拟南芥AtXCD1蛋白的原核表达研究

Prokaryotic Expression of Arabidopsis AtXCD1 Fusion Proteins

[目的]研究拟南芥AtXCD1蛋白的原核表达。[方法]构建XCD1原核表达载体,根据NCBI基因序列设计引物,并以PCR扩增得到大量目的片段XCD1,将XCD1连接到原核表达载体pET-32a+,并对重组质粒进行测序鉴定,将重组质粒转化至大肠杆菌原核表达菌株BL21(DE3),对蛋白表达条件:诱导时间、诱导温度和IPTG浓度等进行优化。IPTG诱导表达获得目的蛋白,对蛋白表达条件:诱导时间、诱导温度和IPTG浓度等进行优化。[结果]XCD1序列全长为1 242 bp,与PCR产物大小一致。蛋白XCD1-pET-32a+较适表达条件为在30℃0.1 mmol/L的IPTG诱导1.5 h。[结论]该结果为进一步蛋白纯化及酶活测定试验奠定了基础。

[Objective] To study prokaryotic expression of Arabidopsis AtXCD1 protein.[Method] To construct the prokaryotic expression plasmid of XCD1,the XCD1 encoding sequence was proliferated by PCR under the direction of the primers designed according to the sequence obtained from NCBI website.Then the cDNA fragments were cloned into prokaryotic expression vectors pET-32a ＋ and sequenced.In addition,recombinant plasmids were transformed into E.coli.BL21 （DE3） for expression.Expression of AtXCD1 was induced to generate their fusion proteins by IPTG,and the protein expression conditions were optimized,such as time,temperature and IPTG concentration.[Result]The whole length of XCD1 sequence is 1 242 bp,the size is the same as PCR products.The proper expression conditions of protein XCD1-pET-32a ＋ is 30 ℃ 0.1mmol/L,IPTG induction for 1.5 h.[Conclusion] The study will lay a foundation for further protein purification and enzyme activity determination.