犬瘟热病毒核衣壳蛋白基因免疫小鼠诱导抗体的研究

Antibody Response in Mice Induced by CDV Nucleocapsid Protein Gene

将犬瘟热病毒(Caninedistemper virus,CDV)核衣壳蛋白基因(Nucleocapsid protein,N)的N蛋白基因克隆到pMD-18T载体,经酶切分析测序获得阳性重组质粒。采用EcoRⅠ和KpnⅠ双酶切后定向克隆进真核表达载体pcDNA中,电泳测序筛选阳性克隆pcDNA-N,转染COS-7细胞,IFA验证结果显示转染的COS-7细胞胞浆中表达了CDV的N蛋白。与弗氏佐剂乳化完全后将重组质粒pcDNA-N腹腔注射8周龄BABL/c小鼠,同时设pcDNA原载体做阴性对照,2周为间隔共免疫3次,三免两周后采血,ELISA检测效价。结果显示:pcDNA-N试验组抗体滴度为101.62±0.164,对照组为100.52±0.56。表明犬瘟热病毒核衣壳蛋白基因免疫小鼠可诱导机体产生特异性体液免疫。

The CDV nucleocapsid protein gene was cloned into pMD-18T vector,restriction analysis was positive for plasmid DNA sequencing.The pMD-18T-N was digested by EcoRⅠand KpnⅠand cloned into the eukaryotic expression vector pcDNA,the positive clones pcDNA-N was screening by electrophoresis sequencing,then transfected COS-7 cells and verified with the IFA.Intraperitoneal injection of 8-week-old BABL /c mice after the plasmid pcDNA-N emμLsified with Freund＇s adjuvant.At same time the original vector as negative control pcDNA.A total of three inocμLations were performed at once every two weeks.Two weeks after the last injection,blood samples were collected to separate serum.ResμLts of ELISA show that titer stimμLated by pcDNA-N was 101.62 ± 0.164.While the control group＇s titer was 100.52 ± 0.56,could not been detected CDV antibody.These experiments provided primary data for in vivo research of dogs.