常规PCR法扩增口蹄疫病毒基因组5’端序列的探索  

Exploration of Foot-and-Mouth Disease Virus Genome's 5'-end Sequences Amplification by Conventional PCR Method

作　　者：朱研 苗书魁 马文戈 李金娜[1] 魏玉荣 汪萍 魏婕 米晓云 黄炯 ZHU Yan;MIAO Shu-kui;MA Wen-ge;LI Jin-na;WEI Yu-rong;WANG Ping;WEI Jie;MI Xiao-yun;HUANG Jiong(College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,China;Institute of Veterinary Medicine/Research Center of Animal Clinical medicine,Xinjiang Academy of Animal Sciences,Urumqi 830011,China)

机构地区：[1]新疆农业大学动物医学学院,乌鲁木齐830052 [2]新疆畜牧科学院兽医研究所/新疆畜牧科学院动物临床医学研究中心,乌鲁木齐830011

出　　处：《新疆农业科学》2018年第3期572-580,共9页Xinjiang Agricultural Sciences

基　　金："十二五"国家科技支撑计划项目"野生动物跨境传播外来动物疫病监测与控制技术研究"(2013BAD12B04);新疆维吾尔自治区科研机构创新发展专项资金项目"口蹄疫疫苗O型种毒质量优化"(2016D4008)~~

摘　　要：【目的】扩增口蹄疫病毒(foot-and-mouth disease virus,FMDV)基因组5’端序列,为获得基因组全长准备基础。【方法】将FMDV O/Akesu/58 CE39A株液氮冻存基因组RNA反转录三次,对c DNA的5’端序列进行扩增、克隆、测序。【结果】以c DNA1与c DNA3为模板,用LA Taq DNA polymerase、EX Taq DNA polymerase、Prime STARHS DNA polymerase及3对引物Ⅰ、Ⅱ、Ⅲ均未扩增出5’端目的片段;以c DNA2为模板,用LA Taq DNA polymerase为扩增酶,两对引物Ⅰ、Ⅱ均能扩增出5’端目的片段,且所测得的序列长度787 bp、797 bp与参考序列核苷酸同源性分别为86.2%和86.3%。【结论】常规PCR法扩增5’端序列较其它技术具有价格便宜、操作简便等优点,但扩增成功率偏低。试验共进行了72次PCR,成功率为6/72,说明FMDV 5’端序列的扩增难度大,应合理设计反转录引物和扩增引物。为许多RNA病毒反向遗传学研究提供了更多的选择。【Objective】To amplify the 5'-end sequences of the FMDV genome.【Method】RNA frozen in liquid nitrogen of FMDV O/Akesu/58 CE39A strain was reverse-transcribed to cDNA,which was amplified,cloned and sequenced for the 5'terminal sequences.【Result】With the cDNA1 and cDNA3 as templates,LA Taq DNA polymerase,EX Taq DNA polymerase,PrimeSTAR HS DNA polymerase,and three pairs of primersⅠ,ⅡandⅢwere used to amplify the 5'end fragment,but failed.Only when LA Taq DNA polymerase were used as the amplification enzyme,the cDNA2 as the template,and two pairs of primersⅠ,Ⅱthe expected fragment of the FMDV 5'terminal fragment could be amplified.And the tested sequences length were 787 bp and 797 bp,homology analysis of nucleotide sequences were 86.2%and 86.3%between the determined and reference sequences.【Conclusion】Compared with other methods,the conventioanl PCR method possesses the advantages of low cost and simple operation in spite of its low efficiency.In this experiment,72 PCR was carried out,and the success rate was 6/72,which indicated that the amplification of FMDV 5'terminal sequences was difficult,and the inversion of the primers and amplification primers should be rationally designed.The experiment has provided an alternative for the research of reverse genetics of a lot of RNA viruses.

关 键 词：口蹄疫病毒 常规PCR 5’端序列 基因扩增 

分 类 号：S855[农业科学—临床兽医学]