【摘要】 利用RT-PCR技术将人源性PLC-γ1中的SH2-SH2-SH3结构域基因扩增并克隆到载体pGEX-2T中,经热激反应转化到大肠杆菌BL21菌株内,在低温(33℃)下、利用IPTG诱导表达构建的重组基因的高效表达体系,利用谷胱甘肽琼脂糖(sepharose)4B纯化得到高表达的重组蛋白。SDS-PAGE和Western blot检测表明,PLC-γ1的SH2-SH2-SH3结构域重组基因可在低温(33℃)条件下在E.coliBL21细胞中稳定完整表达,但是在较高温度下(37℃)会产生SH2-SH2-SH3结构域不完全表达的现象。上述研究结果为人源性重组基因PLC-γ1中SH2-SH2-SH3结构域的深入研究和广泛应用奠定了基础。更多还原

【Abstract】 Phospholipase C gamma-1(PLC-γ1) is a nerve-enriched protein containing SH2-SH2-SH3 domain. It has been demonstrated that the SH2 domain of PLC-γ1 regulates the phosphorylation of PLC -γ1 through interaction with receptor tyrosin kinase , and the SH3 domain of which interacts with sos or vinculin to mediate Ras signaling and regulate actin polymerization. To understand better this potential functional role and produce the monoclonal antibody against SH2-SH2-SH3 of PLC-γ1,using RT-PCR from adult human cortex , the functional domain(SH2-SH2-SH3 domain)of PLC-γ1 was cloned to pGEX-2T fusion vector and transformed into E.coli BL21 host cells. SDS-PAGE and Western blot analysis revealed that the induced recombinant protein by IPTG was expressed successfully at 33℃ but weakly at 37℃. Furthermore, the recombinant protein was degraded at 37℃ . The GST fusion protein extract was immobilised on glutathione sepharose 4B bead and eluted with glutathion. Taken together, the results indicated that a lower culture batch(33℃) was utilized to produce recombinant protein easily. The fragment expression of SH2-SH2-SH3 at higher temperature(37℃), however, suggests that PLC-γ1 degradation was involved not only in eukaryotic cell but also in prokarytic cell(BL21 strain). The identification of cleaved site of SH2-SH2-SH3 domain by peptide sequencing analysis will be done in the future.更多还原