一种新型原核表达载体的构建及应用

目的 利用基因重组技术构建一种新型原核表达载体，使之表达带有His和GST标签的融合蛋白。方法 以质粒pGEX为模板，将PCR扩增出的GST基因插入到载体pQE-30中构建pQE-30-GST，经IPTG诱导GST蛋白表达并用Westernblot鉴定。分别将huNSEl88-354、huPrP23-91和huDoppe124-152基因插入pQE-30-GST，转化E-coliM15并诱导表达融合蛋白。采用NiSepharose4B和(或)Glutathione Sepharose 4B纯化融合蛋白，并用羟胺裂解。结果 重组表达载体pQE-30-GST可有效地表达各种His-GST融合蛋白，并可用两种方法纯化不同大小的融合蛋白；利用羟胺可将目的蛋白肽段与His-GST蛋白有效裂解。结论 新型原核表达载体pQE-30-GST可进行多种蛋白质的表达，表达的蛋白质可经两种方法纯化以提高蛋白质的纯度，融合蛋白可经羟胺裂解获得目的蛋白质单体。

Construction and Application of a New Prokaryotic Expression Vector

Objective To construct a new prokaryotic expression vector that contains both His tag and GST tag by recombinant technology.Methods Using plasmid pGEX as the template, GST encoding gene was amplified by PCR, and cloned into plasmid pQE-30 to generate the recombinant plasmid pQE-30-GST. The expression of His-GST protein was induced by isopropyl-1-thio-b-D-galatoside (IPTG) and verified by Western blot. huNSE188-354, huPrP23-91 and huDoppel24-152 genes were cloned into pQE-30-GST respectively, and the expressions of fusion proteins were conducted after transforming into E. coli M15. The recombinant proteins were purified by Ni Sepharose 4B and/or Glutathione Sepharose 4B. Furthermore, the target proteins were cleaved and released from the fusion forms by hydroxylamine. Results Several proteins in His-GST form were efficiently expressed with vector pQE-30-GST. The expressed proteins were easily purified with both Ni Sepharose 4B and/or Glutathione Sepharose 4B chromatograph. Moreover, the target proteins were sufficiently released from the respective fusion forms with hydroxylamine. Conclusion The newly generated prokaryotic expression vector pQE-30-GST can be used for expression of various proteins that can be purified with both His tag and GST tag to get more highly purified one, and subsequently, cleaved from their fusion forms by more convenient hydroxylamine methodology.