牛种布鲁氏菌VirB12基因的克隆、表达及纯化
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- 英文论文题名：Cloning,Expression and Purification of VirB12 Gene of B.abortus
- 论文作者：唐峰 ; 闫广谋 ; 唐雨顺 ; 刘永华 ; 李冰
- 英文论文作者：TANG Feng ; YAN Guang-mou ; TANG Yu-shun ; LIU Yong-hua ; LI Bing ; Institute of Animal Husbandry and Veterinary Medicine ; Liaoning Medical University ; College of Veterinary Medicine ; Jilin University
- 年：2016
- 作者机构：辽宁医学院畜牧兽医学院;吉林大学动物医学学院;
- 论文关键词：牛种布鲁氏菌 ; VirB12基因 ; 基因克隆 ; 蛋白 ; 纯化 ; 抗原特异性
- 英文论文关键词：B.abortus ; VirB12 gene ; Gene cloning ; Fusion protein ; Purification ; Antigenic Specificity
- 会议召开时间：2016-07-20
- 会议录名称：中国畜牧兽医学会兽医公共卫生学分会第五次学术研讨会论文集
- 语种：中文
- 分类号：S852.61
- 学会代码：SYGW
- 会议名称：中国畜牧兽医学会兽医公共卫生学分会第五次学术研讨会
- 会议地点：中国内蒙古通辽
- 主办单位：中国畜牧兽医学会兽医公共卫生学分会
- 学会名称：中国畜牧兽医学会兽医公共卫生学分会
- 页数：7
- 文件大小：605k
- 原文格式：D
- 会议级别：全国
摘要
目的原核表达牛种布鲁氏菌Vir B12蛋白,并检测其免疫原性。方法提取细菌基因组DNA,根据Gen Bank中的Vir B12序列设计引物,利用PCR方法扩增出Vir B12基因,并克隆到p MD18-T simple载体上,测序,再亚克隆至p ET-30a(+)载体上,转化到大肠杆菌BL21(DE3)中,用IPTG诱导,表达产物经组氨酸结合树脂柱纯化,并对纯化后的表达产物进行免疫印迹免疫印迹鉴定。结果重组质粒经Nde I与Sal I酶切后,可见5422和519 bp的片段,与预期值相符。转化至BL21(DE3)后,37℃诱导4 h,经聚丙烯酰胺凝胶电泳分析,表达出带有多聚组氨酸标签的融合蛋白Vir B12,纯化后的蛋白纯度达94%,经免疫印迹法分析,目的蛋白具有抗原特异性。结论成功表达了布鲁氏菌Vir B12蛋白,纯化蛋白具有抗原特异性。意义近年来,布病给人类健康和公共卫生安全带来严重问题。控制动物布病的发生对降低人类布病的发生具有重要意义。布鲁氏菌Vir B12基因能在感染布病的动物体内会产生Vir B12蛋白,该蛋白在胞内运输过程中发挥重要作用。本试验为进一步研究Vir B12蛋白的结构、功能及相关疫苗的研制奠定了基础。
Objective To clone the Vir B12 gene of B.abortus,highly express in E.coli and purify the expressed product and determine its antigenic specificity.Methods Extract the genomic DNA of B.abortus,amplify Vir B12 gene gene by PCR and clone to p MD18-T simple vector.Identify the constructed recombinant plasmid by sequencing,then subclone to vector p ET-30a(+).Transform the constructed recombinant plasmid to E.coli BL21(DE3) for expression under induction of IPTG.Purify the expressed product by histidine-binding resin column chromatography and identify by Western blot.Results Two gene fragments at lengths of 5422 and 519 bp respectively were obtained by digestion of recombinant plasmid with Nde I and Sal I,which were consistent with the theoretical value.After the transformed E.coli BL21(DE3) was induced at 37℃ for 4 h,SDS-PAGE proved that fusion protein with His-tag label was highly expressed.The fusion protein reached a purity of 94% after purification and showed antigenic specificity as proved by Western blot.Conclusion Vir B12 gene was successfully cloned and expressed,and the expressed product showed antigenic specificity,which laid a foundation of further study on structure and function of Vir B12 and development of the relevant vaccines.
Objective To clone the Vir B12 gene of B.abortus,highly express in E.coli and purify the expressed product and determine its antigenic specificity.Methods Extract the genomic DNA of B.abortus,amplify Vir B12 gene gene by PCR and clone to p MD18-T simple vector.Identify the constructed recombinant plasmid by sequencing,then subclone to vector p ET-30a(+).Transform the constructed recombinant plasmid to E.coli BL21(DE3) for expression under induction of IPTG.Purify the expressed product by histidine-binding resin column chromatography and identify by Western blot.Results Two gene fragments at lengths of 5422 and 519 bp respectively were obtained by digestion of recombinant plasmid with Nde I and Sal I,which were consistent with the theoretical value.After the transformed E.coli BL21(DE3) was induced at 37℃ for 4 h,SDS-PAGE proved that fusion protein with His-tag label was highly expressed.The fusion protein reached a purity of 94% after purification and showed antigenic specificity as proved by Western blot.Conclusion Vir B12 gene was successfully cloned and expressed,and the expressed product showed antigenic specificity,which laid a foundation of further study on structure and function of Vir B12 and development of the relevant vaccines.
引文
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[13]赵岩,李明,胡福泉.细菌的IV型分泌系统[J].生命的化学,2011,31(1):128-133.
[14]张瑞,王秀然,李晓艳,等.布鲁氏菌胞内寄生的研究进展[J].安徽农业科学,2012,40(9):5246-5248.
[15]Rolán G,den Hartigh B,Kahl-Mc Donagh M,et al.Vir B12 is a serological marker of Brucella infection in experimental and natural hosts[J].Clin Vaccine Immunol,2008,15:208-214.
[2]Manthur BG,Amarnath SK.Brucellosis in India-A review[J].Biosci,2008,33:539-547.
[3]伊日盖.布鲁氏菌病的流行病学调查与分子分型研究[D].呼和浩特:内蒙古农业大学2009:2.
[4]Zhang WY,Guo WD,Sun SH,et al.Human brucellosis,Inner Mongolia,China[J].Emerg Infect Dis,2010,16:2001-2003.
[5]Magwedere K,Bishi A,Tjipura-Zaire G,et al.Brucellae through the food chain:the role of sheep,goats and springbok(Antidorcus marsupialis)as sources of human infections in Namibia[J].J S Afr Vet Assoc.2011,82(4):205-212.
[6]Ciesielski F,Griffin DC,Rittig M,et al.Interactions of lipopolysaccharide with lipid membranes,raft models-Asolid state NMR study[J].Biochim Biophys Acta.2013,1828(8):1731-1742.
[7]Lacerda TL,Salcedo SP,Gorvel JP.Brucella T4SS:the VIP pass inside host cells.Brucella T4SS:the VIP pass inside host cells.Curr Opin Microbiol.2013,16(1):45-51.
[8]Celli J,Salcedo S P,Gorvel JP.Brucella coopts the small GTPase Sar1 for intracellular replication[J].Proc Natl Acad Sci U.S.A.,2005,102:1673-1678.
[9]Haag AF,Myka KK,Arnold MF,et al.Importance of Lipopolysaccharide and Cyclicβ-1,2-Glucans in Brucella-Mammalian Infections[J].Int J Microbiol,2010,2010:12.
[10]Seleem MN,Boyle SM,Sriranganathan N.Brucella:A pathogen without classic virulence genes[J].Vet Microbiol,2008,129:1-14.
[11]Sun YH,Rolán HG,den Hartigh AB,et al.Brucella abortus Vir B12 Is Expressed during Infection but Is Not an Essential Component of the Type IV Secretion System[J].Infect Immun,2005,73(9):6048-6054.
[12]Huber B,Scholz H C,Lucero N,et al.Development of a PCR assay for typing and subtyping of Brucella species[J].Int J Med Microbiol,2009,29:563-573.
[13]赵岩,李明,胡福泉.细菌的IV型分泌系统[J].生命的化学,2011,31(1):128-133.
[14]张瑞,王秀然,李晓艳,等.布鲁氏菌胞内寄生的研究进展[J].安徽农业科学,2012,40(9):5246-5248.
[15]Rolán G,den Hartigh B,Kahl-Mc Donagh M,et al.Vir B12 is a serological marker of Brucella infection in experimental and natural hosts[J].Clin Vaccine Immunol,2008,15:208-214.