我国鸡传染性支气管炎病毒地方性流行毒株S1基因的RT-PCR扩增及其克隆与鉴定

Amplification,Cloning and Identificatification of the S1 Genes of Locally Isolated Avian Infectious Bronchitis Virus Strains in China

采用ＲＴ－ＰＣＲ方法，以ＩＢＶＳ１全基因特异性引物分别从我国华东（ＨＤ）、华北（ＨＢ）、华中（ＨＺ）、华南（ＨＮ）、西北（ＸＢ）及东北（ＤＢ）等地的ＩＢＶ流行株基因组中扩增出预期的１．７ｋｂ左右的ＤＮＡ片段。ＰＣＲ产物的ＨａｅＩＩ酶切分析及其与英国ＩＢＶＳ１全基因核酸探针的分子杂交证实所获６个ＩＢＶ流行株的ＰＣＲ产物为ＩＢＶＳ１基因。将此６个毒株的Ｓ１基因ＰＣＲ产物分别进行５’和３’端的ＢａｍＨＩ和ＨｉｎｄＩＩ酶切识别位点的分子修饰之后插入到克隆质粒ｐＵＣ１８的ＢａｍＨＩ／ＨｉｎｄＩＩ位点，在大肠杆菌中实现了目的基因的分子克隆。Ｓ１基因的ＲＦＬＰ分析表明我国ＩＢＶ已有分子水平的变异。

S1 genes of six isolates(designated as HD,HN,HZ,HB,DB and XB,respectively)of Avian Infectious Bronchitis Virus(IBV)were amplified by Reverse Transcription Polymerase Chain Reaction(RT PCR)and proved to be IBV S1 genes by the analysis of HaeIII Restriction Fragment Length Polymophism(RflP).S1 gene PCR products of the six strains were inserted into BamHI/HindIII site of pUC18 to construct recombinants and the cloned DNA fragments were identified as IBV S1 genes by Southern blot.Results demonstrated that the whole S1 genes of IBV isolates could be amplified by RT PCR.The S1 genes of HD and XBA strains had the same HaeIII RFLP type as H52 and M41 standard strains with 0 9kb,0.5kb and 0.3kb fragments,HN and HB strains had the same HaeIII RFLP type as Australia T strain with another 0.65kb,0.55kb,0.35kb and 0.3kb and 0.2kb fragments,whereas HZ and DB strains had variant RfLP type with 0.55kb,0.5kb,0.35kb,and 0.3kb fragments,which showed that there were molecular variation on the S1 genes of IBV isolates in China.