摘要
通过RT—PCR扩增柔嫩艾美球虫YZ株折光体SO7抗原基因，并进行克隆和测序。将不舍信号肽编码区片段的S07基因克隆入表达载体pGEX6p-1谷胱甘肽-S-转移酶基因的下游，构建表达质粒pGEX6p-1-SO7，转化宿主菌BL21，获得重组菌。通过建立生长曲线，对诱导条件的摸索，根据SDS—PAGE确定融合蛋白的最佳表达条件。通过融合蛋白切胶免疫小鼠制备超免疫血清，经间接EL-ISA检测其与E。tenella子孢子抗原反应的特异性。结果显示，诱导时机和诱导时间是影响表达的主要因素，诱导温度、1PTG浓度次之；诱导时机以3h为最佳，诱导时间以4．5h为最佳；诱导温度以37℃最佳，0．008～1．000mmol／L的IPTG对表达量的影响不大。在优化的表达条件下，表达产物主要以包涵体存在，表达量占菌体总蛋白的37．5％。间接ELISA结果表明融合蛋白具有一定的免疫原性，保留了天然蛋白的部分抗原性。
SO7 gene of Eizneria tenella YZ strain was amplified by RT-PCR from total RNA of sporulating oocyst, cloned into pUCm-T, and then sequenced. The encoding region without signal peptide was amplified again by PCR from pUCm-T SO7 containing cDNA of SO7 gene and subcloned into pGEX-6p-1. Positive pGEX-6p-1-SO7 was identified by restriction digestion and sequencing. After induction, SO7 antigen was expressed as fusion protein（GST-SO7, 48 ku ） with glutathione S transferase（GST）. Expression conditions including inducing chance, induction time, temperature and IPTG concentration were determined by SDS-PAGE, and growth curve of recombinant bacteria was also established. GST-SO7 separated by SDS-PAGE was extracted from the gel and used to immunize ICR mice once a week for five times. Antiserum was collected, and its specificity to E. tenella was identified by indirect ELISA. The optimal incubation time for production of GST-SO7 was 3 h after inoculation, the optimal induction time was 4.5 h after induction, the best temperature was 37℃, and the best concentration of IPTG was 0.01 mmol/L. Under the optimal expression conditions, the insoluble fusion protein, which accounted for 37.5% of total bacterial proteins, showed good immunogenicity.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2007年第1期54-60,共7页
Chinese Veterinary Science
基金
扬州大学高层次人才科研启动基金项目(2004-10)
扬州大学科研基金项目(VK0413171)
江苏省高校自然科学研究基金项目(NK0310078)
关键词
柔嫩艾美球虫
重组S07抗原
大肠杆菌
表达条件
Eimeria tenella
recombinant SO7 antigen
Escherichia coli
expression condition
