HBV核心蛋白和B7.1分子嵌合质粒的构建及体外表达的初步研究

Construction of chimeric plasmid pcDNA3/HBc/B7.1 and its expression in vitro

目的 构建HBV核心蛋白和B7.1分子真核表达嵌合质粒 ,并检测其在体外的表达。方法 利用亚克隆技术 ,将HBV核心基因片段和B7.1基因片段构建至真核表达质粒 pcDNA3中 ,测定序列后 ,用脂质体包裹转染细胞 ,采用间接免疫荧光法检测两种蛋白的表达。结果 核酸序列测定证实实验所构建的质粒正确 ,嵌合质粒中所含的HBcAg和B7.1分子的核苷酸序列与HBVadr亚型标准株核心区及鼠B7.1分子的同源性分别为 98.18%和 99.89%。该嵌合质粒在体外转染细胞内可表达HBcAg和B7.1分子。 结论 实验所构建的HBcAg/B7.1嵌合质粒能在体外同时表达HBcAg和B7.1分子。

Objective To construct the chimeric plasmid pcDNA3/HBc/B7.1 and detect the expression of the plasmid in vitro. Methods Using subcloning technique, the cDNA fragments of HBV core gene and mouse co stimulatory molecule B7.1 gene were constructed into the eukaryotic expression plasmid vector pcDNA3. The inserted target genes in the chimeric plasmid were verified by nucleotide sequencing. BHK 21 cell line was transfected with this chimeric plasmid using Lipofecarnin reagent. The expression of HBcAg and B7.1 molecules were detected by indirect immunofluorescence technique.Results The chimeric plasmid pcDNA3/HBc/B7.1 was obtained by subcloning technique. The nucleotide sequences of HBV core gene and co stimulatory molecule B7.1 gene in this chimeric plasmid had high homology with HBV adr standard strain (98.18%) and mouse B7.1(99.89%) respectively. After transfection with this chimeric plasmid, the HBcAg and B7.1 molecules were expressed in BHK 21 cells.Conclusion The constructed chimeric plasmid pcDNA3/HBc/B7.1 can express HBcAg and B7.1 molecules in vitro.