【摘要】 以鹅细小病毒(Gooseparvovirus,GPV)HG5/82株基因组作为PCR反应模板,扩增vp基因3’端长864bp的基因片段,将其克隆到pMD18-TSimple克隆载体后转化入大肠杆菌TG1。筛选阳性质粒,并通过BamHⅠ和HindⅢ将外源基因定向克隆到原核表达载体pET-30a,阳性重组质粒经确证性序列测定,证明外源片断插入到pET-30a的预期位置。将其转入大肠杆菌BL21,经终浓度为0.6mmol/L的IPTG诱导,SDS-PAGE表明外源基因获得表达,融合蛋白分子量约为34kDa。将诱导后的工程菌用6mol/L盐酸胍裂解,经超声处理后离心,利用镍离子亲和树脂对裂解产物的上清进行纯化。用纯化的融合蛋白免疫新西兰白兔制备兔抗该融合蛋白的抗血清。Westernblotting结果表明制备的兔抗血清与该融合蛋白及亲本病毒的结构蛋白都具有反应性。结合前期工作进展对GPVVP蛋白的B细胞线性抗原表位进行定位。更多还原

【Abstract】 An 864bp fragment at the 3’- end of the vp3 gene of Goose parvovirus (GPV) HG5/82 isolate was cloned by RT-PCR, and was cloned into pMD18-T Simple vector. Positive clones were identified by REN digestion and PCR and a recombinant plasmid was digested with BamHⅠ and Hind Ⅲ and subcloned into the prokaryotic expression vector pET-30a. E. coli BL21 were transformed by the recombinant vector and induced by IPTG at a concentration of 0.6mmol/L. It was ascertained that the target fusion peptide with a molecular weight of 34kDa was expressed. The bacteria induced by IPTG were treated with 6mol/L Guanidine hydrochloric acid and ultrasonotor. The fusion protein was purified by using a ProBondTM Resin kit. The antiserum against the fusion protein was raised in rabbits. Western blot analysis indicated that the antiserum specifically recognized the fusion protein as well as VP1、VP2 and VP3 of GPV HG5/82 strain. In addition, a 65kDa protein of HG5/82 strain was also recognized by the antiserum. Presumablely, the protein may be an unknown structural viral protein of GPV. Combined with previous work, it can be seen the locations of B cell linear epitopes were mapped in amino acid 145～198 and amino acid 231～733 of VP1 sequence of GPV HG5/82 strain. This information may be helpful when designing recombinant diagnostic antigens for GPV.更多还原