噬菌体宿主谱改变核酸分析研究  

Extracting and comparing analysis the nucleic acid from E.coli bacteriophage with broad host range

作　　者：彭道荣[1] 徐焰[1] 熊鸿燕[2] 张小宁[1] 苏明权[1] 孙怡群[1] 郝晓柯[1] 

机构地区：[1]解放军第四军医大学西京医院检验科 [2]第三军医大学军队流行病学教研室

出　　处：《中华检验医学杂志》2005年第8期847-849,共3页Chinese Journal of Laboratory Medicine

摘　　要：目的探讨一种高效提取RNA噬菌体核酸的新方法,并通过核酸分析从基因水平了解宿主谱改变对噬菌体核酸的影响。方法采用传统的聚乙二醇(PEG)沉淀蛋白酶消化酸性酚提取法、Tripure提取法以及抗体沉淀盐酸胍一步法3种方法分别提取大肠埃希菌f2噬菌体及其宽宿主谱株的RNA,通过琼脂糖凝胶电泳鉴定其纯度。采用兼并引物逆转录聚合酶链反应(RTPCR)及随机引物随机扩增多态性DNA聚合酶链反应(RAPDPCR)比较分析噬菌体宿主谱改变前后其核酸序列组成的变化。结果抗体沉淀盐酸胍一步法提取的噬菌体核酸具纯度高、条带完整等优点;f2噬菌体及其宽噬株均为6000bp左右的单链RNA噬菌体;两噬菌体基因cDNA扩增出的RAPDDNA片段差异明显,其中26条为可区分的DNA带型;噬菌体f2cDNA在450bp附近出现重复性较好的扩增片段,而其宽噬株cDNA在相同条件下未出现扩增产物。结论抗体沉淀盐酸胍一步法较其他两种RNA病毒核酸提取法具有明显的优势,具有应用价值的新的RNA噬菌体核酸的提取方法;宽宿主谱噬菌体的宿主特异性裂解效应已从基因水平发生了变化。Objective To establish a new assay for extracting nucleic acid from RNA phage. Through analysis test for Nucleic acid investigate the gene change of bacteriophage with broad host range, to study the mechanism of phage host specificity and anti-bacteria effects. Methods Comparing the three kinds of assay to extract the nucleic acid from phage f2 and phage f2 with broad host range by PEG-Protease k- Phenol, Tripure and anti-serum-carbamidine hydrochloride respectively. Then valuated their purity with agarose gel electrophoresis. Comparing analysis the differences of nucleic acid sequence between phage f2 and phage f2 with broad host range with RT-PCR and RAPD-PCR. Rusults The anti-serum-carbamidine hydrochloride-one-step assay is the best one than others. Analysis test for Nucleic acid revealed that two phage were a 6 000 bp , single-stranded RNA bacteriophage. Significant differences in its specificity of RAPD-PCR and RT-PCR were founded before and after host range change; There were 26 RAPD-cDNA fragments that can be difference in two phage RAPD-PCR product; The RT-PCR product of phage f2 was 450 bp cDNA fragment, but the phage with broad host range hadn＇ t PCR product. Conclusion The anti-serum-carbamidine hydrochloride-one-step assay is one kind of new assay who has advantage obviously than other tradition assay to extract the nucleic acid from RNA phage. Analysis test for Nucleic acid indicated that host-specific lyric effect of phage xy-1 had been changed in genic level.

关 键 词：RNA 噬菌体 逆转录聚合酶链反应 随机扩增多态DNA技术 F2噬菌体 核酸分析 宿主谱 多态性DNA聚合酶链反应 分析研究 RNA病毒 

分 类 号：R516[医药卫生—内科学]