肺炎支原体P1蛋白结构基因片段的制备及C末端基因片段克隆的研究

STUDY ON PREPARING THE P1 PROTEIN STRUCTURE GENE AND CLONING THE FRAGMENT OF P1 PROTEIN C END GENE OF MYCOPLASMA PNEUMONIAE IN ESCHERICHIA COLI

目的 制备肺炎支原体P1蛋白结构基因 ,并在大肠杆菌中克隆肺炎支原体P1蛋白C末端基因片段 ,为P1蛋白基因片段的扩增、表达及探讨C末端基因片段功能打基础。方法 PCR扩增方法获取P1结构基因。扩增产物用P1蛋白基因特异引物扩增鉴定。用SalⅠ和EcoRⅠ双酶酶切消化方法制备P1蛋白C末端基因片段 ,并与pUC19DNA连接 ,转入大肠杆菌JM10 9菌株。用X - gal平板及质粒图谱分析方法筛选重组克隆株 ,再用限制性核酸内切酶酶切图谱分析鉴定。 结果 经PCR扩增MPDNA获得一条 5 .0kbDNA片段 ,用P1基因区特异引物扩增鉴定获得阳性结果。重组质粒限制性内切酶指纹图谱显示出两条带 ,一条为 pUC19载体DNA带 ,另一条是 1kb的插入片段。

Aim To obtain the P1 structure gene and to clone the P1 protein C end gene fragment of Mycoplsma pneumoniae in Escherichia coli.This experiment will play a foundation for P1 gene expression.And for explore gene product function of P1 protein C end.Methods P1 protein gene was acquired by PCR method and production was identified by PCR that is specific primer of P1 gene.C end.gene of P1 protein was obtained by digested P1 protein gene with both endonuclease Sal Ⅰand EcoR Ⅰ.1kb DNA fragments was recovered and ligated them with pUC19 vector DNA and then transformed into Escherichia coli JM109 strain.The recombinant clone was screened by X-gal plate and plasmid map,and identified by restricted enzyme mapping analysis method.Result 5.0 kb PCR products was obtained and showed positive result identified by P1 gene specific primer.Endonuclease map of recombinant plasmid showed two band one was pUC19 DNA band and another was 1 kb insert fragment.Conclusion We have obtained P1 protein structure gene and the clone strain containing P1 protein C end gene.