摘要
为获得人A组轮状病毒重组结构蛋白VP7及其高效价的卵黄抗体,使用PCR技术扩增VP7 DNA片段并将其克隆到原核表达载体pET28a上形成重组质粒pET28a-VP7。将重组质粒转化基因工程菌E.Coil BL21(DE3),用0.5 mM IPTG诱导VP7重组蛋白的表达。通过包涵体纯化和蛋白复性后,将VP7重组蛋白与弗氏佐剂混匀免疫产蛋鸡,收集鸡蛋。用水稀释-盐析法纯化卵黄抗体IgY。进一步应用SDS-PAGE、ELISA和点杂交技术,分析该卵黄抗体的纯度、效价和特异性。结果表明在基因工程菌E.coli BL21(DE3)中能够实现VP7重组抗原的表达,将重组抗原VP7免疫产蛋鸡,提取纯化得到滴度为1:100 000的抗VP7卵黄抗体,且特异性的识别野生型A组轮状病毒。
In order to prepare recombinant constitutive protein VP7 of human A Rotavirus and it＇s high titer egg yolk antibody（IgY）,the VP7 DNA fragment is amplified by PCR and cloned into the prokaryotic expression vectors pET28a to form recombinant plasmid pET28a-VP7. After identification by DNA sequencing,the recombinant plasmids of pET28a-VP7 is transformed into E. coli BL21 and induced with 0. 5 mM IPTG. Next,the recombinant VP7 insoluble protein is purified and refolded,and then subjected to immune lay hens combined with CFA,eggs are collected. Then,anti-VP7 IgY antibodies are purified by water dilution-salt outing. Moreover,the purity,titer and specificity of isolated IgY are further analyzed by SDS-PAGE,ELISA and dot blot respectively. The results show that the recombinant VP7 protein can be expressed in E. coli BL21 as inclusion body. After immunization with VP7 recombinant antigen,the isolated anti-VP7 IgY antibodies demonstrate a high titer at 1∶ 100 000,and recognize wild type human A rotavirus by its＇ specificity.
出处
《四川理工学院学报（自然科学版）》
CAS
2014年第3期15-19,共5页
Journal of Sichuan University of Science & Engineering(Natural Science Edition)
基金
新疆生物资源基因工程重点实验室开放课题(XJDX0201-2014-01)
四川省战略新兴产业资助项目(10-046-04-Z11)
四川省教育厅项目(12ZA088)
