定点诱变HBcAg质粒在人永生化B淋巴母细胞系中的稳定表达

Stable expression of site-directed mutated HBcAg in human immortalized Blymphoblastoid cell line

目的:构建携带V60,G87,L97突变位点的HBV全基因组真核细胞表达载体,转染慢性HBV感染者永生化B淋巴母细胞系(LCL),建立相同HLA背景下的慢性HBV感染CTL应答的刺激细胞和靶细胞.方法:用定点突变技术在HBVp3.8Ⅱ质粒C基因区3个氨基酸V60,G87,L97位置进行突变(p3.8Ⅱ-V60,G87,L97),转化E.coliXL1-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EBO-plpp真核细胞表达载体,转染LCL,潮霉素稳定筛选后,鉴定目的基因在转染细胞是能够稳定表达.结果: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,WesternBlot和微粒子免疫荧光法证明,转染的LCL能稳定表达HBV抗原.结论:定点诱变HBcAg质粒在人永生化B淋巴母细胞系中可稳定表达.

AIM： To get the stimulator and target cells of CTL response in chronic hepatitis B patients so as to provide an experimental cellular model for studying the biological characteristics of full-length HBV genome carrying the HBcAg hot-spots mutation V60, G87, L97. METHODS： V60, G87, L97 point mutation was introduced from HBV p3.8 Ⅱ plasmid which contained 1.2 copy HBV genome using site-directed mutagenesis. The HBV genome was amplified by PCR from p3.8 Ⅱ and p3. 8 Ⅱ- V60, G87, L97 plasmid, then the PCR product was inserted into EBOplpp eukaryotic expression vector. The recombinant vectors and the EBO-plpp vector were transfected into lymphoblastoid cells with lipofectamine 2000 and selected with hygromycin. The steady expression of target genes was determined by RT-PCR, Western Blot and microparticle enzyme immunoassay. RESULTS： The DNA sequence analysis indicated that the desired mutation was introduced from wild HBV DNA. The HBsAg, HBeAg and HBcAg can be detected in EBO-HBV transfected cell lysate or culture supernatant. CONCLUSION： The site-directed mutated HBcAg plasmid can be expressed stably in human immortalized B-ympho- blastoid cell line.