【摘要】 目的:在大肠杆菌中表达人金属硫蛋白(MT)-2a与谷胱甘肽S-转移酶(GST)的融合蛋白,并制备兔抗MT-2a抗血清。方法:人MT表达菌株BL21/GST-MT-2a经IPTG诱导、超声裂解及GlutathioneSepharose4B亲和层析纯化后,以可溶性融合蛋白GST-MT-2a免疫新西兰大白兔,制备抗血清。用双向凝胶免疫扩散和ELISA检测抗血清的效价,用Westernblot检测抗血清的特异性。结果:经诱导表达及亲和层析纯化,每L菌液可获得可溶性融合蛋白GST-MT-2a38mg。以此融合蛋白免疫新西兰大白兔制备的抗血清,双向免疫扩散效价>1∶256,ELISA效价>1×10-7。Westernblot检测证明抗血清的特异性良好。结论:人MT-2a在大肠杆菌中得到高效表达,以纯化的GST-MT-2a可溶性融合蛋白免疫家兔得到高效价、高特异性的抗血清,为进一步研究MT-2a蛋白的生物学功能提供了重要的制剂。更多还原

【Abstract】 AIM: To express and purify human MT-2a in prokaryotic cells and to prepare the MT-2a-specific rabbit antiserum. METHODS: GST-MT-2a fusion protein was expressed after IPTG induction and further purified with Glutathione Sepharose 4B. Then the purified GST-MT-2a fusion protein was used to immunize New Zealand rabbits. The titer and specificity of rabbit antiserum were evaluated by double immunodiffusion, ELISA and Western blot. RESU-LTS: GST-MT-2a fusion protein was highly expressed . The final yield of the pure GST-MT-2a was about 38 mg per liter of bacterial culture. Its antiserum with high specificity and potency was also obtained. CONCLUSION: The successful expression of GST-MT-2a fusion protein in E.coli and the preparation of MT-2a specific rabbit antiserum will be valuable for the study on the function of human MT-2a.更多还原