豚鼠抗α-毒素抗体的制备及对酵母展示疫苗的检测  

Preparation of antibody against alpha-toxin in guinea pigs and detection of yeast display vaccine

作　　者：石铭 刘小雪 张静[2] 陈静 邵小光 Shi Ming;Liu Xiaoxue;Zhang Jing;Chen Jing;Shao Xiaoguang(Reproductive and Genetic Medicine Center of Dalian Municipal Women And Children's Medical Center,Dalian 116037,China;Department of Laboratory,Dalian Municipal Center Hospital,Dalian 116033,China;Medical Laboratory Technology,Medical College of Dalian University,Dalian 116622,China)

机构地区：[1]大连市妇女儿童医疗中心生殖与遗传医学中心,116037 [2]大连市中心医院检验科,116033 [3]大连大学医学院检验系,116622

出　　处：《中国医师进修杂志》2019年第10期912-916,共5页Chinese Journal of Postgraduates of Medicine

摘　　要：目的制备豚鼠抗α-毒素血清、纯化抗重组金黄色葡萄球菌α-毒素的IgG并应用十二烷基硫酸钠(SDS)-聚丙烯酰胺凝胶(PAGE)电泳检测,检测酵母表达α-毒素情况.方法免疫豚鼠,给豚鼠静脉注射重组α-毒素,制备抗α-毒素血清,利用HiTrap protein A HP纯化抗重组金黄色葡萄球菌α-毒素IgG,SDS-PAGE非还原性电泳检测纯化的IgG.酶联免疫吸附试验(ELISA)和免疫荧光共聚焦检测酵母表达α-毒素情况.结果成功获得了豚鼠抗α-毒素血清及所需的抗α-毒素抗体.非还原性电泳观察到未纯化的血清中抗α-毒素抗体浓度较低,穿透峰中抗α-毒素抗体含量较少,洗脱峰中抗α-毒素抗体的纯度约为85%.ELISA法检测发现,转化子1、2号与阴性对照组相比较,基本可以判断转化子经过诱导表面展示了α-毒素;免疫荧光共聚焦检测发现,经过抗α-毒素抗体染色,1号酵母转化子表面发出绿色荧光而未诱导培养基的细胞表面没有荧光.结论金黄色葡萄球菌α-毒素在酵母表面展示成功,可以进一步应用于生物医学临床或学术研究领域,同时可为研发酵母展示疫苗提供可靠的鉴定方法.Objective To prepare anti-serum against α-toxin in guinea pigs and purify anti-rHla IgG and identify it by SDS-PAGE observe the expression of Hla on the surface of yeast cells by ELISA and immunofluorescence confocal microscopy. Methods Guinea pigs were immunized with recombinant α-toxins to obtain anti-α-toxin serum and total IgG which contains anti-rHla IgG. The purity of IgG was Identified and evaluated with non-reducing SDS-PAGE. The expression of Hla was evaluated with whole cell ELISA and immunofluorescence confocal. Results The anti-α-toxin antibody of guinea pig was successfully obtained. Anti-α-toxin antibodies was captured by the protein A on the pre-packed column. The concentration of IgG in the unpurified serum was low, the α-rHla content in the flow-out peak was even lower, and the elution fraction contains IgG (α-rHla) with purity of about 85%. Through ELISA, transformant-1 and transformant-2 was identified to be positive with rHla on the surface, compared with that in the negative control. After stain with the purified anti-α-toxin IgG, the 1 yeast transformant showed green fluorescence under immunofluorescence confocal microscope. By contrast, without inducing, the 1 yeast transformant was negative with fluorescence. Conclusions The results show that Staphylococcus aureus Hla is successfully displayed on the surface of yeast, and this research could be further applied in the identification of clinical samples or pre-clinical research. This study provides two reliable methods for the development of yeast-display vaccine.

关 键 词：抗体 α-毒素 免疫荧光共聚焦 酵母表面展示 

分 类 号：R392-33[医药卫生—免疫学]