人源抗HBsAg dsFv抗体靶向干扰素重组质粒的构建与表达  

Construction and expression of human anti-HBs-IFN fusion gene

作　　者：江乐[1] 阎瑾琦[2] 郭炳冉[1] 任杰[3] 于继云[2] 

机构地区：[1]曲阜师范大学生命科学学院,山东曲阜273165 [2]军事医学科学院基础医学研究所,北京100850 [3]解放军总医院泌尿外科,北京100853

出　　处：《解放军医学杂志》2010年第4期391-394,共4页Medical Journal of Chinese People's Liberation Army

基　　金：国家科技重大专项基金(2008ZX10002-003);国家自然科学基金(30772002)

摘　　要：目的构建抗体靶向干扰素的重组表达质粒pEE14.1-dsFvαpr+,并在真核细胞中验证重组质粒的表达情况。方法通过重叠延伸PCR将dsFv抗体重链基因与带正电荷的DNA负载区基因融合,并在抗体轻链α干扰素融合基因3′端连入6×His标签。先在过渡载体pCI-GPI中实现轻、重链复合基因的连接,然后连入pEE14.1中,最终构建重组表达质粒pEE14.1-dsFvαpr+。将重组质粒LipofectamineTM2000瞬时转染到CHO-K1细胞,采用RT-PCR、ELISA和Western blotting方法验证目的基因的表达。结果重组质粒经酶切鉴定和测序验证与设计完全一致。RT-PCR结果显示只有重组质粒转染后的细胞能够扩增出1700bp的目的条带,ELISA检测瞬时转染细胞上清α干扰素的浓度约为1.1ng/ml,Western blotting检测显示重组质粒在转染细胞上清中获得表达。结论实现了抗体靶向干扰素在单一质粒中的高效表达,通过对表达蛋白的电性改造有利于后期治疗慢性乙肝的抗体靶向纳米粒药物的研制。Objective To construct eukaryotic expression plasmid pEE14.1-dsFvαpr＋,and detect the expression of the recombined gene in eukaryotic CHO-K1 cells.Methods The cationic DNA fragment was cloned into the 3＇ of VH gene by overlapping extension PCR,and the 6×His tab was inserted to the 3＇ of VL and human IFN-α gene by the same way.The above mentioned recombinant VH and VL genes were inserted into a pCI-GPI vector first,and then cloned into the pEE14.1 vector to construct the recombinant plasmid pEE14.1-dsFvαpr＋.Finally,the recombinant plasmid was transfected into the CHO-K1 cells by LipofectamineTM 2000,and the expression was detected by RT-PCR,ELISA and Western blotting.Results The enzyme digestion and sequencing analysis showed that the recombinant plasmid was successfully constructed.RT-PCR showed that only the cells with transfected plasmid can generate the specific 1700bp fragment.ELISA analysis showed that the production of IFN-αexpressed in the supernatant of transfected cells was about 1.1ng/ml.Also,Western blotting could reveal the characteristic band of HBsAg dsFvαpr＋ protein.Conclusion The antibody targeting to human IFN-αgenes has been successfully expressed in a single open reading frame.Changing the electricity of the antibody may provide the necessary condition for the study of the a new type of anti-HBV drug in nanoscale in the future.

关 键 词：干扰素Α 肝炎表面抗原 乙型 二硫键稳定性Fv抗体 质粒 基因表达 

分 类 号：R512.620.3[医药卫生—内科学]