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

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

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。

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.