Tenebrio molitor抗冻蛋白基因家族cDNA片段的克隆、序列分析及原核表达  

Cloning,Sequencing and Prokaryotic Expression of cDNAs for the Antifreeze Protein Family from the Beetle Tenebrio molitor

作　　者：刘忠渊[1] 王芸[1] 吕国栋[1] 王贤磊[1] 张富春[1] 马纪[1] 

机构地区：[1]新疆大学生命科学与技术学院分子生物学重点实验室,新疆生物资源基因工程重点实验室,乌鲁木齐830046

出　　处：《遗传》2006年第12期1532-1540,共9页Hereditas（Beijing)

基　　金：科技部重大基础研究前期研究专项(编号:2003CCA01000)资助~~

摘　　要：利用反转录-多聚酶链式反应(RT-PCR)的方法,克隆黄粉甲虫(Tenebriomolitor)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.coliBL21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38kDa。构建真核表达载体pCDNA3-tmafp-XJ430,免疫小鼠,获得的抗血清滴度为1:2000。Westernblotting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 mofitor 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. coil 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.

关 键 词：黄粉甲虫 抗冻蛋白 CDNA片段 序列分析 原核表达 

分 类 号：Q78[生物学—分子生物学]