人乳头瘤病毒16型E_7基因的体外扩增及其克隆和序列分析

PCR Ampligiying,Cloning and Sequencing of E7 Gene of Human Papillomavirus Tape 16

人乳头瘤病毒１６型Ｅ７基因是转化基因。作者设计了一对ＰＣＲ引物，在引物的５′端分别引入ＥｃｏＲＩ和ＨｉｎｄⅢ酶切序列，以ＨＰＶ１６ＤＮＡ为模板成功地扩增出Ｅ７基因。通过ＥｃｏＲＩ和ＨｉｎｄⅢ双酶切位点将Ｅ７基因定向克隆入ｐＵＣ１８载体，经过ＰＣＲ、酶切分析和Ｓｏｕｔｈｅｒｎ印迹杂交鉴定得到Ｅ７重组克隆ｐＨＳＥ７、ＤＮＡ序列分析表明所克隆的Ｅ７基因与已发表的ＨＰＶ１６Ｅ７基因序列完全一致且读框正确。

he primers were designed to amplify E7 gene of HPV 16 by polymerase chain reaction(PCR),according to the sequence of E7 gene of HPV 16.The primers 5′terminal incorporate,re-striction endonuclease sites of EcoRI and Hind Ⅲ,The amplified E7 gene fragment was insertedinto pUC18 vector at EcoRI/HindⅢcloning site,Recombinant DNA formed was transformatedinto E.coli JM 109.Restriction enzyme cleavage and Southern blot analysis revealed that the re-striction endonuclease sites were true,The result of DNA sequencing showed that inserted DNAsequence is well in correspondence with that expected theoretically.Recombinant clone of HPV 16E7 gene obtained was named pHSE7.