Adr亚型HBV中蛋白基因的T载体克隆及序列分析

首都医科大学学报 ›› 1999, Vol. 20 ›› Issue (4): 272-274.

Adr亚型HBV中蛋白基因的T载体克隆及序列分析

赵秀英, 黄德庄, 阎惠平, 贺丽香, 罗朝霞

北京佑安医院肝炎研究所

收稿日期:1998-12-01 修回日期:1900-01-01 出版日期:1999-10-15 发布日期:1999-10-15

The T-vector Cloning of HBV Middle Protein Gene of Adr Subtype and Nucleic Acid Sequence Analysis

Zhao Xiuying, Huang Dezhuang, Yan Huiping, He Lixiang, Luo Zhaoxia

Institute of Viral Hepatitis, Beijing You′an Hospital

Received:1998-12-01 Revised:1900-01-01 Online:1999-10-15 Published:1999-10-15

摘要/Abstract

摘要： 构建含适当限制酶切位点的adr亚型乙肝病毒(HBV)中蛋白基因克隆,比较分析序列变化,用作HBV疫苗表达及诊断试剂的研究。患者血清经蛋白酶K消化,再以酚氯仿抽提,对提取物进行PCR扩增。回收PCR阳性产物,经琼脂糖酶消化后直接克隆至T载体。转化后提取质粒经PCR及酶切鉴定,再行序列分析。结果：10份血清提取物的PCR有3份获阳性产物,分别经T载体克隆后转化DH10b菌,3株阳性克隆经酶切及序列分析显示同属于adr亚型,与国内已发表序列的同源性大于99.6%.提示该克隆是用作HBsAg蛋白表达或探针标记研究的理想克隆。

关键词: HBsAg, 前S2基因, adr亚型, PCR, T载体

Abstract: Tvector clones of HBV surface antigen of adr subtype which contain preS2 region were constructed and nucleic acid sequence was analyzed.Sera from hepatitis B patients were digested by protease Kand HBVgenomes were extracted by phenol and chloroform.The target gene was am plified from HBVgenome and PCR products were collected by agrose gel electrophoresis. The am plified gene was digested with agrase and ligated with Tvector directly.The ligation products were transformed into E.coli DH10b and the target sequence was analyzed.The results showed that 3 positive PCR products were obtained from 10 HBVgenomes ,and the positive clones belonged to HBVadr subtype ,there was more than 99.6 % homology with the published clone adr-1 and these clones provided a perfect contribution to the expression of vaccine and HBVadr subtype specialty.

Key words: HBsAg, preS2 gene, adr subtype, PCR, T-vector

中图分类号:

R512.6

赵秀英;黄德庄;阎惠平;贺丽香;罗朝霞. Adr亚型HBV中蛋白基因的T载体克隆及序列分析[J]. 首都医科大学学报, 1999, 20(4): 272-274.

Zhao Xiuying;Huang Dezhuang;Yan Huiping;He Lixiang;Luo Zhaoxia. The T-vector Cloning of HBV Middle Protein Gene of Adr Subtype and Nucleic Acid Sequence Analysis[J]. Journal of Capital Medical University, 1999, 20(4): 272-274.

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