利用反转录-多聚酶链式反应(RT-PCR)的方法, 克隆黄粉甲虫(Tenebrio molitor)抗冻蛋白基因cDNA片段并进行序列分析和原核表达。同源性分析表明, 获得9条新cDNA片段, 与黄粉甲虫抗冻蛋白基因家族的其他基因序列具有较高的同源性。重组质粒pGEX-4T-1-tmafp-XJ430, 转化E.coli BL21进行原核表达, SDS-PAGE分析结果表明, 抗冻蛋白基因以可溶性融合蛋白表达, 相对分子量为38 kDa。构建真核表达载体pCDNA3-tmafp-XJ430, 免疫小鼠, 获得的抗血清滴度为1:2 000。Western blotting 结果为单一的条带, 证明该抗血清具有针对抗冻蛋白TmAFP-XJ430抗原的专一性。

The partial cDNA sequence coding for the antifreeze proteins in the Tenebrio molitor was obtained by RT-PCR. Sequence analysis revealed nine putative cDNAs with a high degree of homology to Tenebrio molitor antifreeze proteins. The recombinant pGEX-4T-1-tmafp-XJ430 was introduced into E. coli BL21 to induce a GST fusion protein by IPTG. SDS-PAGE of the fusion protein demonstrated that the antifreeze protein migrated at a size of 38 kDa. The immunization was performed by intra-muscular injection of pCDNA3- tmafp-XJ430, and then antiserum was detected by ELISA.The titer of the antibody was 1:2 000. Western blotting analysis showed the antiserum was specific against the antifreeze protein. This finding could lead to further investigation of the properties and function of antifreeze proteins.