卵巢癌抗独特型单链抗体6B11ScFv与鼠HSP70融合蛋白的构建、表达及鉴定  

Construction,expression,and identification of ovarian anti-idiotypic single chain antibody 6B11ScFv and mouse HSP70 fusion protein

作　　者：张果[1] 昌晓红[1] 石菁菁[2] 成夜霞[1] 李巍[3] 叶雪[1] 程洪艳[1] 付天云[1] 魏丽惠[1] 崔恒[1] 

机构地区：[1]北京大学人民医院妇科肿瘤中心,100044 [2]北京市卫生局科教处,100053 [3]首都医科大学附属北京妇产医院,100026

出　　处：《中国医药生物技术》2008年第5期336-342,共7页Chinese Medicinal Biotechnology

基　　金：国家自然科学基金(30772320)

摘　　要：目的构建卵巢癌抗独特型单链抗体6B11ScFv与小鼠热休克蛋白70(mHSP70)融合蛋白6B11ScFv/mHSP70,并初步鉴定其免疫活性。方法根据Genebank上已发表的mHSP70基因序列(NM_010478)合成引物行PCR扩增,以扩增所得mHSP70基因插入pET30a(+)-6B11ScFv质粒后转化入E.coliDH5α,获得pET30a(+)-6B11ScFv/mHSP70重组质粒。将鉴定正确的pET30a(+)-6B11ScFv/mHSP70重组质粒转化入E.coliBL21(DE3),获得E.coliBL21(DE3)[pET30a(+)-6B11ScFv/mHSP70]重组菌株。以异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达并行SDS-PAGE分析,对表达产物进行复性和Ni2+-NTA柱亲和层析纯化后,采用蛋白质印迹法和ELISA方法进行鉴定和免疫活性检测。结果6B11ScFv/mHSP70融合基因测序结果基本与理论序列相符。SDS-PAGE分析显示,重组菌株表达产物相对分子质量与预期相符。复性、亲和层析纯化后的蛋白复性率达20.9%,蛋白纯度达95%以上。蛋白质印迹分析证实6B11ScFv/mHSP70融合蛋白相对分子质量为104000,ELISA检测表明其具有6B11ScFv和mHSP70的免疫活性。结论构建表达的6B11ScFv/mHSP70融合蛋白具有良好的免疫活性,为进一步研究其体内外抗卵巢癌活性奠定了基础。Objective To construct the fusion protein of ovarian anti-idiotypic single chain antibody 6B11ScFv and mouse HSP70 （6B 11ScFv/mHSP70）, and to detect its immunoactivity. Methods The recombinant plasmid pET30a （＋）-6B11ScFv/mHSP70 was constructed by subcloning murine full-length mHSP70 cDNA into the PET30a （＋）-6B11ScFv plasmid, using PCR to introduce SpeI/EcoRI restriction site at each terminal of mHSP70 gene. Then the recombinant plasmid was transformed into E.coli DH5α, and the positive clones were screened and confirmed by restriction digestion and DNA sequencing. The correct plasmid was then transformed into E.coli BL21（DE3） to obtain a reconstructed strain, E.coli BL21 （DE3） [pET30a （＋）-6B 11ScFv/mHSP70]. After being induced by IPTG, the expression of the recombinant was analyzed by SDS-PAGE. Afterwards, we isolated, washed, and solubilized the inclusion body, and then performed dilution refolding and affinity chromatography to obtain soluble and pure target protein. The immunoactivity of this fusion protein was detected and determined by Western blotting and ELISA. Results The sequence of fusion gene 6B11ScFv/mHSP70 was identified. SDS-PAGE showed a relative molecular weight of the recombinant protein similar to the expected. The refolding rate of the protein was 20.9% and its purity reached above 95%. As shown by Western blot, the relative molecular weight of the 6B11ScFv/mHSP70 was 104 000, and ELISA revealed that the recombinant strain had both the immunoactivities of 6B11ScFv and mHSP70. Conclusions The recombinant fusion protein 6B11ScFv/mHSP70 has good immunoactivity. Further researches on its in vivo and in intro activity against ovarian cancer is necessary.

关 键 词：抗体 抗独特型 HSP70热休克蛋白质类 重组融合蛋白质类 卵巢肿瘤 

分 类 号：R392[医药卫生—免疫学]