组质粒后经限制性内切酶酶切 ,PCR及测序加以证实。【结果】分别获得长度约为 12 9bp的DNA片段 (简称为ⅠF2 ) ,与长度约为 189bp的DNA片段 (简称为ⅢF2 ) ,与预期片段大小一致。得到重组质粒 pT Ⅰ及 pT Ⅲ ,并得到证实。【结论】获得的α1(Ⅰ )型及 (Ⅲ )型前胶原基因片段是预期所要得到的正确片段。

Objective Collagen protein is one of major constituents of extracellular matrices. Its overaccumulation can lead to the cicatrization. The aim of this study was to obtain the cloning and identification of the gene fragments of α1(Ⅰ)and(Ⅲ) procollagen genes. Methods In this study, separate three primers were designed and synthesized according to the nucleotide sequence of each of α1(Ⅰ)and(Ⅲ) procollagen genes. By using RT-PCR, the gene fragments (ⅠF2, ⅢF2) within the second exons of the two procollagen genes were separately isolated and cloned into the pGEM-T vector. Results The resulting two recombinant plasmids(pT-Ⅰ pT-Ⅲ) were verified by restriction enzymes, PCR and sequencing. Conclusions The cloned sequences were the gene fragments which we just wanted to obtain them.