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。
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 .
出处
《首都医科大学学报》
CAS
1999年第4期272-274,共3页
Journal of Capital Medical University
