【作者】 张秀峰； 王中华； 刘佳旭； 刘艳华； 母连志； 李国江； 丁壮；

【Author】 ZHANG Xiu-feng1,4,WANG Zhong-hua2,LIU Jia-xu1,LIU Yan-hua3,MU Lian-zhi1,LI Guo-jiang1 4,DING Zhuang1(1.College of Animal Science and Veterinary Medicine,Jilin University,Changchun 130062,China;2.Shangqiu Polytechnic,Shangqiu,Henan 476100,China;3.Qiqihar Teachers College,Qiqihar,161000,China;4.Jilin Agricultural Science and Technology College,Jilin,132101,China)

【机构】 吉林大学畜牧兽医学院； 吉林农业科技学院； 商丘职业技术学院； 齐齐哈尔师范高等专科学校；

【摘要】 应用RT-PCR方法从鸭源Ⅰ型副粘病毒DP1/02株中扩增F基因并克隆入pMD18-T载体,经序列测定、分析,结果显示DP1/02株F基因与NDV国家标准强毒F48E9株的核苷酸、氨基酸序列同源性均达到99%。随后将F基因克隆入真核表达载体pCI-neo,构建真核表达质粒pCI-F,将阳性质粒体外转染Vero细胞,通过间接免疫荧光对真核质粒的表达进行鉴定,利用pCI-F质粒进行动物实验。结果表明构建的真核表达质粒pCI-F能够在Vero细胞中表达,雏鸭免疫14天后在体内可检测到特异性抗体,二次免疫雏鸭后,对NDV强毒的攻毒保护率为73%。本实验为利用F基因构建的核酸疫苗提供了重要的技术支持。更多还原

【Abstract】 In this study,fusion glycoprotein(F)gene of paramyxovirus strain DP1/02 isolated from duck was amplified by reverse transcriptase polymerase chain reaction(RT-PCR)and cloned into pMD18-T vector.The sequence analyses of F gene indicated that the homology of nucleotide acid sequences and amino acid sequences between DP1/02 strain and F48E9 strain,standard virulent strain of Newcastle disease virus F48E9 was 99%.Then,F gene was cloned into the eukaryotic expression vector pCI-neo.The recombinant plasmid pCI-F which was transferred into Vero cells successfully expressing F protein was identified by indirect immunofluorescence.In animal experiment,the ducks were vaccined by plasmid pCI-F,the result showed that the plasmid pCI-F was effective,for the first immunization stimulating duck to produce specific antibody 14 days later.After the second immunization,the specific antibody could protect 73% of the ducks against NDV standard virulent infection.The results of animal experiment suggested that it is useful to construct a eukaryotic plasmid which expressed F gene.更多还原