广东番茄黄化曲叶病毒AC2基因的原核表达及抗血清制备

Prokaryotic Expression and Antiserum Preparation of AC2 Gene of Tomato yellow leaf curl Guangdong virus G3 Isolate

利用PCR方法从广东番茄黄化曲叶病毒分离物G3(Tomato yellow leaf curl Guang dong virus-[G3],TYLCGuV-[G3])全长序列的重组质粒上扩增该病毒的AC2基因,将其构建至原核表达载体pET-28b(+)上,经IPTG诱导,在大肠埃希菌Rosetta(DE3)Ⅲ中高效表达.SDS-PAGE电泳结果显示,目的融合蛋白与预期大小一致,相对分子质量约为19 500.以诱导的融合蛋白为抗原,免疫注射新西兰大耳白兔制备抗血清,间接ELISA测定抗血清效价为1∶16 384.Western-blotting检测结果表明,制备的抗血清有较好的抗原-抗体识别反应,为专化性抗血清.

AC2 gene of Tomato yellow leaf curl Guangdong virus isolate G3 （ TYLCGuV- [ G3 ] ） was amplified from its recombinant plasmid by PCR, and the gene was cloned into prokaryote expression vector pET-28b（ ＋ ）. The expressed fusion protein was highly expressed in Escherichia coli Rosetta（ DE3 ）Ⅲ induced by IPTG, and the molecular mass was about 19 500 by SDS-PAGE analysis. The fusion protein was used as an antigen to immunize the healthy rabbit and the antiserum of AC2 was prepared. The titer measured by ELISA was about 1：16 384. Western-blotting analysis indicated that the antiserum could serologically react with AC2 protein of TYLCGuV-[ G3 ].