奶牛乳房炎链球菌的分离鉴定及抗原基因的克隆和表达
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摘要
奶牛乳房炎是由各种病因引起的乳房炎症,它造成的经济损失非常巨大。尽管各国兽医工作者多年来对该病的预防与治疗研究付出了极大的努力,但仍无满意的结果,至今仍以抗生素治疗为主。由于抗生素残留和耐药菌株的出现造成奶品质下降和治疗困难,所以迫切需要研究新的预防与治疗方法。病原学调查表明,无乳链球菌、停乳链球菌和乳房链球菌是奶牛乳房炎的常见病原菌,但有关疫苗却少见报道且效果不理想,部分原因是因为链球菌血清型多缺乏共同保守性抗原。近几年,链球菌的一些保守性抗原被陆续发现,分别是停乳链球菌的MIG基因,无乳链球菌的SIP基因和无乳链球菌、停乳链球菌和乳房链球菌的GAPC基因,并认为它们可作为基因工程疫苗的候选抗原基因。本文克隆并表达了这些保守性抗原基因,为开发链球菌基因工程疫苗奠定基础。
方法:本实验首先运用THB(Todd-Hewitt Broth)固体选择性培养基和色素试验培养基快速分离无乳链球菌和乳房链球菌,并结合生物梅里埃开发的VITEK全自动细菌鉴定仪进行准确鉴定。然后用PCR的方法分别从停乳链球菌、无乳链球菌和乳房链球菌的基因组DNA中扩增出MIG、SIP和各自的GAPC基因,并构建原核表达载体pET32a(+)-MIG、pET32a(+)-SIP和各自的pET32a(+)-GAPC。用BL21(DE3)/pET系统表达Trix-MIG、Trix-SIP和各自的Trix-GAPC融合蛋白,SDS-PAGE和Western blot分析鉴定表达产物。结果:(1)分离鉴定到一株无乳链球菌和一株乳房链球菌。(2)PCR扩增产物经测序,证实与GenBank中MIG基因(AF354651)、和SIP基困(DQ650634)和无乳链球菌(CP000114)/停乳链球菌(AF375662)/乳房链球菌(AF421900)的GAPC基因序列同源性都在99%左右。SDS-PAGE显示,MIG、SIP和GAPC融合蛋白分别为89kDa、71kDa和55kDa。Western blot分析显示,MIG、SIP和GAPC融合蛋白可分别与停乳链球菌、无乳链球菌和乳房链球菌的多克隆抗血清发生特异性反应。结论:成功分离到一株无乳链球菌和一株乳房链球菌并克隆和表达了停乳链球菌MIG基因、无乳链球菌SIP基因和无乳链球菌/停乳链球菌/乳房链球菌GAPC基因,为基因工程疫苗的研制奠定一定的基础。
Bovine mastitis,an inflammation of the mammary gland,is the most important factor contributing to economic losses in the dairy industry.Although researchers work hard all over the world,there is no good method to control it.The most control method is using antibiotics.It is necessary to create new method to control mastitis,because antibiotics not only have side-effect, also it can decrease milk quality.Certainly,vaccine prevention is the best way for controlling. Pathogenic survey showed that several streptococcal species are capable of causing infections that result in mastitis,including Streptococcus agalactiae,Streptococcus dysgalactiae,and Streptococcus uberis.Vaccines developed to control this disease showed limited protection due in part to the lack of common conserved antigens.In recent years,researchers have found that MIG gene was conserved in all S dysgalactiae strains,SIP gene was conserved in all S dysgalactiae strains,GAPC gene were conserved in all S.agalactiae/S.dysgalactiae/S.uberis strains and believed that these genes were ideal vaccine candidates We cloned these genes,and expressed them in E.coli.They laid a foundation for the further development of Streptococcus genetic engineering vaccines.
Objective:Isolation,identification of the S.agalactiae,S.uberis and cloning,expression of the antigen genes.They laid a foundation for the genetic engineering vaccines.Methods: 1.Use THB Selective solid medium,Islam medium,Vitek 32 microbial analyzing system to Isolation and identification the S.agalactiae and S.uberis strains.2.Amplify MIG gene from the genomic DNA of S.dysgalactiae,SIP gene from the genomic DNA of S.agalactia and GAPC gene from the genomic DNA of S.agalactiae/S.dysgalactiae/S.uberis by PCR and construct the recombinant plasmids pET32a(+)-MIG,pET32a(+)-SIP,and pET-32a(+)-GAPCs.Transform the constructed recombinant plasmids into pET system for expression of Trix-MIG/Trix-SIP / Trix=GAPCs fusion protein.The expressed product were identified by SDS-PAGE and Western blot.Results:The homology of amplified MIG,SIP and GAPC gene sequence to that in GenBank all were 99%.SDS-PAGE showed 89kDa,71kDa and 55kDa.The activity of recombinant protein was analyzed by Western blot.Conclusion:Isolation and identification a S.agalactiae strain and a S.uberis strain.MIG,SIP and GAPCs fusion protein were successful expressed.They laid a foundation for the genetic engineering vaccines.
方法:本实验首先运用THB(Todd-Hewitt Broth)固体选择性培养基和色素试验培养基快速分离无乳链球菌和乳房链球菌,并结合生物梅里埃开发的VITEK全自动细菌鉴定仪进行准确鉴定。然后用PCR的方法分别从停乳链球菌、无乳链球菌和乳房链球菌的基因组DNA中扩增出MIG、SIP和各自的GAPC基因,并构建原核表达载体pET32a(+)-MIG、pET32a(+)-SIP和各自的pET32a(+)-GAPC。用BL21(DE3)/pET系统表达Trix-MIG、Trix-SIP和各自的Trix-GAPC融合蛋白,SDS-PAGE和Western blot分析鉴定表达产物。结果:(1)分离鉴定到一株无乳链球菌和一株乳房链球菌。(2)PCR扩增产物经测序,证实与GenBank中MIG基因(AF354651)、和SIP基困(DQ650634)和无乳链球菌(CP000114)/停乳链球菌(AF375662)/乳房链球菌(AF421900)的GAPC基因序列同源性都在99%左右。SDS-PAGE显示,MIG、SIP和GAPC融合蛋白分别为89kDa、71kDa和55kDa。Western blot分析显示,MIG、SIP和GAPC融合蛋白可分别与停乳链球菌、无乳链球菌和乳房链球菌的多克隆抗血清发生特异性反应。结论:成功分离到一株无乳链球菌和一株乳房链球菌并克隆和表达了停乳链球菌MIG基因、无乳链球菌SIP基因和无乳链球菌/停乳链球菌/乳房链球菌GAPC基因,为基因工程疫苗的研制奠定一定的基础。
Bovine mastitis,an inflammation of the mammary gland,is the most important factor contributing to economic losses in the dairy industry.Although researchers work hard all over the world,there is no good method to control it.The most control method is using antibiotics.It is necessary to create new method to control mastitis,because antibiotics not only have side-effect, also it can decrease milk quality.Certainly,vaccine prevention is the best way for controlling. Pathogenic survey showed that several streptococcal species are capable of causing infections that result in mastitis,including Streptococcus agalactiae,Streptococcus dysgalactiae,and Streptococcus uberis.Vaccines developed to control this disease showed limited protection due in part to the lack of common conserved antigens.In recent years,researchers have found that MIG gene was conserved in all S dysgalactiae strains,SIP gene was conserved in all S dysgalactiae strains,GAPC gene were conserved in all S.agalactiae/S.dysgalactiae/S.uberis strains and believed that these genes were ideal vaccine candidates We cloned these genes,and expressed them in E.coli.They laid a foundation for the further development of Streptococcus genetic engineering vaccines.
Objective:Isolation,identification of the S.agalactiae,S.uberis and cloning,expression of the antigen genes.They laid a foundation for the genetic engineering vaccines.Methods: 1.Use THB Selective solid medium,Islam medium,Vitek 32 microbial analyzing system to Isolation and identification the S.agalactiae and S.uberis strains.2.Amplify MIG gene from the genomic DNA of S.dysgalactiae,SIP gene from the genomic DNA of S.agalactia and GAPC gene from the genomic DNA of S.agalactiae/S.dysgalactiae/S.uberis by PCR and construct the recombinant plasmids pET32a(+)-MIG,pET32a(+)-SIP,and pET-32a(+)-GAPCs.Transform the constructed recombinant plasmids into pET system for expression of Trix-MIG/Trix-SIP / Trix=GAPCs fusion protein.The expressed product were identified by SDS-PAGE and Western blot.Results:The homology of amplified MIG,SIP and GAPC gene sequence to that in GenBank all were 99%.SDS-PAGE showed 89kDa,71kDa and 55kDa.The activity of recombinant protein was analyzed by Western blot.Conclusion:Isolation and identification a S.agalactiae strain and a S.uberis strain.MIG,SIP and GAPCs fusion protein were successful expressed.They laid a foundation for the genetic engineering vaccines.
引文
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