烟草H2A的序列分析、原核表达载体构建及诱导表达

目的]对H2A进行序列分析及蛋白结构域分析,并将其克隆入p GEX4T-1原核表达载体进行诱导表达。方法]以p GADT7-Rec-H2A质粒为模板,通过PCR扩增烟草H2A(3~140 Aa),将其插入原核表达载体p GEX-4T-1中,并将重组载体转入BL21(DE3)中,诱导其表达。结果]构建了烟草H2A基因全长和p GEX4T-1载体大片段连接的融合表达载体,并成功地诱导其表达。结论]该研究为运用Pull Down确定Nt Tkr与候选蛋白之间的体外互作进而确定Nt Tkr与蛋白互作的关键结构域奠定了试验基础。

Sequence Analysis,Construction and Induced Expression of Prokaryotic Expression Vector of Tobacco H2A Gene

Objective]The sequence analysis and protein structure domain analysis of H2A were carried out,and it was cloned into pGEX4T-1 prokaryotic expression vector for inducing expression.Method] Using plasmid pGADT7-Rec-H2A as a template,tobacco H2A (3-140 Aa) was amplified by PCR and inserted into prokaryotic expression vector pGEX-4T-1,then the recombinant vector was transformed into BL21 (DE3) and successfully induced by IPTG.Result] The fusion expression vector that the full length of tobacco H2A gene is large fragment connected to pGEX4T-1 was constructed,and successfully induced its expression.Conclusion] This laid the foundation for the use of Pull Down to determine the interaction between NtTkr and candidate proteins in vitro and thus to determine the key domains of NtTkr interaction with proteins.