绵羊肺炎支原体基因组DNA表达文库的构建与鉴定  

Construction and Identification of Genomic DNA Expressions Library of Mycoplasma ovipneumoniae

作　　者：陈诚[1] 乔军[1] 孟庆玲[1] 刘田莉 胡政香 马玉[1] 才学鹏[2] 陈创夫[1] 

机构地区：[1]石河子大学动物科技学院,新疆石河子832003 [2]中国农业科学院兰州兽医研究所,兰州730046

出　　处：《西北农业学报》2016年第5期646-651,共6页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：国家国际科技合作专项(2014DFR31310);兵团国际科技合作计划(2012BC006)~~

摘　　要：为了筛选绵羊肺炎支原体（Mycoplasma ovipneumoniae,MO）免疫原性相关基因,提取MO基因组DNA,利用限制性内切酶Sau3AⅠ对基因组DNA进行酶切,回收500-3 000bp的基因组DNA片段,用T4DNA连接酶将其与经BamHⅠ酶切并去磷酸化处理的pET28a/b/c载体连接,然后转化至DH5α感受态细胞,通过菌液PCR对插入到载体中的片段大小及插入率进行鉴定。从转化的平板上提取质粒,转化至大肠杆菌BL21（DE3）感受态细胞中,构建MO基因组表达文库。随机挑选单菌落,用pET28a/b/c通用引物进行PCR鉴定,结果显示插入片段大小为300-2 000bp,插入率为90%,表明成功构建了MO基因组表达文库,该文库的构建为进一步筛选MO相关的免疫性基因及潜在候选疫苗靶点奠定前期基础。In order to screen some of the protective antigen genes for Mycoplasma ovipneumoniae （MO） ,the genomic DNA was extracted and partially digested with Sau3A I. The DNA fragments of 500--3 000 bp were purified and ligated into pET28a/b/c expression vectors,which were digested with BamH I and treated with alkaline phosphatase. The ligated products were transformed into competent E. coli DHSa,then the frequency and size of the inserts were analyzed by colony PCR using vector-specific primers. All the colonies on the plates were collected and recombinant plasmid DNA was recovered and further transformed into E. coli BL21 （DE3）. Finally, high quality genomic DNA expression library of Mycoplasma ovipneumoniae was constructed successfully,and 90% DNA inserts were from 300 to 2 000 bp. This study indicated that the construction of genomic DNA expression library of Mycoplasma ovipneumoniae will be beneficial for screening some of the protective antigen genes and laying foundation for development of potential vaccine candidates against Mycoplasma ovipneumoniae.

关 键 词：MO 基因组表达文库 构建 鉴定 

分 类 号：S852.62[农业科学—基础兽医学]