表达Omp31基因重组牛布鲁菌的构建及鉴定
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摘要
为提高布鲁菌疫苗株的免疫保护效果,本试验以牛布鲁菌疫苗株S19为研究对象,将羊布鲁菌Omp31基因克隆、扩增并插入至广宿主质粒pBBR1MCS-2中,构建重组质粒pBBR1MCS-Omp31;通过电转化的方式转入S19感受态细胞中,经抗性基因筛选和PCR验证,获得重组牛布鲁菌S19-Omp31株;该重组菌株连续传25代未发现重组质粒和Omp31基因丢失,表明其遗传稳定性良好;进一步经SDS-PAGE检测可见约26 000相对分子质量的目的条带,采用Western blot法检测显示目的蛋白可与His单克隆抗体及Omp31蛋白高免血清反应。本研究构建的重组牛布鲁菌S19-Omp31株能够稳定表达目的基因,为进一步开展S19-Omp31株的免疫效果评价奠定基础。
In order to improve the immune protective efficacy of Brucella vaccine strain,Brucella abortus S19 strain was designed as the object.The outer membrane protein Omp31 gene of Brucella melitensis 16 Mstrain was cloned,amplified and inserted into the broad-host-range plasmid pBBR1 MCS-2,and recombinant pBBR1 MCS-Omp31 was constructed and electro-transformed into Brucella abortus S19 competent cells.Under resistance gene selection and PCR identification,recombinant Brucella abortus S19-Omp31 strain was obtained,the loss of recombinant plasmid and Omp31 gene was not found in 25 th consecutive generations,indicating that the recombinant strain had good genetic stability.SDS-PAGE analysis showed that the target band of about 26 000,and Western blot analysis showed that the target protein could react to His-tag monoclonal antibody and Omp31 protein hyperimmune serum.The recombinant Brucella abortus S19-Omp31 strain constructed in this study could stably express the Omp31 gene,laid the foundation for the immune effect evaluation of recombinant S19-Omp31 strain.
In order to improve the immune protective efficacy of Brucella vaccine strain,Brucella abortus S19 strain was designed as the object.The outer membrane protein Omp31 gene of Brucella melitensis 16 Mstrain was cloned,amplified and inserted into the broad-host-range plasmid pBBR1 MCS-2,and recombinant pBBR1 MCS-Omp31 was constructed and electro-transformed into Brucella abortus S19 competent cells.Under resistance gene selection and PCR identification,recombinant Brucella abortus S19-Omp31 strain was obtained,the loss of recombinant plasmid and Omp31 gene was not found in 25 th consecutive generations,indicating that the recombinant strain had good genetic stability.SDS-PAGE analysis showed that the target band of about 26 000,and Western blot analysis showed that the target protein could react to His-tag monoclonal antibody and Omp31 protein hyperimmune serum.The recombinant Brucella abortus S19-Omp31 strain constructed in this study could stably express the Omp31 gene,laid the foundation for the immune effect evaluation of recombinant S19-Omp31 strain.
引文
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[12]吴同垒,王真,单晓枫,等.羊种布鲁菌效应蛋白的筛选[J].中国兽医学报,2018,38(5):924-936.
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[15]段开放,邓雨明,崔桂梅,等.布鲁菌DnaK蛋白抑制宿主细胞凋亡[J].中国兽医学报,2018,38(5):528-531.
[2]郑雪琴.国家布鲁菌病防治计划(2016-2020年)[J].北方牧业,2016,41(19):4-8.
[3]OLSEN S,TATUM F,KAPIL S,et al.Bovine brucellosis[J].Vet Clin North Am Food Anim Pract,2010,26(1):15-27.
[4]董炳梅,王金良,唐娜,等.布鲁菌的致病机制与细胞免疫机制研究进展[J].中国人兽共患病学报,2012,28(6):635-639.
[5]杨艳玲,盛雪玲,唐婕,等.羊布鲁菌强毒株16M与疫苗株M5外膜蛋白蛋白质组学研究[J].中国兽医学报,2010,30(10):1334-1342.
[6]COMERCI D J,POLLEVICK G D,VIGLIOCCO AM,et al.Vector development for the expression of foreign proteins in the vaccine strain Brucella abortus S19[J].Infect Immun,1998,66(8):3862.
[7]JULIA V,SABIO Y G,BIGI F,et al.Expression of MPB83from Mycobacterium bovis in Brucella abortus S19induces specific cellular immune response against the recombinant antigen in BALB/c mice[J].Microb Infect,2010,12(14):1236-1243.
[8]ESTEIN S M,CHEVES P C,FIORENTINO M A,et al.Immunogenicity of recombinantOmp31from Brucella melitensis in rams and serum bactericidal activity against B.ovis[J].Vet Microbiol,2004,102(3):203-213.
[9]ESTEIN S M,CASSATARO J,VIZCA NO N,et al.The recombinant Omp31fromBrucella melitensis alone or associated with rough lipopolysaccharide induces protection against Brucella ovis infection in BALB/c mice[J].Microb Infect,2003,5(2):85-93.
[10]CASSATARO J,VELIKOVSKY C A,BRUNO L,et al.Improved immunogenicity of a vaccination regimen combining a DNA vaccine encoding Brucella melitensis outer membrane protein 31(Omp31)and recombinant Omp31 boosting[J].Clin Vaccine Immunol Cvi,2007,14(7):869.
[11]JACQUES I,VERGER J M,LAROUCAU K,et al.Immunological responses and protective efficacy against and Omp31 B.melitensis Rev.1deletion mutants in sheep[J].Vaccine,2007,25(5):794-805.
[12]吴同垒,王真,单晓枫,等.羊种布鲁菌效应蛋白的筛选[J].中国兽医学报,2018,38(5):924-936.
[13]李伍举,吴加金.pBV220载体中外源基因表达水平定量分析[J].病毒学报,1997(2):126-133.
[14]吴同垒,单晓枫,康元环,等.布鲁菌Ⅳ型分泌系统效应蛋白的研究进展[J].中国兽医学报,2018,38(3):602-608.
[15]段开放,邓雨明,崔桂梅,等.布鲁菌DnaK蛋白抑制宿主细胞凋亡[J].中国兽医学报,2018,38(5):528-531.