禽传染性支气管炎病毒纤突蛋白S_1基因的RFLP与序列比较分析

Sequence Comparision and RFLP Analysis of S_1 Gene of Avian Infectious Bronchitis Viruses

利用反转录一套式聚合酶链式反应技术从北京地区禽传染性支气管炎病毒(IBV)分离株 BJ_1、BJ_2、BJ_3株中成功扩增出1054bp 纤突蛋白高变区。利用限制性内切酶 Sin Ⅰ和 Pst Ⅰ的酶切分析证实了RT-nested PCR 的特异性,结合计算机酶切位点分析图谱进行理论 RFLP 分析。将三段 S_1基因分别克隆到pUC19质粒中,获得重组质粒 pUC IBV S_1-BJ_1、pUC IBV S_1-BJ_2、pUC IBV S_1-BJ_3。利用双脱氧链终止法对三个重组质粒进行双向测序,获得三株分离株 S_1基因高变区的序列。将得到的三个序列与标准株 M_(41),Beaudette 株和疫苗株 H_(120)及广东地方分离株 D_(41)株进行核酸序列同源性比较分析。结果表明,三株分离株核酸碱基序列和氨基酸序列与 M_(41)株的同源性比与 H_(120)株和 Beandette 株的同源性高。本研究对于进一步深入探讨禽传染性支气管炎病毒(IBV)分子变异机制研究奠定了良好的基础。

Using Reverse Transcription-nested Polymerase Chain Reaction,the S_1 gene hypervariable section of Avian Infectious Bronchitis Viruses(IBV)strains BJ_1,BJ_2,BJ_3, which were isolated from Beijing areas,were amplified sucessfully.The segment section was consisted of 1 054 bp of 5'-end of Sl gene and was regard as the most variable region among IBV strains.Digestion with SinI,PstI and map of restriction enzymes sites proved that correction and specificity of RT-nested PCR products.Sequence of the Sl gene were obtained from recombinant plasmids pUCIBVS_1-BJ_1,pUCIBVS_1-BJ_2,pUCIBVS_1-BJ_3 by using the dideoxy sequencing technique in two directions.Comparing three isolates with standard strains M_(41),Beaudette,vaccine strain H_(120) and one isolated strain D_(41)from Guangdon province,it was shown that they were more homology in nucleotide sequence with M_(41)than Beaudette and H_(120).The study may be helpful in further researching the molecular variant mechanism of Infectious Bronchitis virus.