禽传染性支气管炎病毒免疫原基因的PCR获取及其酶切鉴定

THE ATTAINMENT OF IBV IMMUNOGEN GENE (S1) BY PCR METHOD AND ITS CHARACTERIZATION BY RESTRICTION ENZYME DIGESTION ANALYSIS

介绍用ＲＴ－ＰＣＲ（反转录－聚合酶链反应）获取禽传染性支气管炎病毒（ＩＢＶ）Ｍ４１株和广东地方致弱株Ｄ４１免疫原（Ｓ１）基因的详细方法，所用引物为ＩＢＶＢｅａｕｄｅｔｅ株Ｓ１基因两侧的对应序列，跨幅为１．７ｋｂ。结果表明，两株病毒所获的ＰＣＲ产物与预期的一致；用此对引物，以ＩＢＶＤ４１株Ｓ１基因ＰＣＲ产物为模板，扩增出一样的ＤＮＡ片段。试验还显示，国内外几家公司有关ＲＴ和ＰＣＲ的分子生物学试剂可以兼用，适用于国内的有关研究；ＰＣＲ仪则以进口的或水浴锅式的为佳。对ＩＢＶＭ４１株Ｓ１基因的ＲＴ－ＰＣＲ产物进行ＨａｅⅢ酶切图谱分析。

Desribes in detail the methods used to obtain the immunogen (S1) gene of IBV strain M41 and an attenuated local Guangdong isolate D41 by the PCR technique. Two synthetic primers were designed corresponding to segments on both flanks of the S1 gene of IBV strain Beaudette, a 1700-base sequence. The result demonstrates that the PCR products of the two IBV strains were the same as expected. Using this pair of primers, the S1 gene used the PCR product of IBV strain D41 as PCR template was recovered. It was also found that the chemicals used for RT and PCR of several companies matched well,and it was better to use the imported PCR machine (PE) and the mechanical arm PCR machine using water bath as heating system too. The HaeⅢ restriction pattern of IBV M41 S1 gene was the same as reported in the literature.