摘要
目的构建携带V60、G87和L97突变位点的HBV全基因组真核表达载体,转染慢性HBV感染者永生化B淋巴母细胞系(LCLs)。方法用定点突变技术将HBVp3.8Ⅱ质粒C基因区3个氨基酸V60、G87、L97进行突变(p3.8Ⅱ-V60,G87,L97),转化E.coli.XL1-Blue感受态细胞扩增筛选,用限制性内切酶SacⅠ和KpnⅠ分别将野生型和突变型p3.8Ⅱ质粒进行双酶切,插入EBV-plpp真核细胞表达载体,转染LCLs,潮霉素稳定筛选后,鉴定目的基因在转染细胞能够稳定表达。结果与结论DNA序列分析表明,野型HBVDNA核心区第60、87、97位氨基酸发生了预期的突变,WesternBlotting和微粒子免疫荧光法证明转染的LCLs能稳定表达HBV抗原,证实成功构建了预期细胞模型。
Objective To provide an cell model of immortalized lymphoblstoid B-cell lines for studying the biological characteristics of full-length hepatitis B virus （HBV） genome carrying the hot-spot mutations V60, G87, and L97. Methods V60, G87, and L97 mutation points were introduced into HBV p3.8 Ⅱ plasmid containing 1.2 copy ofHBV genome by means of site-directed mutagenesis. The HBV genome was amplified by PCR from p3.8 Ⅱ and p3.8 Ⅱ - V60, G87, L97 plasmid, and the PCR product was inserted into EBO-plpp eukaryotic expression vector. The recombinant vectors and the EBO-plpp vector were transfected into immortalized human lymphoblasts with lipofectamine 2000 and selected with hygromycin. Steady expression of the target genes was determined by RT-PCR, Western blotting and microparticle enzyme immunoassay. Results DNA sequence analysis indicated that the desired mutation was introduced into wild-type HBV DNA. HBsAg, HBeAg and HBcAg could be detected in EBO-HBV- transfected cell lysate or culture supematant. Conclusion Transfectants that stably express HBV mutant antigen may provide a cell model to study the biological characteristics of HBV carrying hot-spot mutation in vitro.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2006年第6期725-729,共5页
Journal of Southern Medical University
基金
全军医学科学技术研究"十五"计划重点课题(01Z046)
