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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 2融合蛋白
OBJECTIVE: To clone the amino-terminal PID domain of human DOC 2 (tentatively named nDOC 2) and to express it in prokaryotic cells. Methods: The nDOC 2 gene was amplified from normal human ovarian tissue by RT PCR and cloned into vector pUC19. After confirmed by sequencing, it was subcloned into the prokaryotic expression vector pGEX 4T 1 using BamHI / EcoRI double digestion and transformed into E. coli DH5α strain. The engineered bacteria were used to induce the expression with IPTG, and the expressed products were identified by SDS PAGE. Results: ① The human nDOC 2 gene was successfully cloned by RT PCR, sequencing and restriction enzyme digestion. ② The IPTG-induced recombinant plasmid pGEX 4T nDOC 2 expressed a fusion protein with relative molecular mass (Mr) of about 50000, which was consistent with the expected results. Conclusion: The human DOC 2 amino-terminal PID domain was successfully cloned and the GST nDOC 2 fusion protein was expressed in E.coli DH5α