过表达UGPase基因和BCSP31基因的重组流产布氏杆菌的构建与鉴定
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摘要
为提高流产布氏杆菌S19疫苗株的免疫保护效果,分别扩增流产布氏杆菌UGPase基因和BCSP 31基因,将其插入含有双启动子pR/pL序列的广宿主穿梭质粒pBBR1MCS-PT中,电转至流产布氏杆菌S19感受态细胞并经抗性基因筛选,采用PCR法对重组菌株的遗传稳定性进行检测,利用蛋白分析手段对目的基因的表达情况进行分析。PCR检测结果显示,构建的重组流产布氏杆菌S19-UGPase株和S19-BCSP31株的遗传性状稳定。SDS-PAGE与Western-blot结果显示,在33和31 ku处可见明显条带,且与His标签抗体的反应位置一致,表明目的蛋白获得正确表达。结果表明,构建的过表达重组流产布氏杆菌S19-UGPase株和S19-BCSP31株能够稳定表达目的基因,为下一步开展S19-UGPase株和S19-BCSP31株免疫效果的评价奠定了基础。
In order to improve the immunogenicity of the Brucella abortus S19 strain,UGPase gene and BCSP31 gene of B.abortus S19 strain were amplified and inserted into broad-host-range plasmid pBBR1 MCS-PT which containing dual promoter pR/pL,respectively.Two recombinant plasmids were electro-transformed into B.abortus S19 competent cells,respectively,and recombinant strains were determined through resistance gene selection and their genetic stability was detected by PCR.The target gene expressions were analyzed by using SDS-PAGE and Western-blot.The PCR results showed that recombinant B.abortus S19-UGPase strain and S19-BCSP31 strain were constructed with stable genetic traits.SDS-PAGE and Western-blot results showed two target proteins of about 33 ku and 31 ku reacted with His-tag antibody respectively,indicating that these target proteins correctly expressed.In summary,these recombinant Brucella strains can stably express UGPase gene and BCSP31 gene,which laid the foundation for the immune effect evaluation of recombinant S19-UGPase strain and S19-BCSP31 strain.
In order to improve the immunogenicity of the Brucella abortus S19 strain,UGPase gene and BCSP31 gene of B.abortus S19 strain were amplified and inserted into broad-host-range plasmid pBBR1 MCS-PT which containing dual promoter pR/pL,respectively.Two recombinant plasmids were electro-transformed into B.abortus S19 competent cells,respectively,and recombinant strains were determined through resistance gene selection and their genetic stability was detected by PCR.The target gene expressions were analyzed by using SDS-PAGE and Western-blot.The PCR results showed that recombinant B.abortus S19-UGPase strain and S19-BCSP31 strain were constructed with stable genetic traits.SDS-PAGE and Western-blot results showed two target proteins of about 33 ku and 31 ku reacted with His-tag antibody respectively,indicating that these target proteins correctly expressed.In summary,these recombinant Brucella strains can stably express UGPase gene and BCSP31 gene,which laid the foundation for the immune effect evaluation of recombinant S19-UGPase strain and S19-BCSP31 strain.
引文
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[2] OLSEN S,TATUM F,KAPIL S,et al.Bovine brucellosis[J].Veterinary Clinics of North America Food Animal Practice,2010,26(1):15-27.
[3]丁家波,毛开荣,陈小云,等.8株布鲁氏菌BCSP31基因的序列分析[J].中国兽药杂志,2006,40(3):13-16.DING Jia-bo,MAO Kai-rong,CHEN Xiao-yun,et al.Sequence analysis on BCSP31 genes of 8 different Brucella species[J].Chinese Journal of Veterinary Drug,2006,40(3):13-16.(in Chinese)
[4] BOUNAADJA L,ALBERT D,CHENAIS B,et al.Real-time PCR for identification of Brucella spp.:A comparative study of IS711,bcsp31 and per target genes[J].Veterinary Microbiology,2009,137(1):156-164.
[5] ZHANG L,WU X A,ZHANG F L,et al.Soluble expression and purification of Brucella cell surface protein(BCSP31)of Brucella melitensis and preparation of anti-BCSP31 monoclonal antibodies[J].Molecular Biology Reports,2012,39(1):431-438.
[6] YANG Y,WANG L,YIN J,et al.Immunoproteomic analysis of Brucella melitensis and identification of a new immunogenic candidate protein for the development of brucellosis subunit vaccine[J].Molecular Immunology,2011,49(1-2):175-184.
[7] DECKER D,LINDBERG S,ERIKSSON J,et al.A luminescence-based assay of UDP-sugar producing pyrophosphorylases[J].Analytical Methods,2013,6(1):57-61.
[8]卜昭阳,杨艳玲,郎需龙,等.羊布鲁菌16M UGPase基因缺失株的构建及其特性分析[J].中国生物制品学杂志,2017,30(7):690-694.BU Zhao-yang,YANG Yan-ling,LANG Xu-long,et al.Construction and characteristics of UGPase gene-deleted mutant of Brucella melitensis 16M[J].Chinese Journal of Biologicals,2017,30(7):690-694.(in Chinese)
[9] YU D H,HU X D,CAI H.A combined DNA vaccine encoding BCSP31, SOD, and L7/L12 confers high protection against Brucella abortus 2308 by inducing specific CTL responses[J].DNA&Cell Biology,2007,26(6):435-443.
[10]潘文,王佳莹,赵明秋,等.布氏杆菌bp26基因缺失株的构建[J].中国兽医科学,2011,41(3):280-286.PAN Wen,WANG Jia-ying,ZHAO Ming-qiu,et al.Construction of unmarked bp26 gene-deleted strains of Brucella spp.[J].Chinese Veterinary Science,2011,41(3):280-286.(in Chinese)
[11]张敏,韩先干,田明星,等.流产布氏杆菌rfbD基因缺失株的构建及其生物学特性[J].中国兽医科学,2013,43(5):446-452.ZHANG Min,HAN Xian-gan,TIAN Ming-xing,et al.Construction and biological characterization of rfbD gene-deleted Brucella abortus mutant strainΔrfbD[J].Chinese Veterinary Science,2013,43(5):446-452.(in Chinese)
[12]刘翠翠,王佳莹,马思思,等.表达牛分枝杆菌esat-6和cfp-10基因的重组流产布氏杆菌S19疫苗株的构建及毒力评估[J].中国兽医科学,2015,45(4):395-401.LIU Cui-cui,WANG Jia-ying,MA Si-si,et al.Construction and assessment of virulence of Brucella abortus S19 mutant strains expressing esat-6and cfp-10 genes from Mycobacterium bovis[J].Chinese Veterinary Science,2015,45(4):395-401.(in Chinese)
[13] VEMULAPALLI R,YONGQUN H,CRAVERO S,et al.Overex pression of protective antigen as a novel approach to enhance vaccine efficacy of Brucella abortus strain RB51[J].Infection and Immunity,2000,68(6):3286.
[14] RAJASEKARAN P,SURENDRAN N,SELEEM M N,et al.Overexpression of homologous anti gens in a leucine auxotroph of Brucella abortus strain RB51protects mice against a virulent B.suis challenge[J].Vaccine,2011,29(17):3106-3110.
[15] COMERCI D J,POLLEVICK G D,VIGLIOCCO A M,et al.Vector development for the expression of foreign proteins in the vaccine strain Brucella abortus S19[J].Infection and Immunity,1998,66(8):3862.
[16] SABIO Y GARCIA J V,BIGI F,ROSSETTI O,et al.Expression of MPB83 from Mycobacterium bovis in Brucella abortus S19 induces specific cellular immune response against the recombinant antigen in BALB/c mice[J].Microbes&Infection,2010,12(14-15):1236-1243.
[17]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,1997,13(2):126-133.LI Wu-ju,WU Jia-jin.Quantitative analysis on expression level of foreign gene in pBV220 vector[J].Chinese Journal of Virology,1997,13(2):126-133.(in Chinese)