摘要
目的构建His-CSP/pGAPZαA重组表达质粒,转化并筛选阳性His-CSP/pGAPZαA/GS115重组酵母菌,通过发酵和层析纯化获得高纯度His-CSP蛋白。方法根据葛兰素史克公司RTS’S/AS01疫苗在GenBank公布的基因序列,选择去除14个NANP重复序列,并在其N末端加6个His标签作为目的基因。人工合成的目的基因克隆到毕赤酵母表达载体pGAPZαA,构建His-CSP/pGAPZαA重组表达质粒,转化酵母菌并筛选阳性转化子。发酵阳性His-CSP/pGAPZαA/GS115重组酵母菌并收获表达产物,经过Ni^(2+)柱亲和层析、阴离子交换层析和分子筛层析纯化His-CSP目的蛋白,采用SDS-PAGE和Western blot鉴定纯化蛋白。结果成功构建了His-CSP/pGAPZαA重组表达质粒,筛选到阳性His-CSP/pGAPZαA/GS115重组酵母菌,ELISA检测到表达上清中的His-CSP蛋白。层析纯化产物SDS-PAGE分析纯度>90%,Western blot分析显示纯化蛋白能被相应抗体识别。结论在毕赤酵母中成功表达了His-CSP蛋白,并通过发酵和纯化得到高纯度His-CSP蛋白,为建立CSP的ELISA检测方法奠定了基础。
Objective To construct a recombinant plasmid containing the His-CSP/pGAPZαA gene,to transform the recombinant plasmid into Pichia,to screen positive recombinant yeast,and to ferment and purify the protein on a chromatography column in order to yield highly pure His-CSP protein.Methods Based on the genetic sequence of the GSK RTS’S/AS01 vaccine published in GenBank,the 14 NANP repeat sequence was removed and 6 His at the N terminal of the antisense sequence of the genetic sequence was tagged as the target gene.The synthetic target gene was cloned into the expression vector Pichia pastoris,the recombinant expression plasmid His-CSP/pGAPZαA was constructed,and then the yeast was transformed and positive transformants were screened.The recombinant yeast containing the His-CSP/pGAPZαA/GS115 gene was fermented and the expression product was collected.The His-CSP protein was purified through Ni^(2+)column affinity chromatography,anion exchange chromatography,and molecular sieve chromatography.The protein was identified with DS-PAGE and Western blotting.Results The recombinant expression plasmid His-CSP/pGAPZαA was successfully constructed,the positive recombinant yeast His-CSP/PGAPZαa/GS115 was screened,and the expression of His-CSP protein in supernatant was successfully detected using ELISA.After purification using chromatography,SDS-PAGE was used to analyze the purity of the product,which was more than 90%.Western blot analysis indicated that the purified protein was recognized by corresponding antibodies.Conclusion His-CSP protein was successfully expressed in Pichia.Highly pure His-CSP protein was obtained through fermentation and purification.These findings have provided a foundation for studying CSP detection kits in the future.
作者
王婉如
刘宇阳
高雪峰
杨林鹏
李宣钟
陈勇
WANG Wan-ru;LIU Yu-yang;GAO Xue-feng;YANG Lin-peng;LI Xuan-zhong;CHEN Yong(Lanzhou Institute of Biological Products Co.,Ltd.,Center for Gansu Provincial Vaccine Engineering Research,Lanzhou 730046,Gansu,China)
出处
《中国病原生物学杂志》
CSCD
北大核心
2021年第11期1307-1311,共5页
Journal of Pathogen Biology
