摘要
目的 :克隆人DOC 2氨基端PID结构域 (暂命名为nDOC 2 ) ,并在原核细胞中表达。方法 :用RT PCR从正常人卵巢组织中扩增nDOC 2基因 ,并克隆到载体 pUC 19中。经测序证实后 ,用BamHI/EcoRI双酶切 ,亚克隆到原核表达载体 pGEX 4T 1,并转化E .coliDH5α菌株。取工程菌 ,用IPTG诱导表达 ,对表达产物进行SDS PAGE鉴定。结果 :①经RT PCR、测序和酶切鉴定 ,成功地克隆了人nDOC 2基因。②经IPTG诱导的重组质粒pGEX 4T nDOC 2表达出相对分子质量 (Mr)约为 5 0 0 0 0的融合蛋白 ,与预期的结果相符。结论 :成功克隆到人DOC 2氨基端PID结构域 ,并在E .coliDH5α中表达出GST nDOC
AIM: To clone the PID domain of human DOC 2 (nDOC 2) and express it in E.coli DH5α. METHODS: The cDNA fragment encoding the PID domain of nDOC 2 was amplified by RT PCR from normal human ovarian tissue and cloned to pUC19. The DNA fragment from the pUC19 nDOC 2 digested with Bam H I and Eco R I was ligated to the Bam H I/ Eco R I digested prokaryotic expression vector pGEX 4T 1. The expression of fusion protein was induced with IPTG and the expressed product was identified by SDS PAGE. RESULTS: ①The sequencing and endonucleases digestion analysis showed that the fragment of nDOC 2 gene was insert into vectors pUC19 and pGEX 4T 1; ②SDS PAGE showed the nDOC 2 gene had been expressed in E.coli DH5α. CONCLUSION: The PID domain of nDOC 2 was expressed successfully in prokaryote, whic makes preparation for further researching the function of DOC 2 and preparing antibodies to DOC 2 protein.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2003年第2期140-141,144,共3页
Chinese Journal of Cellular and Molecular Immunology
基金
陕西省自然科学基金资助项目 (No.2 0 0 2C2 - 1 2 )
