辣根过氧化物酶基因与血管内皮生长因子基因的连接及克隆

Linkage and Cloning of Genes Encoding Horseradish Peroxidase and Vascular Endothelial Growth Factor

目的连接辣根过氧化物酶(HRP)基因与血管内皮生长因子(VEGF)基因并进行克隆。方法用RT-PCR法从人肺肿瘤组织获得VEGF165基因,从含有HRPC3基因的质粒pMDC3EX中扩增得到HRPC3基因,将两种目的基因通过连接肽相应DNA序列进行连接,再以带有特定酶切位点的引物进行PCR,然后将目的基因插入到毕赤酵母表达载体pPIC9K中,构建重组质粒,在大肠杆菌中克隆,并进行序列分析。再将重组质粒转入毕赤酵母中,筛选阳性克隆,提取基因组DNA,采用PCR法鉴定。结果用PCR扩增的VEGF165基因和HRPC3基因片段与预期相符,将二者连接后,克隆到pPIC9K质粒中,经DNA测序证明重组基因与公开发表的相同DNA同源性为97.3%。经PCR鉴定,目的基因已转入毕赤酵母中。结论已成功将辣根过氧化物酶基因与血管内皮生长因子基因连接并克隆。

Objective To hnk and clone the genes encoding horseradish peroxidase and vascular endothelial growth factor for expression of fusion protein. Methods Amplify VEGF165 gene from human hmg tumor tissue by RT-PCR, and HRPC3 gene from recombinant plasmid pMDC3EX by PCR. Link the two amplified genes by a DNA sequence corresponding to linking peptide for amplification by PCR using the primers with specific restriction sites. Digest the PCR product with restriction endonuclease, and insert the obtained target gene fragment into Pichia pastoris expression vector pPIC9K.Transform the recombinant plasmid to E. coli And screen positive clones for sequencing,then transform to P/ch/a pastor/s, screen positive clones, extract genomic DNA an identify by PCR. Results The amplified VEGF165 and HRPC3 genes were consistent with those expected. The DNA sequence of positive clones of transformed E. coli showed a homology of 97.3 % to that reported.PCR proved that the linked target gene was successfully transformed to Pichia pastoris an cloned. Conclusion The genes encoding horseradish peroxidase and vascular endotyelia growth factor were successfully linked and cloned.