【作者】 张明；

【导师】 文心田； 曹三杰；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2009， 硕士

【摘要】 猪传染性胸膜肺炎（PCP）是由胸膜肺炎放线杆菌（APP）引起的高度接触性呼吸道疾病,每年给我国的畜牧产业带来巨大的经济损失。本试验设计用APP血清3型菌S1421株为材料,用氨苄青霉素抗性基因替换其毒力蛋白的激活基因ApxⅢC,对猪传染性胸膜肺炎放线杆菌ApxⅢC基因缺失重组转移载体的构建进行了研究,内容如下:1、APP基因缺失apxⅢC^-/Amp^r+重组转移载体的左、右同源臂及氨苄抗性基因的克隆与鉴定根据NCBI发表的APP血清3型的全基因组序列,参照ApxⅢC上游和下游的部分基因,分别设计引物扩增1553 bp和1537 bp的序列作为重组转移载体的左右同源臂。将左同源臂（包含部分C基因）PCR扩增后直接TA克隆到pBS-TⅡ上;筛选获得的正向克隆命名为pBSLⅡ。将右同源臂TA克隆到pMP-19 simple载体上,命名为pMD-Ra。左、右同源臂经测序后与NCBI上公布的序列同源性均达到了99.99%。根据pMD-19 simple载体的全基因序列,设计了一对引物扩增氨苄抗性基因,大小为861 bp。将其TA克隆到pMD-19 simple载体上,鉴定正确后命名为pMD-Amp。用HindⅢ和XbaⅠ分别双酶切卡那抗性的pVAX1质粒载体和pBSL-Amp,前者回收大片段后者回收小片段,将此二片段定向连接后转化入大肠杆菌中,复壮后涂布于含有氨苄青霉素和卡那霉素的LB/X-gal平板中,挑选白斑抽取质粒进行酶切鉴定。HindⅢ和XbaⅠ双酶切后得到一条3000 bp和近2400 bp的条带。用引物Amp（U）和Amp（L）扩增氨苄抗性基因,得到一条近900 bp的条带,与预期结果相符。证明氨苄抗性基因可用ApxⅢC基因的启动子启动表达。氨苄抗性基因经测序与宝生物公布的序列同源性达到了99.9%。2、APP基因缺失apxⅢC^-/Amp^r+重组转移载体的构建用BamHⅠ和XbaⅠ双酶切pBSLⅡ和pMD-Amp,分别胶回收大片段和小片段;连接后得到中间载体pBSL-Amp。用XbaⅠ和SacⅡ双酶切载体pBSL-Amp和pMD-Ra,分别回收大片段和小片段;连接后得到重组转移载体pBSL-Amp-R。载体经酶切和PCR鉴定正确。更多还原

【Abstract】 Porcine contagious pleuropneumonia（PCP） induced by Actinobacillus pleuropneumoniae（APP）,which is a highly contagious and often fatal disease of swine.It is currently considered to be one of the most economically important diseases of swine in our country.In this research,we got Actinobacillus pleuropneumoniae serovar 3 strain S1421 as material,the apxⅢC gene of the bacterium would be replaced by ampicillin resistance gene so that ApxⅢcould not be activated.In this research,we constructed a gene deleted vector of Actinobacillus Pleuropneumoniae as pBSL-Amp-R.The paper as follows.1,Clone and identification of left,right homologization brach and ampieillin resistance gene of APP gene deleted vector pBSL-Amp-RBy the complete genome sequence of APP serovar 3,two pairs of primers were designed and synthesized of apxⅢoperon in Actinobacillus pleuropneumoniae serovar3（APP-3） to amplify 1,553 bp in the 5’ region of apxⅢC gene as left homologization brach and 1,537 bp in the 3’ region of apxⅢC gene as right homologization brach.Left homologization brach was cloned into pBS-TⅡto generate plasmid in which the norientation plasmid was called pBSLⅡ.Right homologization brach was cloned into pMD19-T Simple to generate plasmid pMD19-RA.The homology of left and right homologization brach got 99.99%aligned by the sequence announced by NCBI.One pair of primers was designed and synthesized on published sequence of pMD-19-Simple vector to amplify ampicillin resistance gene.Ampicillin resistance gene was cloned into pMD19-T-Simple to generate plasmid pMD19-Amp.The vector pVAX1 which got the Kan resistance and the vector pBSL-Amp were digested by HindⅢand XbaⅠ,and generate a plasmid.Transforme the plasmid into the E.coli to identify.We found it got the ampicillin resistance.Pick up the white bot colony and drew the plasmid,digested by HindⅢand XbaⅠ,we got two fragment as 3000bp and 2400bp,the plasmid was PCR amplification by the primer of amp（u） and amp（l）,we got the fragment nearly 900bp.The ampicillin resistance gene would promoted by the operon of ApxⅢC.The homology of ampicillin resistance gene got 99.9%aligned by the sequence announced.2,Construction of the Gene DeLeted ApxⅢC^-/Amp^r+ Vector of Actinobacillus PleuropneumoniaeThe ampicillin resistance gene generated from digestion of pMD 19-Amp with BamHⅠand XbaⅠwas cloned into pBSLⅡbetween enzyme sites of BamHⅠand XbaⅠto generate interim plamid pBSL-Amp.Right homologization brach generated from digestion of pMD 19-R with XbaⅠand SacⅡwas cloned into pBSL-Amp between enzyme sites of XbaⅠand SacⅡto generate recombinant transfer plamid pBSL-Amp-RA.Enzyme digestion indicated the vector pBSL-Amp-RA was successfully constructed.更多还原