CAJ | 학술논문

目的构建人Hexastatin蛋白原核表达载体,大量表达并纯化Hexastatin蛋白,为其在体内外的活性研究奠定基础.方法提取人食管癌EC9706细胞总RNA,通过RT-PCR方法扩增人Hexastatin基因,导入pMD18-T载体测序,然后克隆至pMAL-c4x表达载体,IPTG诱导表达,并利用Amylose亲和树脂纯化融合蛋白,产物进行Western blot 鉴定.结果经PCR扩增成功获得了687 bp的人Hexastatin基因,测序正确.重组人Hexastatin基因在BL21菌体中获得高效可溶性表达,表达的可溶性蛋白占总蛋白量的24.8%,纯化后的MBP-Hexastatin融合蛋白纯度可达90%,Western blot证实表达蛋白为目的蛋白.结论人Hexastatin基因克隆、表达及纯化的成功,为进一步在体内外研究其生物活性奠定基础,同时也为肿瘤的生物治疗提供了新的方法.
목적 구건인Hexastatin단백원핵표체재체,대량표체병순화Hexastatin단백,위기재체내외적활성연구전정기출.방법 제취인식관암EC9706세포총RNA,통과RT-PCR방법확증인Hexastatin기인,도입pMD18-T재체측서,연후극륭지pMAL-c4x표체재체,IPTG유도표체,병이용Amylose친화수지순화융합단백,산물진행Western blot 감정.결과 경PCR확증성공획득료687 bp적인Hexastatin기인,측서정학.중조인Hexastatin기인재BL21균체중획득고효가용성표체,표체적가용성단백점총단백량적24.8%,순화후적MBP-Hexastatin융합단백순도가체90%,Western blot증실표체단백위목적 단백.결론 인Hexastatin기인극륭、표체급순화적성공,위진일보재체내외연구기생물활성전정기출,동시야위종류적생물치료제공료신적방법.
purpose To construct the expression vector of Hexastatin gene,to express and to purify the recombinant protein for further activity research.Methods The human Hexastatin gene was isolated by RT-PCR from EC9706 cells total RNA and cloned into pMDl8-T for sequencing.Then the Hexastatin gene was subcloned into pMAL-c4x expression vector and induced to express by IPTG.The recombinant fusion protein was purified with Amylose Resin Heads.Results RT-PCR product was about 687 bp and its sequence was the same as that of Hexastatin reported.The recombinant protein was expressed in E.coli BL21 with high level and the soluble protein accounted for 24.8% of the total bacterial protein,The purification of recombinant protein purified with Amylose Resin Heads reached more than 90%.Conclusion The cloning,expression and purification of human Hexastatin have laid a foundation for its anti-angiogenesis therapy for tumor.