鹅源新城疫病毒HN基因在Bac to Bac系统中的表达及抗原性检测

血凝素-神经氨酸酶蛋白(Haemagglutinin Neuramindase,HN)是鹅源新城疫病毒诱导体液免疫的重要靶抗原。经pMD18-T-HN阳性质粒扩增了HN基因,扩增的片段亚克隆到转座载体pFastHTb中构建重组转座载体pFastHTb-HN。将其转化到DH10Bac感受态细胞中,得到重组杆粒rBacmid-HN,重组杆粒rBacmid-HN转染sf9昆虫细胞,收获的重组病毒盲传两代。PCR鉴定重组病毒,获得的重组杆状病毒命名为rBv-HN。经间接免疫荧光(IFA)和SDS-PAGE鉴定,获得的重组杆状病毒rBv-HN在sf9昆虫细胞中获得表达,且表达的蛋白大小为75ku左右,与理论值相当。Western blot和Dot-ELISA检测结果表明,表达的蛋白可与鸡抗NDV血清发生特异性抗原反应,证实所表达的蛋白具有良好的反应原性。

Bac to Bac expression and antigenicity detection of HN gene of NDV of goose

Haemagglutinin Neuramindase(HN) protein is the important antigen of Newcastal Deases Virus of goose which can induce humoral immunity.Amplified HN gene according to the pMD18-T-HN,amplified the baculovirus recombinant transfer vector pFastHTb-HN were constructed by subclonning the amplified fragment into the transfer vector.The recombinant transfer vectors was transformed into the receptor cells and the recombinant bacmid was acquired.We transfected the recombinant rBacmid-HN into insect sf9 cells and harvested.The virus was further amplified by propagation in sf9 cells,the recombinant virus identified by PCR,and named as rBv-HN.The recombinant baculovirus were shown to be efficiently expressed in sf9 cells by detecting with indirect immunofluorescent assay and SDS-PAGE.The protein of approximately 75 ku was detected which in accord with theoretical values.Specific antigen responses to the expressed proteins with chick anti-NDV serum were demonstrated by Western blot and Dot-ELISA.