布鲁氏菌vceA基因缺失株的构建及生物学特性研究
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摘要
试验旨在构建布鲁氏菌Ⅳ型分泌系统vceA基因缺失株,分析其生长特征及其在人胚胎滋养层细胞(HPT-8)中的存活能力。以布鲁氏菌S2308为模板,PCR扩增vceA基因上、下游同源臂,以pUC19K质粒为模板扩增kan基因,然后利用融合PCR技术将3段基因进行融合,再将此片段与pMD19-T载体连接,电转至大肠杆菌DH5α感受态细胞中,构建ΔvceA基因缺失株(S2308ΔvceA),通过卡那抗性对缺失株进行连续筛选,并检测其遗传稳定性。在相同培养条件下,观察亲本株S2308和S2308ΔvceA的生长变化趋势,以侵染复数(MOI)100∶1侵染HPT-8细胞,通过CFU计数检测亲本株S2308和缺失株S2308ΔvceA在细胞内的生存能力。结果显示,试验成功获得片段大小为372、510、1 093 bp的vceA基因上、下游同源臂和kan基因;且成功构建pMD19-T-vceA-kan自杀载体,将其电转入大肠杆菌DH5α感受态细胞,构建基因缺失株,连续传代10次未发现基因回复突变;在体外相同培养条件下,缺失株S2308ΔvceA与亲本株S2308生长趋势相似,均在12 h达到对数生长期,30 h进入平台期;侵染HPT-8细胞12 h时,缺失株S2308ΔvceA在细胞中的数量显著低于亲本株S2308(P<0.05)。综上所述,本试验成功构建并获得了具有良好遗传稳定性的布鲁氏菌vceA基因缺失株,该缺失株在体外培养条件下与亲本株生长趋势相似;但该缺失株在HPT-8细胞内的存活能力显著变弱。
This study was aimed to construct Brucella 2308(S2308) vceA gene mutants then analyse its growth character and survival ability in human embryonic trophoblast cells(HPT-8).The upstream,downstream homologous arms of vceA gene and kan gene were amplified by PCR method with wild S2308 strain as template.The fusion PCR technology was used to fuse the three genes fragments and connected to pMD19-T vector and then electric transported to E. coli DH5α competent cells to construct S2308ΔvceA strain.The genetic stability of the mutant strain was tested by screening continuously with kanar resistance,and the growth rates between the S2308ΔvceA and S2308 strains were compared.The HPT-8 cells were infected with S2308ΔvceA and S2308 strains at a multiplicity of infection of 100∶ 1 and the intracellular survival ability was determined by CFU counting.The results showed that the target gene fragments of upstream homologous arms(372 bp),downstream homologous arms(510 bp) of vceA gene and kan gene(1 093 bp) were amplified respectively.The mutants was successfully constructed and the virulence not recover after culture at least 10 generations.The growth curves of S2308ΔvceA and S2308 strains were similar under the same condition.These two strains reached the logarithm growth phase at 12 h,entered in platform period after 30 h.However,the survival rates of the S2308ΔvceA strain was significantly lower than its parent strain S2308 after the infection of HPT-8 cells at 12 h(P<0.05).In conclusion,This experiment successfully constructed and obtained a vceA gene deletion strain of Brucella with good genetic stability,and the growth trend of this deleted strain was similar to parent strain under the condition of in vitro culture.However,the viability of the deleted strain in HPT-8 cells was significantly weakened.
This study was aimed to construct Brucella 2308(S2308) vceA gene mutants then analyse its growth character and survival ability in human embryonic trophoblast cells(HPT-8).The upstream,downstream homologous arms of vceA gene and kan gene were amplified by PCR method with wild S2308 strain as template.The fusion PCR technology was used to fuse the three genes fragments and connected to pMD19-T vector and then electric transported to E. coli DH5α competent cells to construct S2308ΔvceA strain.The genetic stability of the mutant strain was tested by screening continuously with kanar resistance,and the growth rates between the S2308ΔvceA and S2308 strains were compared.The HPT-8 cells were infected with S2308ΔvceA and S2308 strains at a multiplicity of infection of 100∶ 1 and the intracellular survival ability was determined by CFU counting.The results showed that the target gene fragments of upstream homologous arms(372 bp),downstream homologous arms(510 bp) of vceA gene and kan gene(1 093 bp) were amplified respectively.The mutants was successfully constructed and the virulence not recover after culture at least 10 generations.The growth curves of S2308ΔvceA and S2308 strains were similar under the same condition.These two strains reached the logarithm growth phase at 12 h,entered in platform period after 30 h.However,the survival rates of the S2308ΔvceA strain was significantly lower than its parent strain S2308 after the infection of HPT-8 cells at 12 h(P<0.05).In conclusion,This experiment successfully constructed and obtained a vceA gene deletion strain of Brucella with good genetic stability,and the growth trend of this deleted strain was similar to parent strain under the condition of in vitro culture.However,the viability of the deleted strain in HPT-8 cells was significantly weakened.
引文
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[9] 相彩霞.布鲁氏菌VceC蛋白对山羊滋养层细胞内质网应激反应和性腺激素分泌的影响[D].杨凌:西北农林科技大学,2017. XIANG C X.The endoplasmic reticulum stress and gonadal hormonesecretory influence of Brucella VceC on goat trophoblast cells[D].Yangling:Northwest A & F University,2017.(in Chinese)
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[14] 赵红庆,熊衍文,徐建国.细菌Ⅳ型分泌系统的研究进展[J].疾病监测,2012,27(11):913-917. ZHAO H Q,XIONG Y W,XU J G.Progress in research of bacterial type Ⅳ secretion system[J].Disease Surveillance,2012,27(11):913-917.(in Chinese)
[15] KWOK T,ZABLER D,URMAN S,et al.Helicobacter exploits integrin for type Ⅳ secretion and kinase activation[J].Nature,2007,449(7164):862-866.
[16] MIRAGLIA M C,COSTA FRANCO M M,RODRIGUEZ A M,et al.Glial cell-elicited activation of brain microvasculature in response to Brucella abortus infection requires ASC inflammasome-dependent IL-1β production[J].Journal of Immunology,2016,196(9):3794-3805.
[17] 史静雪,刘来珍,李敏,等.布鲁氏菌Ⅳ型分泌系统蛋白基因VceA的原核表达、纯化及功能分析[J].石河子大学学报(自然科学版),2017,35(1):83-88. SHI J X,LIU L Z,LI M,et al.Prokaryotic expression,purification and function analysis of Brucella Ⅳ secretion system protein VceA gene[J].Journal of Shihezi University(Natural Science),2017,35(1):83-88.(in Chinese)
[18] 胡森,郑孝辉,王加兰,等.马耳它布氏杆菌bp26基因缺失株的构建及鉴定[J].中国预防兽医学报,2009,31(8):583-586. HU S,ZHENG X H,WANG J L,et al.Development of a mutant Brucella melitens is as vaccine in mice[J].Chinese Journal of Preventive Veterinary Medicine,2009,31(8):583-586.(in Chinese)
[19] DEL GIUDICE M G,UGALDE J E,CZIBENER C.A lysozyme-like protein in Brucella abortus is involved in the early stages of intracellular replication[J].Infection & Immunity,2013,81(3):956-964.
[20] 易继海,王月丽,王震,等.布鲁氏菌胞内存活机制与巨噬细胞极化关系研究进展[J].中国畜牧兽医,2018,45(4):1082-1088. YI J H,WANG Y L,WANG Z,et al.Research progress on the relationship between intracellular survival mechanism of Brucella and macropohage polarization[J].China Animal Husbandry & Veterinary Medicine,2018,45(4):1082-1088.(in Chinese)
[21] 马树立,李芳,徐彪,等.胎盘各组织来源间充质干细胞生物学特性比较[J].国际免疫学杂志,2013,36(5):397-401. MA S L,LI F,XU B,et al.The comparison of biological characteristics of mesenchy of mesenchymal stem cells derived from difference placenta tissues[J].International Journal of Immunology,2013,36(5):397-401.(in Chinese)
[2] DAWSON C E,STUBBERFIELD E J,PERRETT L L,et al.Phenotypic and molecular characterisation of Brucella isolates from marine mammals[J].BMC Microbiology,2008,8(1):224.
[3] ZHANG W Y,GUO W D,SUN S H,et al.Human Brucellosis,inner Mongolia,China[J].Emerging Infectious Diseases,2010,16(12):2001-2003.
[4] ROUX M,ROLAN H G,SANTOS R L,et al.Brucella requires a functional type Ⅳ secretion system to elicit innate immune responses in mice[J].Cellular Microbiology,2007,9(7):1851-1869.
[5] RUSSELL A B,HOOD R D,BUI N K,et al.Type Ⅵ secretion delivers bacteriolytic effectors to target cells[J].Nature,2011,475(7356):343-347.
[6] BASLER M,PILHOFER M,HENDERSON G P,et al.Type Ⅵ secretion requires a dynamic contractile phage tail-like structure[J].Nature,2012,483(7388):182-186.
[7] DE JONG M F,SUN Y H,DEN HARTIGH A B,et al.Identification of VceA and VceC,two members of the VjbR regulon that are translocated into macrophages by the Brucella type Ⅳ secretion system[J].Molecular Microbiology,2010,70(6):1378-1396.
[8] HASHEMIFAR I,YADEGAR A,JAZI F M,et al.Molecular prevalence of putative virulence-associated genes in Brucella melitensis and Brucella abortus isolates from human and livestock specimens in Iran[J].Microbial Pathogenesis,2017,105:334-339.
[9] 相彩霞.布鲁氏菌VceC蛋白对山羊滋养层细胞内质网应激反应和性腺激素分泌的影响[D].杨凌:西北农林科技大学,2017. XIANG C X.The endoplasmic reticulum stress and gonadal hormonesecretory influence of Brucella VceC on goat trophoblast cells[D].Yangling:Northwest A & F University,2017.(in Chinese)
[10] LACERDA T L S,SALCEDO S P,GORVEL J P.Brucella T4SS:The VIP pass inside host cells[J].Current Opinion in Microbiology,2013,16(1):45-51.
[11] VILLAMIL GIRALDO A M,SIVANESAN D,CARLE A,et al.Type Ⅳ secretion system core component VirB8 from Brucella binds to the globular domain of VirB5 and to a periplasmic domain of VirB6[J].Biochemistry,2012,51(18):3881-3890.
[12] DE JONG M F,TSOLIS R M.Brucellosis and type Ⅳ secretion[J].Future Microbiology,2012,7(1):47-58.
[13] 肖启程,田一男,彭广能,等.布鲁氏菌Ⅳ型分泌蛋白及其参与免疫应答的研究进展[J].中国人兽共患病学报,2017,33(11):1029-1035. XIAO Q C,TIAN Y N,PENG G N,et al.Reseaech advance on Brucella Ⅳ secretion system proteins and its immune response[J].Chinese Journal of Zoonoses,2017,33(11):1029-1035.(in Chinese)
[14] 赵红庆,熊衍文,徐建国.细菌Ⅳ型分泌系统的研究进展[J].疾病监测,2012,27(11):913-917. ZHAO H Q,XIONG Y W,XU J G.Progress in research of bacterial type Ⅳ secretion system[J].Disease Surveillance,2012,27(11):913-917.(in Chinese)
[15] KWOK T,ZABLER D,URMAN S,et al.Helicobacter exploits integrin for type Ⅳ secretion and kinase activation[J].Nature,2007,449(7164):862-866.
[16] MIRAGLIA M C,COSTA FRANCO M M,RODRIGUEZ A M,et al.Glial cell-elicited activation of brain microvasculature in response to Brucella abortus infection requires ASC inflammasome-dependent IL-1β production[J].Journal of Immunology,2016,196(9):3794-3805.
[17] 史静雪,刘来珍,李敏,等.布鲁氏菌Ⅳ型分泌系统蛋白基因VceA的原核表达、纯化及功能分析[J].石河子大学学报(自然科学版),2017,35(1):83-88. SHI J X,LIU L Z,LI M,et al.Prokaryotic expression,purification and function analysis of Brucella Ⅳ secretion system protein VceA gene[J].Journal of Shihezi University(Natural Science),2017,35(1):83-88.(in Chinese)
[18] 胡森,郑孝辉,王加兰,等.马耳它布氏杆菌bp26基因缺失株的构建及鉴定[J].中国预防兽医学报,2009,31(8):583-586. HU S,ZHENG X H,WANG J L,et al.Development of a mutant Brucella melitens is as vaccine in mice[J].Chinese Journal of Preventive Veterinary Medicine,2009,31(8):583-586.(in Chinese)
[19] DEL GIUDICE M G,UGALDE J E,CZIBENER C.A lysozyme-like protein in Brucella abortus is involved in the early stages of intracellular replication[J].Infection & Immunity,2013,81(3):956-964.
[20] 易继海,王月丽,王震,等.布鲁氏菌胞内存活机制与巨噬细胞极化关系研究进展[J].中国畜牧兽医,2018,45(4):1082-1088. YI J H,WANG Y L,WANG Z,et al.Research progress on the relationship between intracellular survival mechanism of Brucella and macropohage polarization[J].China Animal Husbandry & Veterinary Medicine,2018,45(4):1082-1088.(in Chinese)
[21] 马树立,李芳,徐彪,等.胎盘各组织来源间充质干细胞生物学特性比较[J].国际免疫学杂志,2013,36(5):397-401. MA S L,LI F,XU B,et al.The comparison of biological characteristics of mesenchy of mesenchymal stem cells derived from difference placenta tissues[J].International Journal of Immunology,2013,36(5):397-401.(in Chinese)
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