多杀性巴氏杆菌中recN基因的克隆、原核表达及生物信息学分析
|
摘要
试验旨在对多杀性巴氏杆菌recN基因进行克隆和原核表达,并对其表达蛋白进行生物信息学分析。参照GenBank中recN基因序列(登录号:CP003313.1)设计1对引物,通过PCR扩增获得目的基因片段,构建pET-28a(+)-recN重组质粒并转化E.coli DH5α感受态细胞,提取质粒进行酶切鉴定,将鉴定正确的重组质粒转化E.coli BL21(DE3)感受态细胞,经IPTG诱导表达,对融合蛋白进行SDS-PAGE及Western blotting鉴定。结果表明,试验成功克隆了大小约为1 677 bp的recN基因序列,通过诱导表达的His-Tag融合蛋白大小约为66.94 ku,主要以包涵体形式存在。经生物信息学分析,recN蛋白的分子式为C_(2735)H_(4428)N_(786)O_(855)S_(16),消光系数为24 785,不稳定系数为43.99,属于不稳定蛋白;理论等电点(pI)为5.62,为酸性蛋白;总平均亲水性为-0.316,与试验表达的包涵体蛋白性质相同,即同为疏水性蛋白;recN蛋白在哺乳动物网织红细胞的半衰期为30 h,在酵母(体内)中的半衰期>20 h,在大肠杆菌(体内)中的半衰期>10 h;二级结构主要以α-螺旋(64.87%)及无规则卷曲(21.00%)为主;经疏水性分析,预测该蛋白有3个高疏水性区域和9个高亲水性区域。本试验结果为进一步探究多杀性巴氏杆菌recN基因的功能提供了参考依据。
This experiment was aimed to study the clone and prokaryotic expression of recN gene of Pasteurella multocida,and analyze its protein by bioinformatics method.A pair of primers was designed according to the recN gene sequence in GenBank(accession No.:CP003313.1),and the target gene fragment was obtained by PCR.The recombinant plasmid pET-28 a(+)-recN was constructed and transformed into E.coli DH5α competent cells,and the plasmid was extracted for restriction enzyme digestion.The correct recombinant plasmid was transformed into E.coli BL21(DE3) competent cells.The fusion protein was induced by IPTG,and identified by SDS-PAGE and Western blotting.The results showed that the recN gene with a size of about 1 677 bp was successfully cloned in this experiment.The size of the His-Tag fusion protein induced by expression was about 66.94 ku,and it mainly existed in the form of inclusion body.According to bioinformatics analysis,the recN protein had the molecular formula C_(2735)H_(4428)N_(786)O_(855)S_(16),the extinction coefficient was 24 785,the instability coefficient was 43.99,which was unstable protein;The theoretical isoelectric point(pI) was 5.62,which was acidic protein;The total average hydrophilicity was-0.316,which was the same hydrophobic protein as the experimentally expressed inclusion body protein;The half-life of reticulocytes in mammalian was estimated to be 30 h,the half-life in yeast and E.coli(in vivo) was more than 20 and 10 h,respectively.The secondary structure prediction was mainly alpha helix(64.87%) and random coil(21.00%);The hydrophobic analysis results showed that recN protein had 3 highly hydrophobic sexual regions and 9 highly hydrophilic regions.This results provided a reference basis for further exploration of the function of recN gene of Pasteurella multocida.
This experiment was aimed to study the clone and prokaryotic expression of recN gene of Pasteurella multocida,and analyze its protein by bioinformatics method.A pair of primers was designed according to the recN gene sequence in GenBank(accession No.:CP003313.1),and the target gene fragment was obtained by PCR.The recombinant plasmid pET-28 a(+)-recN was constructed and transformed into E.coli DH5α competent cells,and the plasmid was extracted for restriction enzyme digestion.The correct recombinant plasmid was transformed into E.coli BL21(DE3) competent cells.The fusion protein was induced by IPTG,and identified by SDS-PAGE and Western blotting.The results showed that the recN gene with a size of about 1 677 bp was successfully cloned in this experiment.The size of the His-Tag fusion protein induced by expression was about 66.94 ku,and it mainly existed in the form of inclusion body.According to bioinformatics analysis,the recN protein had the molecular formula C_(2735)H_(4428)N_(786)O_(855)S_(16),the extinction coefficient was 24 785,the instability coefficient was 43.99,which was unstable protein;The theoretical isoelectric point(pI) was 5.62,which was acidic protein;The total average hydrophilicity was-0.316,which was the same hydrophobic protein as the experimentally expressed inclusion body protein;The half-life of reticulocytes in mammalian was estimated to be 30 h,the half-life in yeast and E.coli(in vivo) was more than 20 and 10 h,respectively.The secondary structure prediction was mainly alpha helix(64.87%) and random coil(21.00%);The hydrophobic analysis results showed that recN protein had 3 highly hydrophobic sexual regions and 9 highly hydrophilic regions.This results provided a reference basis for further exploration of the function of recN gene of Pasteurella multocida.
引文
[1] JABLONSKI L,SRIRANGANATHA N,BOYLE S M,et al.Conditions for transformation of Pasteurella multocida by electroporation[J].Microbial Pathogenesis,1992,12(1):62-68.
[2] 许泽芳,谢永兴,郭树源,等.巴氏杆菌诊断技术研究进展[J].水禽世界,2015,3:38-41. XU Z F,XIE Y X,GUO S Y,et al.Progress in the diagnosis of Pasteurella[J].Waterfowl World,2015,3:38-41.(in Chinese)
[3] BOYCE J D,CHUNG J Y,ADLER B.Pasteurella multocida capsule:Composition,function and ge-netics[J].Journal of Biotechnology,2000,83(1-2):153-160.
[4] 范国寿,梁建明.黑山羊巴氏杆菌病的诊断与治疗[J].山东畜牧兽医,2016,10:23-24. FAN G S,LIANG J M.Black goat pasteurellosis diagnosis and treatment[J].Shandong Journal of Animal Science and Veterinary Medicine,2016,10:23-24.(in Chinese)
[5] 赵恒章,王天有,李国旺.鸡霍乱组织灭活苗的研究[J].安徽农业科学,2005,33(5):853-854. ZHAO H Z,WANG T Y,LI G W.Chicken cholera tissue inactivated vaccine[J].Anhui Agricultural Sciences,2005,33(5):853-854.(in Chinese)
[6] LLOYD R G.Linkage distortion following conjugational transfer of sbcC+ to recBC sbcBC strains of Escherichia coli[J].Journal of Bacteriology,1991,173(18):5694-5698.
[7] NAGEL R,CHAN A.RecBC and RecF recombination pathways and the induced precise excision of Tn10 in Escherichia coli[J].Mutation Research,1999,433(2):99-107.
[8] YOUSSEF M M,Al-OMAIR M A,PICKSLEY S M.Genetic characterization of Escherichia coli RecN protein as a member of SMC family of proteins[J].Arabian Journal of Chemistry,2014,7(3):327-334.
[9] 李宝宝,聂鑫,杨小健,等.羊种布鲁氏菌dhbC基因的克隆、原核表达及生物信息学分析[J].中国畜牧兽医,2017,44(7):1947-1953. LI B B,NIE X,YANG X J,et al.Cloning,prokaryotic expression and bioinformatics analysis of dhbC gene of Brucella sinensis[J].China Journal of Animal Husbandry & Veterinary Medicine,2017,44(7):1947-1953.(in Chinese)
[10] 徐开莲,朱华培,赵天靖,等.羊种布鲁氏菌Omp10基因的克隆及原核表达[J].中国畜牧兽医,2014,41(12):122-125. XU K L,ZHU H P,ZHAO T J.et al.Cloning and prokaryotic expression of the Omp10 gene of Brucella serrata[J].China Journal of Animal Husbandry & Veterinary Medicine,2014,41(12):122-125.(in Chinese)
[11] 刘云杰,初瑞琦.牛多杀性巴氏杆菌病诊断与防治[J].饲料博览,2018,4:64. LIU Y J,CHU R Q.Diagnosis and prevention of pasteurellosis in cattle[J].Feed Review,2018,4:64.(in Chinese)
[12] TURNI C,BLACKALL P J.Serovar profiling of Haemophilus parasuis on Australian farms by sampling live pigs[J].Australian Veterinary Journal,2010,88(7):255-259.
[13] SUBAAHARAN S,BLACKALL L L,BLACKALL P J.Development of a multi-locus sequence typing scheme for avian isolates of Pasteurella multocida[J].Veterinary Microbiology,2010,141:354-361.
[14] 覃岚,李忠全,虞顺刚.羊巴氏杆菌病的诊治与防控[J].贵州畜牧兽医,2017,41:47-48. QIN L,LI Z Q,YU S G.Diagnosis and control of sheep pasteurellosis[J].Guizhou Animal Husbandry and Veterinary Medicine,2017,41:47-48.(in Chinese)
[15] 陈品安,汪金义.羊巴氏杆菌病的流行特点及综合防治措施[J].畜牧与饲料科学,2013,31(1):110-111. CHEN P A,WANG J Y.Epidemic characteristics of sheep pasteurellosis and comprehensive prevention and treatment measures[J].Livestock and Feed Science,2013,31(1):110-111.(in Chinese)
[16] 刘玉华,陈光明.鸭巴氏杆菌病油佐剂灭活苗的研制[J].浙江农业科学,2017,58(12):2256-2258. LIU Y H,CHEN G M.Development of inactivated vaccine for oil adjuvant of duck pasteurellosis[J].Zhejiang Agricultural Sciences,2017,58(12):2256-2258.(in Chinese)
[17] 黄怡,刘忠令.细菌耐药机制的研究现状[J].国外医学(微生物分册),2000,23(1):24-26. HUANG Y,LIU Z L.Research status of bacterial resistance mechanism[J].Foreign Medicine(Micro biology),2000,23(1):24-26.(in Chinese)
[18] 肖国生,曹三杰,黄小波.猪胸膜肺炎放线杆菌、肺炎支原体和多杀性巴氏杆菌联合检测芯片的研制及应用[J]. 畜牧兽医学报,2014,41(1):77-85. XIAO G S,CAO S J,HUANG X B.Development and application of a combined detection chip for Actinobacillus pleuropneumoniae,Mycoplasma pneumoniae and Pasteurella multocida[J].Journal of Animal Husbandry and Veterinary Medicine,2014,41(1):77-85.(in Chinese)
[19] BUKAU B.Regulation of the Eseheriehia coli heat-shock response[J].Molecular Microbiology,1993,9(4):671-680.
[20] MARIANA S,HIRST R.The immunogenicity and pathogenicity of Pazteurella multocida isolated from poultry in Indonesia[J].Veterinary Microbiology,2000,72(1-2):27-36.
[21] HAIDINGER W,MAYR U B,SZOSTAK M P,et al.Eseherichia coli ghost production by expression lysis gene E and staphylococcal nuclease[J].Appl Environ Microbiol,2003,69(10):6106-6113.
[22] ZHURAVEL D V,BOREIKO A V.Regularities of excising transposon Tn10 in rec-mutant E.coli cells exposed to gamma radiation[J].Radiatsionnaia Biologiia,Radioecologiia,2002,42(6):636-638.
[23] 田雪,王娉,赵勇胜,等.克罗诺杆菌种间鉴定recN基因方法建立[J].中国公共卫生,2015,31(1):63-66. TIAN X,WANG P,ZHAO Y S,et al.Establishment of a method for identifying recN gene between Cronobacter species[J].China Public Health,2015,31(1):63-66.(in Chinese)
[2] 许泽芳,谢永兴,郭树源,等.巴氏杆菌诊断技术研究进展[J].水禽世界,2015,3:38-41. XU Z F,XIE Y X,GUO S Y,et al.Progress in the diagnosis of Pasteurella[J].Waterfowl World,2015,3:38-41.(in Chinese)
[3] BOYCE J D,CHUNG J Y,ADLER B.Pasteurella multocida capsule:Composition,function and ge-netics[J].Journal of Biotechnology,2000,83(1-2):153-160.
[4] 范国寿,梁建明.黑山羊巴氏杆菌病的诊断与治疗[J].山东畜牧兽医,2016,10:23-24. FAN G S,LIANG J M.Black goat pasteurellosis diagnosis and treatment[J].Shandong Journal of Animal Science and Veterinary Medicine,2016,10:23-24.(in Chinese)
[5] 赵恒章,王天有,李国旺.鸡霍乱组织灭活苗的研究[J].安徽农业科学,2005,33(5):853-854. ZHAO H Z,WANG T Y,LI G W.Chicken cholera tissue inactivated vaccine[J].Anhui Agricultural Sciences,2005,33(5):853-854.(in Chinese)
[6] LLOYD R G.Linkage distortion following conjugational transfer of sbcC+ to recBC sbcBC strains of Escherichia coli[J].Journal of Bacteriology,1991,173(18):5694-5698.
[7] NAGEL R,CHAN A.RecBC and RecF recombination pathways and the induced precise excision of Tn10 in Escherichia coli[J].Mutation Research,1999,433(2):99-107.
[8] YOUSSEF M M,Al-OMAIR M A,PICKSLEY S M.Genetic characterization of Escherichia coli RecN protein as a member of SMC family of proteins[J].Arabian Journal of Chemistry,2014,7(3):327-334.
[9] 李宝宝,聂鑫,杨小健,等.羊种布鲁氏菌dhbC基因的克隆、原核表达及生物信息学分析[J].中国畜牧兽医,2017,44(7):1947-1953. LI B B,NIE X,YANG X J,et al.Cloning,prokaryotic expression and bioinformatics analysis of dhbC gene of Brucella sinensis[J].China Journal of Animal Husbandry & Veterinary Medicine,2017,44(7):1947-1953.(in Chinese)
[10] 徐开莲,朱华培,赵天靖,等.羊种布鲁氏菌Omp10基因的克隆及原核表达[J].中国畜牧兽医,2014,41(12):122-125. XU K L,ZHU H P,ZHAO T J.et al.Cloning and prokaryotic expression of the Omp10 gene of Brucella serrata[J].China Journal of Animal Husbandry & Veterinary Medicine,2014,41(12):122-125.(in Chinese)
[11] 刘云杰,初瑞琦.牛多杀性巴氏杆菌病诊断与防治[J].饲料博览,2018,4:64. LIU Y J,CHU R Q.Diagnosis and prevention of pasteurellosis in cattle[J].Feed Review,2018,4:64.(in Chinese)
[12] TURNI C,BLACKALL P J.Serovar profiling of Haemophilus parasuis on Australian farms by sampling live pigs[J].Australian Veterinary Journal,2010,88(7):255-259.
[13] SUBAAHARAN S,BLACKALL L L,BLACKALL P J.Development of a multi-locus sequence typing scheme for avian isolates of Pasteurella multocida[J].Veterinary Microbiology,2010,141:354-361.
[14] 覃岚,李忠全,虞顺刚.羊巴氏杆菌病的诊治与防控[J].贵州畜牧兽医,2017,41:47-48. QIN L,LI Z Q,YU S G.Diagnosis and control of sheep pasteurellosis[J].Guizhou Animal Husbandry and Veterinary Medicine,2017,41:47-48.(in Chinese)
[15] 陈品安,汪金义.羊巴氏杆菌病的流行特点及综合防治措施[J].畜牧与饲料科学,2013,31(1):110-111. CHEN P A,WANG J Y.Epidemic characteristics of sheep pasteurellosis and comprehensive prevention and treatment measures[J].Livestock and Feed Science,2013,31(1):110-111.(in Chinese)
[16] 刘玉华,陈光明.鸭巴氏杆菌病油佐剂灭活苗的研制[J].浙江农业科学,2017,58(12):2256-2258. LIU Y H,CHEN G M.Development of inactivated vaccine for oil adjuvant of duck pasteurellosis[J].Zhejiang Agricultural Sciences,2017,58(12):2256-2258.(in Chinese)
[17] 黄怡,刘忠令.细菌耐药机制的研究现状[J].国外医学(微生物分册),2000,23(1):24-26. HUANG Y,LIU Z L.Research status of bacterial resistance mechanism[J].Foreign Medicine(Micro biology),2000,23(1):24-26.(in Chinese)
[18] 肖国生,曹三杰,黄小波.猪胸膜肺炎放线杆菌、肺炎支原体和多杀性巴氏杆菌联合检测芯片的研制及应用[J]. 畜牧兽医学报,2014,41(1):77-85. XIAO G S,CAO S J,HUANG X B.Development and application of a combined detection chip for Actinobacillus pleuropneumoniae,Mycoplasma pneumoniae and Pasteurella multocida[J].Journal of Animal Husbandry and Veterinary Medicine,2014,41(1):77-85.(in Chinese)
[19] BUKAU B.Regulation of the Eseheriehia coli heat-shock response[J].Molecular Microbiology,1993,9(4):671-680.
[20] MARIANA S,HIRST R.The immunogenicity and pathogenicity of Pazteurella multocida isolated from poultry in Indonesia[J].Veterinary Microbiology,2000,72(1-2):27-36.
[21] HAIDINGER W,MAYR U B,SZOSTAK M P,et al.Eseherichia coli ghost production by expression lysis gene E and staphylococcal nuclease[J].Appl Environ Microbiol,2003,69(10):6106-6113.
[22] ZHURAVEL D V,BOREIKO A V.Regularities of excising transposon Tn10 in rec-mutant E.coli cells exposed to gamma radiation[J].Radiatsionnaia Biologiia,Radioecologiia,2002,42(6):636-638.
[23] 田雪,王娉,赵勇胜,等.克罗诺杆菌种间鉴定recN基因方法建立[J].中国公共卫生,2015,31(1):63-66. TIAN X,WANG P,ZHAO Y S,et al.Establishment of a method for identifying recN gene between Cronobacter species[J].China Public Health,2015,31(1):63-66.(in Chinese)