棉铃虫核多角体病毒泛素基因的克隆表达及抗体制备

Cloning and Expression of Ubiquitin Gene of Helicoverpa armigera Single-nucleocapsid Nucleopolyhedrovirus and its Antiserum Preparation

昆虫杆状病毒和痘病毒是目前已知唯一编码泛素基因的病毒。通过PCR方法,克隆了棉铃虫核多角体病毒(HaSNPV)泛素基因(Ubiquitin,Ubi)。序列分析表明,该基因编码区全长252bp,编码83个氨基酸残基,预计分子量为9.24kDa。将泛素基因克隆到原核表达载体pET28a上,构建重组质粒pETUbi,转化至大肠杆菌BL21(DE3)感受态细胞中,IPTG诱导表达融合蛋白。用Histag抗体检测目的蛋白,Westenblot实验证明所表达的蛋白是带有Histag的重组融合蛋白。通过改变IPTG浓度和诱导时间对表达条件进行了优化。利用NI琼脂糖凝胶亲和层析柱纯化目的蛋白,SDSPAGE鉴定为单一条带,同时用提纯蛋白制备了特异性抗体,为进一步的研究打下基础。

Baculoviruses and entomopoxvirus are the viruses which have been reported so far having ubiquitin gene. The ubiquitin gene of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus(HaSNPV) was amplified by PCR. Nucleotide analysis showed that it was 252bp,encoded 83 amino acids with a predicted size of 9.24kDa. The PCR product containing ubiquitin gene was inserted into pET-28a expressive vector. The Ubi-His-tag fusion protein was expressed in E.coli BL21(DE3).The fusion protein was identified by Western blotting with His-tag antibody. The expressions conditions including the contentrations of IPTG and the inducing times were optimized to achieve optional expression. The antibody anti the HaSNPV ubiquitin was produced in rabbit with purified ubiquitin fusion protein , and further study on the function of HaSNPV UBI is underway.