宫颈癌相关新癌基因hWAPL的原核表达和免疫原性分析

Prokaryotic expression and immunogenicity of hWAPL,a norel oncogene related to cervical cancer

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。

AIM： To construct the prokaryotic expression plasmid of hWAPL, induce the expression of the protein and prepare polyclonal antibody. METHODS： The hWAPL cDNA was amplified by RT-PCR from HeLa cells derived RNA and then cloned to pMD18-T according to A-T. The DNA fragment was isolated and linked to prokaryotic expression vector pET28a. The recombinant protein was induced by IPTG and identified by SDS-PAGE. Then it was injected into mice to prepare polyclonal antibody. The immunogenicity and specificity of the recombinant protein were identified by ELISA and Western blot. RESULTS： The sequencing, PCR and endonucleases digestion results showed that the hWAPL fragment was correctly inserted into pMD18-T and pET28a vectors. SDS-PAGE showed 25 000 fusion hWAPL protein was expressed in BL21 cells. The titer of polyclonal antibody was 1： 3 200 by indirect ELISA. The 25 000 fusion hWAPL protein was detected by SDS-PAGE and Western blot. CONCLUSION： The hWAPL protein can be expressed by prokaryotic vector pET28a. Furthermore, the expression protein can be used as immunogen to generate antibodies with specificity.