hβ_2GPⅠ第一功能区基因单体和二串联体的克隆和表达  

Cloning and expression of monomer and dimmer of hβ_2GPⅠ first domain gene

作　　者：李明辉[1] 谭岩[1] 姜艳芳[1] 刘力华[1] 方艳秋[1] 许淑芬[1] 段秀梅[1] 付嘉[1] 

机构地区：[1]吉林大学第一医院中心实验室,吉林长春130021

出　　处：《吉林大学学报（医学版）》2006年第2期203-206,共4页Journal of Jilin University：Medicine Edition

基　　金：吉林省科技厅科研基金资助课题(20030419);国家博士点学科专项基金资助课题(20040183066);吉林省杰出青年基金资助课题(20050113)

摘　　要：目的:构建h2βGPⅠ(h2βGPI)第一功能区基因单体及二串联体的原核表达载体PQE30-β2GP-ⅠDI和PQE30-β2GPⅠ-DI2,并在大肠杆菌M15中进行高效表达。方法:应用PCR技术扩增hβ2GPⅠ第一功能区基因的单体β2GPⅠ-DI,并根据同尾酶特性构建hβ2GPⅠ第一功能区基因二串联体β2GPⅠ-DI2,将获得的单体和二串联体分别与PGEM-T easy载体连接测序,测序正确后分别克隆于原核表达载体PQE30中,构建成重组表达载体PQE30-β2GPⅠ-DI和PQE30-β2GPⅠ-DI2,在大肠杆菌M15中以l mmol.L-1IPTG诱导蛋白表达,经Western blotting鉴定目的蛋白的抗原性。结果:表达了hβ2GPⅠ第一功能区基因的单体及二串联体的蛋白,相对分子质量约为9 000和17 000,其表达量均可达菌体总蛋白的25%,并具有hβ2GPⅠ的抗原性。结论:成功构建了hβ2GPⅠ第一功能区基因的单体及二串联体的原核表达载体,并诱导了蛋白的高效表达。Objective To clone and express the genes of hβ2GPⅠ-DI and hβ2GPⅠ-DI2 in E. coli M15. Methods Target gene ~2GP I-DI was cloned by PCR, the PCR products were connected into dimmer, the monomer and dimmer were inserted into PGEM-T easy vector and sequenced. Then they were inserted into an expression vector POE30 and expressed in E. coli M15. The recombinant proteins were identified by Western blotting. Results The expected protein of hβ2GPⅠ -DI and hβ2GPⅠ -DI2 recombinant protein were synthesized, their relative molecular mass were 9 000 and 17 000. The two proteins could be recognized by hβ2GPⅠ mAb with Western blotting. Conclusion The monomer and dimmer of hβ2GPⅠ first domain gene are constructed successfully.

关 键 词：人β2GPⅠ 遗传载体 基因表达 

分 类 号：Q78[生物学—分子生物学]