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

 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.coli DH5α,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.coli BL21（DE3）,获得 E.coli BL21（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 104 000,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。

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

Objective To construct the fusion protein of ovarian anti-idiotypic single chain antibody 6B11ScFv and mouse HSP70 (6B11ScFv/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 (+)-6B11ScFv/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.