抗乙型肝炎病毒表面抗原的IgG全抗体表达载体的构建

目的构建抗乙型肝炎病毒表面抗原(HBsAg)的IgG全抗体杆状病毒表达载体。方法用PCR方法扩增抗HBsAg抗体Fab片段的轻链(L)及重链Fd段(VH+CH1)基因片段,将杆状病毒载体pAC-k-Fc与L基因连接,重组为过渡表达载体pAC-k-L-Fc,再与Fd基因连接,构建重组表达载体pAC-HBs-Fc,并进行酶切鉴定及DNA测序分析。确定正确后,转染昆虫细胞sf9,用免疫荧光检测IgG的表达。结果PCR扩增的片段约650bp,与预期值一致。载体pAC-k-L-Fc和pAC-HBs-Fc的酶切片段和DNA序列与预期结果一致。转染sf9细胞呈阳性荧光反应,未转染细胞呈阴性荧光反应。结论已成功构建表达载体pAC-HBs-Fc,为表达抗人HBsAg的IgG全抗体奠定了基础。

Construction of Expression Vector for Complete IgG Antibody against HBsAg

Objective To construct a baculovirus expression vector for complete IgG antibody against HBsAg.Methods Amplify L and Fd(VH CH1) genes from plasmid L3rd6 containing the gene encoding Fab fragment of antibody against HBsAg by PCR.Insert the amplified L gene into baculovirus expression vector pAC-k-Fc to construct transient expression vector pAC-k-L-Fc,in which Fd gene was then inserted.After identification by restriction analysis and DNA sequencing,the constructed recombinant plasmid pAC-HBs-Fc was transfected to sf9 cells,and the expressed product was identified by IFA.Results The length of amplified fragments were about 650 bp,which were identical to those expected.Both the lengths and DNA sequences of gene fragments extracted from recombinant plasmids pAC-k-L-Fc and pAC-HBs-Fc were also consistent with those expected.The sf9 cells transfected with pAC-HBs-Fc was positive in IFA,while those untransfected were negative.Conclusion The baculovirus expression vector pAC-HBs-Fc for complete IgG antibody against HBsAg was successfully constructed.