CAJ | 학술논문

目的旨在克隆表达纯化GPS2蛋白,并制备抗血清用于研究GPS2的功能.方法用RTPCR得到GPS2基因,并克隆到pET-28a原核表达载体,用Ni2+-NTA树脂纯化GPS2后,免疫兔得到抗血清.结果成功构建了pET-28a-GPS2原核表达质粒,并实现重组蛋白在大肠杆菌中的高效表达,Ni2+-NTA树脂亲和层析纯化了GPS2蛋白,经过透析复性后得到质量浓度约为300μg/ml的GPS2纯化蛋白；制备了GPS2蛋白的特异性多克隆抗血清,ELISA测定抗血清效价远远高于1:12 800,Western Blot表明该抗血清能够特异性识别结合GPS2蛋白.结论 GPS2在E.coli高效表达,其纯化产物可用于制备抗血清,用于GPS2的功能分析.
목적 지재극륭표체순화GPS2단백,병제비항혈청용우연구GPS2적공능.방법 용RTPCR득도GPS2기인,병극륭도pET-28a원핵표체재체,용Ni2+-NTA수지순화GPS2후,면역토득도항혈청.결과성공구건료pET-28a-GPS2원핵표체질립,병실현중조단백재대장간균중적고효표체,Ni2+-NTA수지친화층석순화료GPS2단백,경과투석복성후득도질량농도약위300μg/ml적GPS2순화단백；제비료GPS2단백적특이성다극륭항혈청,ELISA측정항혈청효개원원고우1:12 800,Western Blot표명해항혈청능구특이성식별결합GPS2단백.결론 GPS2재E.coli고효표체,기순화산물가용우제비항혈청,용우GPS2적공능분석.
Objective The aim was to construct the recombinant plasmid of pET-28a-G-protein pathway suppressor 2 (GPS2) GPS2,express GPS2 protein in E.coli,and obtain specific polyclonal antiserum of GPS2.Methods GPS2 gene was obtained and the amplified fragment was then cloned into E.coli expression vector pET-28a to construct recombinant plasmid.The recombinant plasmid was transformed into E.coli expression strain BL21(DE3).IPTG induces the expression protein GPS2 protein,and the induction conditions were optimized.The induced product was purified by Ni2+ affinity chromatography,and the purified product was dialyzed with buffer for refolding.The purified protein can be used as antigen,injected to immunize male New Zealand white rabbit to get polyclonal antiserum.The titer and specificity of the rabbit antiserum were detected by ELISA and Western Blotting.Results The E.coli expression vector pET-28a-GPS2 was constructed successfully and the recombinant protein was efficiently expressed and purified.The purified protein was used to immunize male New Zealand white rabbit to get polyclonal antiserum and the ELISA and Western Blot results showed that the high titer of specific polyclonal antiserum.Conclusion GSP2 could be highly expressed in E.coli.Antiserum of GPS2 protein can be obtained by the purified recombinant to analyze its function.