人类8型疱疹病毒K1基因真核表达载体的构建

Construction of Eukaryotic Expression Vectors of Human Herpesvirus 8 K1 Gene

为构建人类8型疱疹病毒(Human herpesvirus 8,HHV-8)K1基因的真核表达载体,采集卡波氏肉瘤(Kaposi’ssarcoma,KS)患者血清,采用柱式病毒DNA抽提试剂盒抽提血清中的病毒DNA,采用套式PCR技术检测HHV-8的特异性片断KS330233以判断是否得到了HHV-8 DNA,采用PCR技术扩增HHV8 K1基因并纯化回收,酶切、连接入真核表达载体pcDNA3.1,转化大肠杆菌DH5α,挑取阳性克隆pcDNA3.1/HHV8-K1扩增后,提取质粒,进行双酶切和DNA测序鉴定。结果显示采集了经典型KS血清3例,提取了血清HHV-8DNA,扩增了特异性片断KS330233,测序结果正确提示我们得到了HHV8 DNA,扩增了HHV8 K1基因,构建了pcDNA3.1/HHV8-K1真核表达载体,经限制性内切酶酶切鉴定及测序分析证实了其序列的正确性。由此可知:成功构建了pcDNA3.1/HHV8-K1真核表达载体,为进一步研究该基因的功能奠定良好的实验基础。

to construct eukaryotic expression vectors of human herpesvirus 8 K1 from 3 Kaposi sarcoma patients, extract HHV-8 DNA. HHV-8 DNA was detected gene. We collected blood serum by neated Polymerase chain reaction （PCR） to amplify the special fragments KS330233. The HHV-8 K1 gene fragments were amplified from the HHV-8 genome by PCR and were inserted into eukaryotic expression vector pcDNA3.1. The positive clone vectors were transformed into E. Coli DH5α. Then we amplified and identified the positive clone by digestion and sequencing. The eukaryotic expression vectors of Human herpesvirus 8 K1 gene were constructed successfully. The construction of the eukaryotic expression vectors of Human herpesvirus 8 K1 gene provide a solid foundation for further experimental studies on the function of the gene.