摘要
根据已发表的猪传染性胸膜肺炎放线杆菌(APP)血清5型核苷酸序列设计并合成了一对引物,从自行分离的APP菌株PCR扩增apxⅡCA基因,将该片段与pBluescriptSK(+)载体连接,得到质粒pSK CA。另外,从质粒pIC neo中得到的Ampr基因经XbaⅠ酶切消化后,克隆到pSK CA中的XbaⅠ位点,获得了重组转移质粒pSK CAmpr。pSK CAmprDNA通过电转化导入亲本菌,电转化后的产物涂布于TM平板,可获得突变株。提取突变株和亲本菌基因组DNA,用PCR检测含有apxⅡC基因的胸膜肺炎放线杆菌染色体区域。从突变株基因组DNA扩增得到的一条PCRDNA片段比从亲本菌基因组DNA扩增得到的一条DNA片段大1 3kb,增大的片段与所插入的Ampr基因大小相符合。用合成的Ampr基因引物从突变株基因组DNA中也能扩增到一条1 3kb的特异性DNA片段。同时South ernblot鉴定有特异性带。这表明我们所构建的突变株是成功的。测定了突变株的遗传稳定性及突变株对动物的安全性,为进一步研究用其它报告基因置换Ampr基因后,研制单基因工程疫苗及以APP毒力缺失突变菌株为载体,表达外源基因的APP基因工程菌的研究奠定了坚实的基础。
The primers were designed and synthesized on published porcine Actinobacillus pleuropneumoniae (APP) serovar 5 nueclea sequence to amplify apxⅡCA gene of isolated APP strains(strain WH).The amplified 3.4 kb DNA fragment was cloned into pBluescript SK(+) to generate plasmid pSK- CA.In addition,the Amp^r gene from plasmid of pⅠC- neo was digested by XbaⅠ and cloned into the unique XbaⅠ site of pSK- CA to generate recombinant transfer plasmid pSK- CAmp^r.The linearized plasmid pSK- CAmp^r DNA was electroporated into A.pleuropneumoniae parent strain (strain Wh,serovar 7).Products of the electroporation were plated onto TM agar containing ampicillin(Amp^r).After 2~3 days,the recombinant strains were selected.Genomic DNA was extracted from HBC- Amp^r mutants and the parent strain.PCR was used to confirm the region of the A.pleuropneumoniae chromosome containing the apxⅠC gene.The PCR product obtained by using WHC- Amp^r genomic DNA (1.75 kb) was approximately 1.25 kb larger in size than that obtained with the parent(0.5 kb).This increased in size corresponded to the size of the Amp^r gene.The genetic stability of mutant strains was measured.A 1.25 kb DNA fragment was also amplified from genomic DNA of mutant strains by the inserted Amp^r gene primers.Southern Blotting was used to prove mutant strains,indicating that successful construction of mutant strains.In addition,the stability and the safety of mutant strains to animals was also detected,which provide a strong basis for further researching the vector and genetic engineering vaccine.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2004年第2期186-191,共6页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
湖北省科技攻关项目 (2 0 0 1AA2 0 1B0 2 )
国家自然科学基金资助 (3 0 2 0 0 0 1 1 )
