鸡传染性支气管炎病毒中国流行株免疫原S1基因的分子克隆、序列分析及其DNA免疫的初步研究  

Cloning,Sequencing and the S1 Gene DNA Vaccine of Infectious Bronchitis Virus QD Isolate in China

作　　者：江国托[1] 步志高[1] 刘思国[1] 康丽娟[1] 祁贤 卢景良[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室

出　　处：《生物工程学报》1999年第3期304-309,共6页Chinese Journal of Biotechnology

基　　金：国家攀登计划B类资助课题

摘　　要：对来源于我国华东地区的鸡传染性支气管炎病毒流行株ＱＤ免疫原Ｓ１基因ｃＤＮＡ进行了克隆、序列分析和ＤＮＡ免疫的初步研究。ＲＴＰＣＲ扩增ＱＤ毒株的Ｓ１基因，将其５′和３′端分别进行分子修饰后插入克隆载体ｐＵＣ１８的ＢａｍＨⅠ／ＨｉｎｄⅢ位点，在大肠杆菌中实现了目的基因的克隆；利用英国ＩＢＶ毒株Ｓ１全基因核酸探针与ＱＤ毒株Ｓ１基因的重组克隆质粒分子杂交后，采用ＨａｅⅢ，ＰｖｕⅡ和ＸｂａⅠ等限制酶对此流行毒株Ｓ１基因ｃＤＮＡ进行了酶切分析；在测定ＱＤ毒株Ｓ１基因５′端高变区核苷酸序列并以此与ＩＢＶＭ４１，Ｈ１２０，６／８２及Ｂｅａｕｄ等参考毒株序列对比分析的基础上，构建了ＱＤ株Ｓ１基因ＤＮＡ免疫表达质粒，肌肉注射免疫小鼠后，鸡胚病毒中和试验的结果表明，ＩＢＶＳ１基因ＤＮＡ免疫表达质粒能诱导小鼠产生病毒特异的中和抗体，具有良好的免疫原性，初步显示基因疫苗在鸡传染性支气管炎防治上应用前景。One local strain of avian infectious bronchitis virus (IBV) designated as QD from the east of China was identified and used in this test.The gene encoding S1 glycoprotein of the isolate was obtained by RT PCR,cloned in E.coli, and identified by Southern blot,Restriction enzyme fragment length polymorphism analysis (RFLP) and sequencing.To investigate the probability of DNA va ccine in controlling IB,cDNA of S1 gene was transferred from pUCQDS1 to pSY538 for adding end code TAA at it's 3′ end.After that,the fragment containing intact open reading fragment of S1 gene was inserted into pSY β LacZ at downstream of the SV40 enhancer and promotor and upstream of SV40 polyA signal to replace the LacZ gene and the plasmid was designated as S1 gene DNA vaccine expressive plasmid pSYQDS1.Soluted in PBS,the plasmid was injected into quadriceps muscular of mice at a dose of 300μg.Four weeks post intramuscular injection,the seum of the immunized mice was collected to neutralize IBV on SPF chicken embryos.Results demonstrated that the S1 gene DNA vaccine plasmid pSYQDS1 could efficiently induce the neutralizing antibodies in mice.

关 键 词：鸡 传染性支气管炎 病毒 免疫原S1基因 分子克隆 

分 类 号：S852.65[农业科学—基础兽医学] S858.315.3[农业科学—兽医学]