金黄色葡萄球菌SirH主要抗原表位区的表达及其抗血清制备

摘要

为在大肠埃希菌中表达金黄色葡萄球菌Sir H主要抗原表位区并制备相应纯化重组蛋白的抗血清，通过生物信息学软件分析Sir H的主要抗原表位区，PCR扩增Sir H主要抗原表位区的编码序列，插入表达载体pGEX-4T-1中，构建重组表达载体pGEX4T1-sirH。该载体经酶切和DNA测序鉴定正确后，转化大肠埃希菌BL21，IPTG诱导表达，GST 亲和层析纯化获得重组蛋白后，采用Western blot方法检测目的蛋白的反应原性。用纯化的重组蛋白免疫新西兰大白兔制备多克隆抗体，间接ELISA检测抗血清效价。结果显示，PCR扩增的SirH主要抗原表位区的编码序列长度约759 bp ，SDS-PAGE初步测定表达蛋白的分子质量约为54 ku ，与理论值相符。亲和层析纯化获得了目的蛋白，Western blot结果证实该纯化蛋白能与乳房炎患病奶牛恢复期的血清发生反应。应用纯化蛋白免疫新西兰大白兔后制备的抗血清效价在1∶409600以上。结果表明，Sir H主要抗原表位区能刺激机体产生较强的体液免疫应答，Sir H是一个有潜力的疫苗候选抗原。%In order to express the major antigenic epitope region of candidate protective antigen SirH from S .aureus and prepare the polyclonal antibodies against its expressed products ,bioinformatics softwares were used to analyze the major antigenic epitope region of SirH .A DNA fragment about 759 bp was ampli-fied by PCR , and then the amplified gene fragment was inserted into pGEX-4T-1 vector to build pGEX4T1-sirH .Subsequently ,the plasmid pGEX4T1-sirH was transformed into E .coli BL21 for expres-sion under IPTG induction .SDS-PAGE analysis indicated that the recombinant protein with the predicted molecular mass about 54 000 was expressed .Western blot confirmed that the purified recombinant protein could be recognized by cow serum infected with S .aureus .At last ,the polyclonal antibodies against SirH were prepared by immunizing New Zealand rabbits with the purified recombinant SirH .Indirect ELISA was used for the detection of antibody titer .The result showed that antibody titer of the rabbit serum reached 1∶409 600 .In conclusion ,the major antigenic epitope region of SirH could stimulate strong hu-moral immune response in rabbits .SirH is a potential vaccine candidate antigen for S .aureus infection .