论文部分内容阅读
目的:构建全人源鼻咽癌双价抗独特型抗体原核表达载体,并对其产物作初步鉴定。方法:利用分子克隆技术依次将G22,I50插入到原核表达载体pET25b(+)中,经菌落PCR,双酶切验证并测序。阳性重组子转化入表达菌株E.coliBL21(DE3),经IPTG诱导表达后,表达的重组蛋白经His-tag纯化试剂盒纯化后复性。用Westernblot方法鉴定G22-I50双价蛋白的表达,ELISA方法分析其蛋白活性,并进一步观察其对外周血单个核细胞的刺激增殖情况。结果:构建的原核表达载体pET25b-G22-I50经测序验证,序列正确。双价蛋白G22-I50以包涵体形式高效表达,经纯化后纯度达90%以上。Westernblot鉴定表达的蛋白相对分子质量(Mr)约42000,与预期相符。ELISA鉴定复性后的蛋白已恢复活性,并能在体外刺激外周血单个核细胞的增殖。结论:成功获得了有活性的双价抗独特型抗体,为临床对鼻咽癌进行主动免疫治疗提供了理论基础。
OBJECTIVE: To construct a prokaryotic expression vector for full-length nasopharyngeal carcinoma bivalent anti-idiotypic antibody and to identify its products. METHODS: G22 and I50 were inserted into the prokaryotic expression vector pET25b (+) by molecular cloning in turn. After colony PCR and double enzyme digestion, the sequences were sequenced. The positive recombinant was transformed into E.coli BL21 (DE3). After induced by IPTG, the recombinant protein was purified by His-tag purification kit and renatured. Western blot was used to identify the expression of G22-I50 double-stranded protein. ELISA was used to analyze the protein activity of G22-I50. The proliferation of peripheral blood mononuclear cells was further observed. Results: The prokaryotic expression vector pET25b-G22-I50 was verified by sequencing and the sequence was correct. The bivalent protein G22-I50 is highly expressed in the form of inclusion bodies, and its purity is over 90% after purification. The relative molecular mass (Mr) of the expressed protein by Western blot was about 42,000, which was in line with the expectation. The protein renatured by ELISA has been restored to activity and stimulated the proliferation of peripheral blood mononuclear cells in vitro. Conclusion: The active bivalent anti-idiotypic antibody was successfully obtained, which provided a theoretical basis for the clinical active immunotherapy of nasopharyngeal carcinoma.