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目的:用基因工程手段获得有活性的抗独特型抗体I50,并在体外鉴定其活性。方法:以fuse5-I50为模板,用PCR方法扩增出抗独特型抗体I50基因,并将其插入到pET25b(+)中构建原核表达载体pET25b-I50。含有重组质粒pET25b-I50的菌株E.coli BL21(DE3)经异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导能够得到有效表达。采用Western印迹鉴定I50蛋白的表达,采用透析复性方法恢复I50蛋白的活性,并用Dot-ELISA法,淋巴细胞增殖实验鉴定其活性。结果:成功的构建了原核表达载体pET25b-I50,并经IPTG诱导获得了I50蛋白,该蛋白以包涵体形式高效表达,经纯化后纯度达90%以上。Western印迹鉴定表达的蛋白相对分子质量约15000,与预期相符。Dot-ELISA法鉴定复性后的蛋白已恢复活性,并能够刺激淋巴细胞的增殖,且呈剂量依赖关系。结论:成功获得了有活性的I50抗独特型抗体,为其应用于鼻咽癌的治疗鉴定了基础。
OBJECTIVE: To obtain active anti-idiotypic antibody I50 by genetic engineering and identify its activity in vitro. Methods: The fuseI50-I50 was used as a template to amplify the anti-idiotypic antibody I50 gene by PCR and inserted into pET25b (+) to construct prokaryotic expression vector pET25b-I50. The E. coli BL21 (DE3) strain containing the recombinant plasmid pET25b-I50 was efficiently expressed by induction with isopropyl-β-D-thiogalactopyranoside (IPTG). The expression of I50 protein was identified by Western blotting. The activity of I50 protein was restored by dialysis refolding method. The activity of I50 protein was identified by Dot-ELISA and lymphocyte proliferation assay. Results: The prokaryotic expression vector pET25b-I50 was successfully constructed and the I50 protein was induced by IPTG. The protein was highly expressed in inclusion bodies and the purity was over 90% after purification. Western blot identification of the expressed protein relative molecular mass of about 15000, in line with expectations. Dot-ELISA showed that the refolded protein had recovered activity and stimulated lymphocyte proliferation in a dose-dependent manner. CONCLUSION: The active I50 anti-idiotypic antibody was successfully obtained and its application in the treatment of nasopharyngeal carcinoma was identified.