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目的:构建hWAPL原核表达载体,诱导hWAPL蛋白表达并制备其多克隆抗体。方法:RT-PCR技术扩增hWAPL cDNA片段,连接到pMD18-T载体,测序正确后亚克隆入原核表达载体pET28a并转化BL21菌株,用SDS-PAGE电泳鉴定重组菌的诱导表达情况,制备hWAPL蛋白并免疫BALB/c小鼠,ELISA法测定其抗体效价,Western blot鉴定表达hWAPL蛋白的免疫原性。结果:①经PCR、双酶切鉴定和测序,证实hWAPL正确插入pMD18-T载体,序列正确。②经IPTG诱导的pET28a-hWAPL重组菌表达出相对分子质量(Mr)约为25000的蛋白,与预期蛋白Mr相符。③免疫小鼠后测得的平均抗体效价为1∶3200。④Western blot结果显示Mr约25000处有反应蛋白条带。结论:pET28a载体能够表达hWAPL蛋白,表达蛋白具有良好的免疫原性,其免疫小鼠后获得的多克隆抗体具有高效价和特异性。
OBJECTIVE: To construct prokaryotic expression vector hWAPL, induce hWAPL protein expression and prepare its polyclonal antibody. Methods: The hWAPL cDNA fragment was amplified by RT-PCR and ligated into pMD18-T vector. After sequencing, the recombinant plasmid was subcloned into prokaryotic expression vector pET28a and transformed into BL21 strain. The recombinant plasmid was identified by SDS-PAGE electrophoresis. The hWAPL protein The BALB / c mice were immunized and the antibody titers were determined by ELISA. The immunogenicity of hWAPL protein was identified by Western blot. Results: ①The PCR products were identified by restriction enzyme digestion and sequencing. The correct insertions of hWAPL into pMD18-T vector were confirmed. ② The recombinant protein pET28a-hWAPL induced by IPTG expressed a protein with a relative molecular mass (Mr) of about 25000, consistent with the expected protein Mr. ③ The average antibody titer measured after immunization was 1:3200. Western blot results showed Mr about 25000 reactive protein bands. CONCLUSION: The pET28a vector can express hWAPL protein and the expressed protein has good immunogenicity. The polyclonal antibody obtained from the immunized mouse has high titer and specificity.