牛支原体pdhc基因的克隆、表达及其亚细胞定位
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摘要
试验旨在研究牛支原体(Mycoplasma bovis,M.bovis)武威株二氢硫辛酰胺转乙酰酶(PDHc-E2)基因序列特征及其在牛支原体细胞中的位置。参照GenBank中牛支原体HB0801株pdhc基因(登录号:CP002058.1)设计引物,应用PCR扩增获得牛支原体武威株pdhc基因,在测序及序列分析的基础上,应用Overlap PCR完成点突变后将其克隆至pET-28a(+)中,构建原核表达载体pET-pdhc。pET-pdhc转化大肠杆菌Rosetta(DE3)感受态细胞后经IPTG诱导获得融合蛋白,将纯化蛋白免疫新西兰兔制备多抗血清,应用iELISA和Western blotting对牛支原体武威株PDHc-E2在细胞内的分布进行初步研究。结果显示,牛支原体武威株pdhc基因CDS全长735bp,编码244个氨基酸,与国内牛支原体分离株HB0801、Hubei-1、CQ-W70、NM2012等基因序列完全一致,与国际标准株PG45同源性为99.2%,与无乳支原体(M.agalactiae)同源性为90.9%~91.2%,与加利福尼亚支原体(M.californicum)ST6株的同源性仅为78.4%,基因序列非常保守;通过Overlap PCR将该基因中4个编码色氨酸的TGA密码子突变为TGG,且完成点突变后的基因在大肠杆菌中成功表达,重组蛋白大小约为29ku,主要以可溶性形式存在,iELISA结果显示,重组蛋白PDHc-E2具有较高的免疫原性,可刺激新西兰兔产生高水平的抗体,血清效价高达1∶100 000;亚细胞定位结果表明,制备的多抗血清与重组蛋白PDHc-E2、牛支原体全菌蛋白、牛支原体膜蛋白、牛支原体胞浆蛋白均能发生特异性结合,说明该蛋白在牛支原体细胞膜和细胞质中均有分布,为膜相关蛋白,但在细胞质中的分布多于细胞膜。本研究结果为进一步研究牛支原体的生物学功能提供了理论依据。
To study the sequences characteristics of pdhc gene in M.bovis Wuwei strain and its location in M.bovis cells,the primers of pdhc gene were designed according to M.bovis HB0801 strain(accession No.:CP002058.1)in GenBank,and the pdhc gene of M.bovis Wuwei strain was amplified by PCR.Based on sequencing and gene analysis,the prokaryotic expression vector pETpdhc was constructed,and then transformed into Escherichia coli Rosetta(DE3).After induced by IPTG,the M.bovis fusion protein PDHc-E2 was purified and New Zealand rabbits(Oryctolagus cuniculus)was immunized to prepare anti-serum against M.bovis rocombinant protein PDHcE2.Subsequently,the distribution of PDHc-E2 in M.bovis cells was analyzed using iELISA and Western blotting.The results showed that the full-length sequence of pdhc gene CDS of M.bovis Wuwei strain was 735 bp and encoded 244 amino acids.It had 100.0% homology with the domestic M.bovis isolates,such as HB0801,Hubei-1,CQ-W70 and NM2012,and had 99.2% homology with the international standard strain PG45,the homology with Mycoplasma agalactiae(M.aga-lactiae)were 90.9%to 91.2%,the homology with the ST6 strain of Mycoplasma californicum(M.californicum)was only 78.4%,and the gene sequence was very conservative.By Overlap PCR,four TGA codons encoding tryptophan in this gene mutated to TGG;After point mutation the pdhc gene successfully expressed in Escherichia coli.The molecular weight of recombinant protein was approximately 29 ku,and it mainly existed in the form of soluble protein.iELISA results showed that recombinant protein PDHc-E2 had a high immunogenicity,and could stimulate New Zealand rabbit to produce high levels of antibodies,the iELISA titer of antiserum was approximately up to 1∶100 000.The results of subcellular localization showed that the preparation of polyclonal antibody could bind specifically to recombinant protein PDHc-E2,total protein,membrane protein and cytoplasm of M.bovis,which indicated that the PDHc-E2 of M.bovis was distributed in both the membrane and the cytoplasm,and was a membrane-related protein,but the distribution in the cytoplasm was more than in the cell membrane.The results of this study laid a theoretical basis for further investigation on biological functions of M.bovis.
To study the sequences characteristics of pdhc gene in M.bovis Wuwei strain and its location in M.bovis cells,the primers of pdhc gene were designed according to M.bovis HB0801 strain(accession No.:CP002058.1)in GenBank,and the pdhc gene of M.bovis Wuwei strain was amplified by PCR.Based on sequencing and gene analysis,the prokaryotic expression vector pETpdhc was constructed,and then transformed into Escherichia coli Rosetta(DE3).After induced by IPTG,the M.bovis fusion protein PDHc-E2 was purified and New Zealand rabbits(Oryctolagus cuniculus)was immunized to prepare anti-serum against M.bovis rocombinant protein PDHcE2.Subsequently,the distribution of PDHc-E2 in M.bovis cells was analyzed using iELISA and Western blotting.The results showed that the full-length sequence of pdhc gene CDS of M.bovis Wuwei strain was 735 bp and encoded 244 amino acids.It had 100.0% homology with the domestic M.bovis isolates,such as HB0801,Hubei-1,CQ-W70 and NM2012,and had 99.2% homology with the international standard strain PG45,the homology with Mycoplasma agalactiae(M.aga-lactiae)were 90.9%to 91.2%,the homology with the ST6 strain of Mycoplasma californicum(M.californicum)was only 78.4%,and the gene sequence was very conservative.By Overlap PCR,four TGA codons encoding tryptophan in this gene mutated to TGG;After point mutation the pdhc gene successfully expressed in Escherichia coli.The molecular weight of recombinant protein was approximately 29 ku,and it mainly existed in the form of soluble protein.iELISA results showed that recombinant protein PDHc-E2 had a high immunogenicity,and could stimulate New Zealand rabbit to produce high levels of antibodies,the iELISA titer of antiserum was approximately up to 1∶100 000.The results of subcellular localization showed that the preparation of polyclonal antibody could bind specifically to recombinant protein PDHc-E2,total protein,membrane protein and cytoplasm of M.bovis,which indicated that the PDHc-E2 of M.bovis was distributed in both the membrane and the cytoplasm,and was a membrane-related protein,but the distribution in the cytoplasm was more than in the cell membrane.The results of this study laid a theoretical basis for further investigation on biological functions of M.bovis.
引文
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[6]辛九庆,李媛,郭丹,等.国内首次从患肺炎的犊牛肺脏中分离到牛支原体[J].中国预防兽医学报,2008,30(9):661-664.XIN J Q,LI Y,GUO D,et al.First isolation of Mycoplasma bovis from calf lung with pneumonia in China[J].Chinese Journal of Preventive Veterinary Medicine,2008,30(9):661-664.(in Chinese)
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[14]NADAL I,RICO J,PREZMARTINEZ G,et al.Diacetyl and acetoin production from whey permeate using engineered Lactobacillus casei[J].Journal of Industrial Microbiology&Biotechnology,2009,36(9):1233-1237.
[15]GRNDEL A,JACOBS E,DUMKE R.Interactions of surface-displayed glycolytic enzymes of Mycoplasma pneumoniae with components of the human extracellular matrix[J].International Journal of Medical Microbiology,2016,306(8):675-685.
[16]高翔,邢小勇,冯娜,等.牛支原体武威株fba基因的克隆、表达及亚细胞定位[J].农业生物技术学报,2017,25(2):274-281.GAO X,XING X Y,FENG N,et al.Cloning,expression and subcellular localization of fbagene of Mycoplasma bovis Wuwei strain[J].Journal of Agricultural Biotechnology,2017,25(2):274-281.(in Chinese)
[17]陈忠琼,范媛,周蓓,等.牛支原体膜蛋白6种提取方法的比较研究[J].中国兽医学报,2013,33(6):855-860.CHEN Z Q,FAN Y,ZHOU B,et al.The comparative study on six kinds of extraction methods for membrane proteins of Mycoplasma bovis[J].Chinese Journal of Veterinary Science,2013,33(6):855-860.(in Chinese)
[18]陈曦,朱洪梅,赵刚,等.牛支原体HB0801株VspX蛋白单克隆抗体的制备与鉴定[J].中国畜牧兽医,2017,44(3):868-878.CHEN X,ZHU H M,ZHAO G,et al.Preparation and identification of the monoclonal antibodies against VspX protein in Mycoplasma bovis strain HB0801[J].China Animal Husbandry&Veterinary Medicine,2017,44(3):868-878.(in Chinese)
[19]庄金秋,张颖,梅建国,等.牛支原体检测方法研究进展[J].中国动物检疫,2016,33(11):66-70.ZHUANG J Q,ZHANG Y,MEI J G,et al.Research progress on detection methods of Mycoplasma bovis[J].China Animal Health Inspection,2016,33(11):66-70.(in Chinese)
[20]MULONGO M,PRYSLIAK T,PEREZ-CASAL J.Vaccination of feedlot cattle with extracts and membrane factions from two Mycoplasma bovis isolates results is strong humoral immune responses but does not protect against an experimental challenge[J].Vaccine,2013,31(10):1406-1412.
[21]季文恒,吴娅琴,翟肖辉,等.牛支原体疫苗的研究进展[J].中国畜牧兽医,2018,45(3):763-769.JI W H,WU Y Q,ZHAI X H,et al.Advances on the research of vaccine against Mycoplasma bovis[J].China Animal Husbandry&Veterinary Medicine,2018,45(3):763-769.(in Chinese)
[22]GAUTIER-BOUCHARDON A V,FERRS,GRAND D L,et al.Overall decrease in the susceptibility of Mycoplasma bovis to antimicrobials over the past 30years in France[J].PLoS One,2014,9(2):e87672.
[23]邹晓辉,李媛,汪洋,等.牛支原体表面一种新的膜蛋白(P33)的特性研究[J].中国预防兽医学报,2013,35(4):255-258.ZOU X H,LI Y,WANG Y,et al.Function study of a new surface protein(P33)in Mycoplasma bovis[J].Chinese Journal of Preventive Veterinary Medicine,2013,35(4):255-258.(in Chinese)
[24]吴金迪.牛支原体表面一种新的膜蛋白(P41)的鉴定及特性研究[D].哈尔滨:东北农业大学,2014.WU J D.Characterization of a new surface protein(P41)in Mycoplasma bovis[D].Harbin:Northeast Agricultural University,2014.(in Chinese)
[25]赵阳,陈创夫,剡根强,等.牛支原体新疆分离株重组P81膜蛋白的表达及鉴定[J].中国病原生物学杂志,2017,12(10):947-951.ZHAO Y,CHEN C F,YAN G Q,et al.An experiment to express and identify membrane protein P81from Mycoplasma bovis isolates from Xinjiang[J].Journal of Pathogen Biology,2017,12(10):947-951.(in Chinese)
[26]TAN L,HU M,YU S,et al.Characterization of the chaperonin GroEL in Mycoplasma gallisepticum[J].Archives of Microbiology,2015,197(2):235-244.
[27]GUO Y,ZHU H,WANG J,et al.TrmFO,a fibronectin-binding adhesin of Mycoplasma bovis[J].International Journal of Molecular Sciences,2017,18(8):1732.
[2]HALE H H,HELMBOLDT C F,PLASTRIDGE W N,et al.Bovine mastitis caused by a Mycoplasma species[J].Cornell Veterinarian,1962,52(4):582-591.
[3]ASKAA G,ERNH.NOTE:Elevation of Mycoplasma agalactiae subsp.bovis to species rank:Mycoplasma bovis(Hale et al.)comb.nov[J].International Journal of Systematic Bacteriology,1976,26(3):323-325.
[4]NICHOLAS R A,AYLING R D.Mycoplasma bovis:Disease,diagnosis,and control[J].Research in Veterinary Science,2003,74(2):105-112.
[5]CASWELL J L,ARCHAMBAULT M.Mycoplasma bovis pneumonia in cattle[J].Animal Health Research Reviews,2007,8(2):161-186.
[6]辛九庆,李媛,郭丹,等.国内首次从患肺炎的犊牛肺脏中分离到牛支原体[J].中国预防兽医学报,2008,30(9):661-664.XIN J Q,LI Y,GUO D,et al.First isolation of Mycoplasma bovis from calf lung with pneumonia in China[J].Chinese Journal of Preventive Veterinary Medicine,2008,30(9):661-664.(in Chinese)
[7]郭雨丝,陈颖钰,赵刚,等.牛支原体病研究进展[J].中国奶牛,2015,14:36-41.GUO Y S,CHEN Y Y,ZHAO G,et al.A review of Mycoplasma bovis[J].China Dairy Cattle,2015,14:36-41.(in Chinese)
[8]李大伟.牛支原体PCR诊断方法的建立及流行病学调查[D].呼和浩特:内蒙古农业大学,2011.LI D W.Establishment of the PCR method for detecting Mycoplasma bovis and epidemiological survey of Mycoplasma bovis disease[D].Hohhot:Inner Mongolia Agricultural University,2011.(in Chinese)
[9]PATEL M S,ROCHE T E.Molecular biology and biochemistry of pyruvate dehydrogenase complexes[J].Faseb Journal Official Publication of the Federation of American Societies for Experimental Biology,1990,4(14):3224-3233.
[10]CORONA L,CILLARA G,TOLA S.Proteomic approach for identification of immunogenic proteins of Mycoplasma mycoides subsp.capri[J].Veterinary Microbiology,2013,167(3-4):434-439.
[11]ROCHA T S,TRAMUTA C,CATANIA S,et al.Cloning,expression,and antigenic characterization of recombinant protein of Mycoplasma gallisepticum expressed in Escherichia coli[J].Poultry Science,2015,94(4):621-627.
[12]JAN G,LE H M,FONTENELLE C,et al.Biochemical and antigenic characterization of Mycoplasma gallisepticum membrane proteins P52and P67(pMGA)[J].Archives of Microbiology,2001,177(1):81-90.
[13]EASLON E,TSANG F,DILOVA I,et al.The dihydrolipoamide acetyltransferase is a novel metabolic longevity factor and is required for calorie restrictionmediated life span extension[J].Journal of Biological Chemistry,2007,282(9):6161-6171.
[14]NADAL I,RICO J,PREZMARTINEZ G,et al.Diacetyl and acetoin production from whey permeate using engineered Lactobacillus casei[J].Journal of Industrial Microbiology&Biotechnology,2009,36(9):1233-1237.
[15]GRNDEL A,JACOBS E,DUMKE R.Interactions of surface-displayed glycolytic enzymes of Mycoplasma pneumoniae with components of the human extracellular matrix[J].International Journal of Medical Microbiology,2016,306(8):675-685.
[16]高翔,邢小勇,冯娜,等.牛支原体武威株fba基因的克隆、表达及亚细胞定位[J].农业生物技术学报,2017,25(2):274-281.GAO X,XING X Y,FENG N,et al.Cloning,expression and subcellular localization of fbagene of Mycoplasma bovis Wuwei strain[J].Journal of Agricultural Biotechnology,2017,25(2):274-281.(in Chinese)
[17]陈忠琼,范媛,周蓓,等.牛支原体膜蛋白6种提取方法的比较研究[J].中国兽医学报,2013,33(6):855-860.CHEN Z Q,FAN Y,ZHOU B,et al.The comparative study on six kinds of extraction methods for membrane proteins of Mycoplasma bovis[J].Chinese Journal of Veterinary Science,2013,33(6):855-860.(in Chinese)
[18]陈曦,朱洪梅,赵刚,等.牛支原体HB0801株VspX蛋白单克隆抗体的制备与鉴定[J].中国畜牧兽医,2017,44(3):868-878.CHEN X,ZHU H M,ZHAO G,et al.Preparation and identification of the monoclonal antibodies against VspX protein in Mycoplasma bovis strain HB0801[J].China Animal Husbandry&Veterinary Medicine,2017,44(3):868-878.(in Chinese)
[19]庄金秋,张颖,梅建国,等.牛支原体检测方法研究进展[J].中国动物检疫,2016,33(11):66-70.ZHUANG J Q,ZHANG Y,MEI J G,et al.Research progress on detection methods of Mycoplasma bovis[J].China Animal Health Inspection,2016,33(11):66-70.(in Chinese)
[20]MULONGO M,PRYSLIAK T,PEREZ-CASAL J.Vaccination of feedlot cattle with extracts and membrane factions from two Mycoplasma bovis isolates results is strong humoral immune responses but does not protect against an experimental challenge[J].Vaccine,2013,31(10):1406-1412.
[21]季文恒,吴娅琴,翟肖辉,等.牛支原体疫苗的研究进展[J].中国畜牧兽医,2018,45(3):763-769.JI W H,WU Y Q,ZHAI X H,et al.Advances on the research of vaccine against Mycoplasma bovis[J].China Animal Husbandry&Veterinary Medicine,2018,45(3):763-769.(in Chinese)
[22]GAUTIER-BOUCHARDON A V,FERRS,GRAND D L,et al.Overall decrease in the susceptibility of Mycoplasma bovis to antimicrobials over the past 30years in France[J].PLoS One,2014,9(2):e87672.
[23]邹晓辉,李媛,汪洋,等.牛支原体表面一种新的膜蛋白(P33)的特性研究[J].中国预防兽医学报,2013,35(4):255-258.ZOU X H,LI Y,WANG Y,et al.Function study of a new surface protein(P33)in Mycoplasma bovis[J].Chinese Journal of Preventive Veterinary Medicine,2013,35(4):255-258.(in Chinese)
[24]吴金迪.牛支原体表面一种新的膜蛋白(P41)的鉴定及特性研究[D].哈尔滨:东北农业大学,2014.WU J D.Characterization of a new surface protein(P41)in Mycoplasma bovis[D].Harbin:Northeast Agricultural University,2014.(in Chinese)
[25]赵阳,陈创夫,剡根强,等.牛支原体新疆分离株重组P81膜蛋白的表达及鉴定[J].中国病原生物学杂志,2017,12(10):947-951.ZHAO Y,CHEN C F,YAN G Q,et al.An experiment to express and identify membrane protein P81from Mycoplasma bovis isolates from Xinjiang[J].Journal of Pathogen Biology,2017,12(10):947-951.(in Chinese)
[26]TAN L,HU M,YU S,et al.Characterization of the chaperonin GroEL in Mycoplasma gallisepticum[J].Archives of Microbiology,2015,197(2):235-244.
[27]GUO Y,ZHU H,WANG J,et al.TrmFO,a fibronectin-binding adhesin of Mycoplasma bovis[J].International Journal of Molecular Sciences,2017,18(8):1732.