摘要
采用加端聚合酶链反应技术，从湖北地区一宫颈癌患者癌组织ＤＮＡ中分离出人乳头瘤病毒１６型（ＨＰＶ１６）Ｅ７基因，并在ｐＵＣ１８载体中克隆。经限制性核酸内切酶分析和ＤＮＡ序列分析，确认了含ＨＰＶ１６Ｅ７重组克隆质粒，命名ｐＨＰＶ１６Ｅ７─ＨＢ。ＤＮＡ序列分析表明，ＨＰＶ１６Ｅ７─ＨＢ基因全长２９４ｂｐ（与报道的标准株基因长度相同），但其核苷酸顺序中有两处发生了Ｃ→Ｔ突变，即第４３位密码子ＣＡＡ变为ＴＡＡ，第７６位ＣＧＴ变为ＴＧＴ；前者使谷氨酰胺密码子变为终止密码，即无义奕变（ｎｏｎｓｅｎｓｅｍｕｔａｔｉｏｎ）。这种突变发生在２９４个碱基的ＤＮＡ扩增产物之中，不像是ＰＣＲ本身的错配，而很可能是湖北株与标准株之间的结构差异。
he technique of add-on PCR was used for isolating HPV16 E7 gene from cervical carcinomabiopsy DNA in Hubei province.The E7 gene was inserted into vector pUC18.By restriction en-zyme cleavage and nucleotide sequence detection,a new recombinant plasmid was constructed andnamed it pHPV16 E7-HB. The result of nucleotide sequencing showed that the overall length ofHPV16 E7-HB gene is 294 bp,it is the same as the standard strain.But there were two C→Tmutation in HPV16 E7-HB.Anonsense mutation was identifed at codon 43.a Gln codon(CAA)was converted into ochre termination codon (TAA).The mutations occurring in 294 bp ofDNA PCR production seemed to be a structure difference between HB strain and standard strainrather than a mismatch of PCR itself.
出处
《中国病毒学》
CSCD
1996年第3期220-224,共5页
Virologica Sinica
基金
湖北省科委"八五"科技攻关项目
