昆虫抗冻蛋白DAFP的原核表达、纯化和抗血清制备

目的:制备昆虫抗冻蛋白DAFP特异性的鼠抗血清。方法:根据GenBank已发表的Dendroides canadensis抗冻蛋白基因序列(gi:2737939),人工合成加拿大拟步甲虫(Dendroides canadensis)的抗冻蛋白基因(dafp),并克隆到原核表达载体pGEX-4T-1和pMAL-p2x中,在大肠杆菌中进行表达。表达的融合蛋白经Glutathione Sepharose4B纯化后,免疫小鼠制备抗DAFP的抗血清,抗体的效价及特异性分别采用ELISA和Western blot进行检测。结果:SDS-PAGE检测表明,人工合成的dafp基因在大肠杆菌中可表达相对分子质量(Mr)为38000的可溶性融合蛋白。以该融合蛋白免疫小鼠所制备的抗体,其ELISA滴度为1∶5000,Western blot证实BL21/pGEX-4T-1-dafp菌和TB1/pMAL-p2x-dafp菌的表达产物可特异性地与该抗体结合。结论:在大肠杆菌中成功地表达了DAFP的融合蛋白并制备出抗融合蛋白的鼠抗血清,为进一步研究DAFP的特性奠定了基础。

Prokaryotic expression and purification of antifreeze protein DAFP and preparation of its antiserum

AIM: To prepare the Dendroides canadensis antifreeze proteins (DAFP) specific mouse antiserum. METHODS: According to the published antifreeze protein gene (gi: 2737939) from Dendroides canadensis in GenBank, one isoform of the gene was chemically synthesized and cloned into the expression vector pGEX-4T-1 and pMAL-p2x respectively and expressed in E.coli. The expressed fusion protein was purified through Glutathione Sepharose 4B column, and then used to immunize the mice for preparing the specific the antibody. The titer and specificity of the antibody were analyzed by ELISA and Western blot, respectively. RESULTS: SDS-PAGE analysis showed that the DAFP was expressed in E.coli. The relative molecular masses of expressed fusion protein was 38,000. The titer of antibody was 1:5,000. Western blot analysis showed the expressed protein of BL21/pGEX-4T-1-dafp and TB1/pMAL-p2x-dafp could react specifically with DAFP antibody. CONCLUSION: The DAFP was expressed in E.coli and its specific antibody was prepared successfully.