鸡传染性支气管炎病毒中国流行株免疫原基因的分子克隆与基因分型

MOLECULAR CLONING AND GENETYPING OF THE IMMUNOGENCITIVE GENE OF AVIAN INFECTIOUS BRONCHITIS VIRUS ISOLATES IN CHINA

从我国华中、华东、华南、华北、东北及西北等地分离鸡传染性支气管炎病毒流行株，经病毒的病原性、形态结构、理化特性、病毒抗原的特异性检测、组织嗜性、结构多肽及血清型分析等多方面的鉴定，对来源于我国不同地区的流行毒株免疫原Ｓ１基因进行了分子克隆及基因分型；ＲＴ－ＰＣＲ扩增病毒Ｓ１基因，将Ｓ１基因５′和３′端分别进行分子修饰之后插入克隆载体ｐＵＣ１８的ＢａｍＨⅠ／ＨｉｎｄⅢ位点，在Ｅｃｏｌｉ中实现了目的基因的克隆；利用英国病毒株Ｓ１全基因核酸探针与流行株Ｓ１基因的重组克隆质粒分子杂交及目的基因５′端高变区核苷酸序列分析鉴定后，采用ＨａｅⅢ、ＰＶＵⅡ和ＸｂａⅠ等限制性核酸内切酶酶切分析不同流行株Ｓ１基因ｃＤＮＡ。结果表明，我国流行的鸡传染性支气管炎病毒分为６个主要的基因型；试验发现除与Ｍ４１和澳大利亚Ｔ株相近基因型毒株之外，我国流行的毒株存在明显的分子水平的变异，这与病毒血清型鉴定的结果一致，鸡传染性支气管炎病毒中国流行毒株免疫原基因Ｓ１的分子克隆及基因分型为该病毒分子流行病学及病毒遗传变异的主要分子基础的深入研究奠定了基础。

vian Infectious Bronchitis Virus(IBV) isolates were isolated from the middle area, east, south, north, northeast and northwest of China, identified by the detection of viral pathogenicity, viral structure, physchemistry property, viral antigen detection, tissue tropism, polypdptide analysis and serotyping. The gene encoding immunogenicitive S1 glycoprotein of IBV isolates from different area in China were cloned and genetyped. The S1 gene of IBVs were amplified by RTPCR. After modified on 5′ and 3′ end, the S1 genes were inserted into BamHⅠ/HidⅢ site of pUC18 cloning vector, and cloned in Ecoli cells. Identified by Southern blot and sequencing of the 5′ end of S1 gene(including hypervariant region), the cDNAs of each IBV S1 gene were digesed by HaeⅢ,PVUⅡ and XbaⅠ. Results demonstrated that there were mainly six genetypes of IBV isolates in China. Including M41 and Australia T strain genetypes, there were great molecular variant of IBV S1 gene in China.