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目的表达、纯化ZNF382融合蛋白及制备兔多克隆抗体。方法通过PCR扩增ZNF382编码区,将其克隆到pRSETC载体中,重组载体酶切鉴定,测序,转化大肠杆菌BL21(DE3)并用IPTG进行诱导表达,原核表达产物经鉴定、纯化后,免疫新西兰大白兔。通过ELISA和Wester nblot检测抗血清的效价和特异性。结果成功构建了pRSETC-ZNF382原核表达重组质粒,His6-ZNF382融合蛋白被强烈诱导表达,以包涵体的形式存在,其相对分子质量约为64×103,纯化后免疫产生的多克隆抗体效价约为1∶10000,与ZNF382原核表达蛋白特异性结合。结论获得了大量高效价、高特异性的兔抗人ZNF382多克隆抗体。
Objective To express and purify ZNF382 fusion protein and prepare rabbit polyclonal antibody. Methods The coding region of ZNF382 was amplified by PCR, cloned into pRSETC vector, identified by restriction enzyme digestion, sequenced and transformed into E.coli BL21 (DE3) and induced by IPTG. The prokaryotic expression product was identified and purified, then immunized New Zealand white rabbit. Titers and specificities of antisera were tested by ELISA and Wester nblot. Results The recombinant plasmid pRSETC-ZNF382 was successfully constructed. His6-ZNF382 fusion protein was strongly induced and expressed in the form of inclusion bodies. The relative molecular mass was about 64 × 103. The titer of the purified polyclonal antibody after immunization was about 1: 10000, and prokaryotic expression of ZNF382 specific binding. Conclusion A large number of high titer, high specificity rabbit anti-human ZNF382 polyclonal antibody was obtained.