创伤弧菌溶血素基因在大肠杆菌中的高效表达和鉴定
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摘要
目的:创伤弧菌(Vibrio vulnificus,Vv)自然存在于近海和海湾的海水和海底沉积物中,属低度嗜盐菌。人通过进食生的牡蛎,经胃肠道粘膜或破损的皮肤接触海水而感染创伤弧菌。创伤弧菌感染后病情发展迅速,最终发展为感染性休克、多器官衰竭而死亡,死亡率高达70%。创伤弧菌的致病性与许多毒力因子有关,包括溶血素、脂多糖、荚膜多糖、弹性组织蛋白激酶和金属蛋白激酶等。研究发现胞外溶血素基因(Vibriovulnificus cytolysin,vvc)是重要的致病因子之一,参与了细菌入侵宿主的毒力机制。本文通过构建创伤弧菌溶血素基因(vvc)的融合表达载体,并实现创伤弧菌溶血素在大肠杆菌中的高效表达,为今后的进一步研究提供依据。
方法:根据GenBank公布的创伤弧菌vvc基因核苷酸序列自行设计了一对引物,并在5'-端与3'-端分别增加BamHⅠ和HindⅢ酶切位点,应用PCR技术从一株创伤弧菌基因组中扩增了vvc基因,PCR产物经BamHⅠ和HindⅢ双酶切回收后,定向插入pET-32a(+)原核表达载体,提取pET32a(+)-vvc重组质粒,进行双酶切并测序,将测定结果与GenBank中已报道的序列进行同源性分析。结果正确后将构建的重组质粒转化于E.coli BL21(DE3)工程菌株中,IPTG诱导表达,表达产物行SDS-PAGE并进行Westernblot鉴定。
结果:构建了pET-32a(+)-vvc融合表达载体,DNA测序证明,获得的vvc基因长度为1311bp,与GenBank中报道的创伤弧菌溶血素基因序列完全一致。SDS-PAGE分析表明,表达的产物为71kDa左右的融合蛋白,该条带出现于经离心处理的沉淀样品中,显示重组的融合蛋白以包涵体的形式获得成功表达。并用灰度扫描软件分析显示,BL21(DE3)菌株融合蛋白表达量约占菌体总蛋白的32%。Western blot结果显示,含有重组质粒pET32a(+)-vvc的BL21(DE3)的诱导表达产物,只与鼠抗Vv血清发生结合,并在Mr为71kDa处可见明显的杂交信号,表明鼠抗Vv抗体能与目的融合蛋白发生特异性结合。
结论:从创伤弧菌的总DNA中克隆了胞外蛋白vvc基因,成功地构建了pET32a(+)-vvc重组质粒,并在大肠杆菌中实现了高效表达和鉴定。本研究为进一步深入了解VVC蛋白的生物学活性,以及诊断试剂盒的制备、阐明该菌的致病机制奠定了基础。
Objective:Vibrio vulnificus is an estuarine gram-negative,halophilic bacterium that is naturally present in coastal waters and the gulf of the water and seabed sediments.Vibrio vulnificus is a new food poisoning,its occurrence and the consumption of raw or cooked shellfish processing at the end of oysters,and can be through the damaged skin or gastrointestinal mucous membrane into the body and infection.The infection caused by this bacterium has attracted special interest because of its powerful rapid development.It can be extended the eventual development of the septic shock diseases,multi-functional organ failure, and then died.Mortality from this infection is more than 70%and death may occur within 1 to 2 days after the first signs of illness.A variety of endotoxins and exotoxins,including a cytolytic hemolysin,lipopolysaccharide,polysaccharide capsules,an elastolytic protease,and aphospholipase A2,have been implicated as virulence factors for this organism.Although a definite role of cytolysin in bacetrial infection is controversial,Vibrio vulnificus cytolysin is still one of the prime candidates in the pathogenesis of disease.To construct expression vector of Vibrio vulnificus cytolysin genes so that we can expression and identification them in the E.colis,which can provide a basis for further study.
Method:In this paper,the cloning and expressing of the cytolysin of Vibrio vulnificus were discussed.A pair of prmiers were designed according to the GeneBank published nucleotide sequence of we.The action site of BamHⅠwas added to 5' terminal of upper primer and HindⅢto the lower one.Vvc gene was cloned by polymerase chain reaction. This result was tested by 1%agarose electrophoresis,and the length of polymerase chain reaction production accorded with the anticipation.With the cleavage of BamHⅠand HindⅢ,the target fragment was inserted oriently into pET-32a(+)prokaryotic expression vector. After enzyme restriction and sequence analysis,the nucleotide data had been further analyzed. The recombinant plasmid was transformed into E.coli BL21(DE3).After 1.0 mmol/L IPTG induce expressed 6 hours,we used SDS-PAGE to analyze and mouse-anti-vv antibody to have Western blot identification.
Results:The nucleotide sequences of the cleavage of enzyme restriction was matched with the NCBI GeneBank reported.It analysis showed that this sequences were composed of 1311bp encoding mature peptide of 437 amino acids.SDS-PAGE analysis suggested that the VVC protein had efficient expressed,the molecular mass of fusion protein was 71kDa.Gray scanning software suggested that fusion protein was about account of 32%of total protein in VVC.Antibody against three Vibrios was got through acquisition mice serum.Then these serums was diluted with 1:200.The western blot result suggested that the prokaryotic expression VVC fusion protein can particularly combined with Vibrio vulnificus anti-serum.
Conclusion:We not only successful gained the vvc genes,but also constructed a recombinant expression plasmid,pET32a(+)-vvc plasmid,which was efficiently expressed in E.coli BL21(DE3).Expressed VVC protein can be used for understanding its biological activity,preparing its diagnostic kits and laying the foundation on its pathogenic mechanism.
方法:根据GenBank公布的创伤弧菌vvc基因核苷酸序列自行设计了一对引物,并在5'-端与3'-端分别增加BamHⅠ和HindⅢ酶切位点,应用PCR技术从一株创伤弧菌基因组中扩增了vvc基因,PCR产物经BamHⅠ和HindⅢ双酶切回收后,定向插入pET-32a(+)原核表达载体,提取pET32a(+)-vvc重组质粒,进行双酶切并测序,将测定结果与GenBank中已报道的序列进行同源性分析。结果正确后将构建的重组质粒转化于E.coli BL21(DE3)工程菌株中,IPTG诱导表达,表达产物行SDS-PAGE并进行Westernblot鉴定。
结果:构建了pET-32a(+)-vvc融合表达载体,DNA测序证明,获得的vvc基因长度为1311bp,与GenBank中报道的创伤弧菌溶血素基因序列完全一致。SDS-PAGE分析表明,表达的产物为71kDa左右的融合蛋白,该条带出现于经离心处理的沉淀样品中,显示重组的融合蛋白以包涵体的形式获得成功表达。并用灰度扫描软件分析显示,BL21(DE3)菌株融合蛋白表达量约占菌体总蛋白的32%。Western blot结果显示,含有重组质粒pET32a(+)-vvc的BL21(DE3)的诱导表达产物,只与鼠抗Vv血清发生结合,并在Mr为71kDa处可见明显的杂交信号,表明鼠抗Vv抗体能与目的融合蛋白发生特异性结合。
结论:从创伤弧菌的总DNA中克隆了胞外蛋白vvc基因,成功地构建了pET32a(+)-vvc重组质粒,并在大肠杆菌中实现了高效表达和鉴定。本研究为进一步深入了解VVC蛋白的生物学活性,以及诊断试剂盒的制备、阐明该菌的致病机制奠定了基础。
Objective:Vibrio vulnificus is an estuarine gram-negative,halophilic bacterium that is naturally present in coastal waters and the gulf of the water and seabed sediments.Vibrio vulnificus is a new food poisoning,its occurrence and the consumption of raw or cooked shellfish processing at the end of oysters,and can be through the damaged skin or gastrointestinal mucous membrane into the body and infection.The infection caused by this bacterium has attracted special interest because of its powerful rapid development.It can be extended the eventual development of the septic shock diseases,multi-functional organ failure, and then died.Mortality from this infection is more than 70%and death may occur within 1 to 2 days after the first signs of illness.A variety of endotoxins and exotoxins,including a cytolytic hemolysin,lipopolysaccharide,polysaccharide capsules,an elastolytic protease,and aphospholipase A2,have been implicated as virulence factors for this organism.Although a definite role of cytolysin in bacetrial infection is controversial,Vibrio vulnificus cytolysin is still one of the prime candidates in the pathogenesis of disease.To construct expression vector of Vibrio vulnificus cytolysin genes so that we can expression and identification them in the E.colis,which can provide a basis for further study.
Method:In this paper,the cloning and expressing of the cytolysin of Vibrio vulnificus were discussed.A pair of prmiers were designed according to the GeneBank published nucleotide sequence of we.The action site of BamHⅠwas added to 5' terminal of upper primer and HindⅢto the lower one.Vvc gene was cloned by polymerase chain reaction. This result was tested by 1%agarose electrophoresis,and the length of polymerase chain reaction production accorded with the anticipation.With the cleavage of BamHⅠand HindⅢ,the target fragment was inserted oriently into pET-32a(+)prokaryotic expression vector. After enzyme restriction and sequence analysis,the nucleotide data had been further analyzed. The recombinant plasmid was transformed into E.coli BL21(DE3).After 1.0 mmol/L IPTG induce expressed 6 hours,we used SDS-PAGE to analyze and mouse-anti-vv antibody to have Western blot identification.
Results:The nucleotide sequences of the cleavage of enzyme restriction was matched with the NCBI GeneBank reported.It analysis showed that this sequences were composed of 1311bp encoding mature peptide of 437 amino acids.SDS-PAGE analysis suggested that the VVC protein had efficient expressed,the molecular mass of fusion protein was 71kDa.Gray scanning software suggested that fusion protein was about account of 32%of total protein in VVC.Antibody against three Vibrios was got through acquisition mice serum.Then these serums was diluted with 1:200.The western blot result suggested that the prokaryotic expression VVC fusion protein can particularly combined with Vibrio vulnificus anti-serum.
Conclusion:We not only successful gained the vvc genes,but also constructed a recombinant expression plasmid,pET32a(+)-vvc plasmid,which was efficiently expressed in E.coli BL21(DE3).Expressed VVC protein can be used for understanding its biological activity,preparing its diagnostic kits and laying the foundation on its pathogenic mechanism.
引文
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3.周丽萍,应斌宇,董海艳,等.创伤弧菌引起小鼠致死性感染的实验研究[J].中华检验医学杂志,2002,25(1):42-44.
4.Roland FP.Leg gangrene and endotoxin shock due to Vibrio parahaemolyticus-an infection acquired in New England coastal waters[J].NEngl J Med,1970,282:1306.
5.Hollis DG,Weaver RE,Baker CN.Thornsberry C.Halophilic Vibrio species isolated from blood cultures[J].J Clin Microbiol,1976,3:425-431.
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7.Jackson JK,Murphree RL,Tamplin ML.Evidence that mortality from Vibrio vulnificus inf- ection results from single strains among heterogeneous populations in shellfish[J].J Clin Microbiol,1997,35(8):2098-2101.
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9.Nakafusa J,Misago N,Miura Y,et al.The importance of serum creatine phosphokinase level in the early diagnosis,and as a prognostic factor,of Vibrio vulnificus infection[J].Br J Dermatol,2001,145(2):280-284.
10.Chang JJ,Sheen IS,Peng SM,et al.Vibrio vulnificus infection-report of 8 cases and review of cases[J].Changgeng Yi Xue Za Zhi,1994,17(4):339-346.
11.Hlady WG,Mullen RC,Hopkin RS.Vibrio vulnificus from raw oysters.Leading cause of re- ported deaths from foodbome illness in Florida[J].J Fla Med Assoc,1993,80(8):536-538.
12. Lerstloompleephunt N, Tantawichien T, Sitprija V. Renal failure in Vibrio vulnificus infecti- on[J]. Ren Fail, 2000,22(3):337-343.
13. AU A, Mehra MR, Stapleton DD, et al. Vibrio vulnificus sepsis in solid organ transplantati- on: a medical nemesis [J]. J Heart Lung Transplant, 1995,14 (3):598-600.
14. Hlady Wg, Klontz Kc. The epidemiology of Vibrio infections in Florida, 1981-1993.[J]. In- fect Dis, 1996,173(5): 1176-1183.
15. Lowenstein CJ, Dinerman JL, Snyder SH. Nitric oxide: A physiologic messenger[J]. Ann Intern Med, 1994,120,227-237.
16. Xie Q, Nathan C. (1994) The high-output nitric oxidepathway: Role and regulation. [J]. Leu- kocyte Biol,1994,56, 576-582.
17. Moncada S, Palmer RM,Higgs EA.Nitric oxide:Physiology, pathophysiology, and pharmacy- ology. Pharmacol.Rev.199 43, 109-142.
18. Kang MK Jhee FC.et al. Induction of Nitric Oxide Synthase Expression by Vibrio vulnificus Cytolysin[J]. Bioch Bioph Res Comm,2002,290,1090-1095.
19. Cohen J. Immunol.1993,14,126-130.
20. Kwon KB, Yang JY.et al. Vibrio vulnificus Cytolysin Induces Superoxide Anion-initiated Apoptotic Signaling Pathway in Human ECV304 Cells[J].Biol Chem,2001.27(50):47518 -47523.
21. Rho HW, Choi MJ. Cytotoxic mechanism of vibrio vulnificus cytolysin in CPAE cells[J]. Life Sci.2002,70:1923-1934.
22. Lee YR, Park KH. A Calcium-Calmodulin Antagonist Blocks Experimental Vibrio vulnificus Cytolysin-Induced Lethality in an Experimental Mouse Model[J].Infe Immu. 2004, 72(10): 6157-6159.
23. Kim HR, Rho HW, Jeong MH, et al.Hemolytic mechanism of cytolysin produced from V. vulnificus[J]. Life Sci 1993;53:571-578.
24. Kim BS, Kim JS. Cholesterol induce oligomerization of Vibrio vulnificus cytolysin specifica- lly[J].Eexp Mole Med,2002,34(3): 239-242.
25. Shao CP, Hor LI. Metalloprotease is not essential for Vibrio vulnificus virulence in mice[J].Infect Immun,2000,68(6):3569-3573.
26.Banner CR,Remove JS,Fryer L,et al.A rapid method for labeling rabbit immunoglobulin with fluorescent for use in detection of fish pathogens[J].Bull eur Assoc Fish Patyol,1992,2:35-37.
27.Baxa DV,Dawai K,Kusuda R,et al.Detection of Flexibacter mantims by fluorescnt antibo- dy technique in experimentino tally infected black sea bream fry[J].Fish pathology,1998,23(3):29-32.
28.张晓华,徐怀恕.中国对虾弧菌病的间接荧光抗体诊断技术[J].海洋湖沼,1997,28(6):604-610.
29.叶林,俞开康.皱纹盘鲍幼鲍溃烂病病原菌的ELISA检测法[J].中国水产科学,1998,5(2):118-122.
30.Robertson AW,Xu HS,Austin B.An enzyme linked immunosorbent assay (ELISA)for the detection,of Vibrio harveyi in penaeid shrimp and water[J].Journal of Microbiological Method- s,1998,34:31-39.
31.Guangign T,Tetsuya I,Koichiro Y,et al.Analysis of functional domains of Vibrio parahem- ohuticus thermostable direct hemolysin using monoclonal antibodies[J].FEMS Microbiology letter,1997,150:289-296.
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2.Powell J L,Strauss K A,Wiley C,et al.Inflammctory cytokine response to Vibrio vulnificus elicited by peripheral blood monoclear cells from chronic alcohol users is associated with biom- aekers of cellular oxidativestress[J].Infect Immunity,2003,71(7):4212-4216.
3.周丽萍,应斌宇,董海艳,等.创伤弧菌引起小鼠致死性感染的实验研究[J].中华检验医学杂志,2002,25(1):42-44.
4.Roland FP.Leg gangrene and endotoxin shock due to Vibrio parahaemolyticus-an infection acquired in New England coastal waters[J].NEngl J Med,1970,282:1306.
5.Hollis DG,Weaver RE,Baker CN.Thornsberry C.Halophilic Vibrio species isolated from blood cultures[J].J Clin Microbiol,1976,3:425-431.
6.Farmer JJ Ⅲ.Vibrio('Beneckea')vulnificus.The bacterium associated with sepsis,Septica- emia,and the sea[J].Lancet,1979,ⅱ:903.
7.Jackson JK,Murphree RL,Tamplin ML.Evidence that mortality from Vibrio vulnificus inf- ection results from single strains among heterogeneous populations in shellfish[J].J Clin Microbiol,1997,35(8):2098-2101.
8.Klolntz KC,Lieb S,Schreiber M,et al.Syndromes of Vibrio vulnificus infection-clinical and epidemiologic features in Florida cases,1981-1987[J].Ann Intern Med,1988,109:318-323.
9.Nakafusa J,Misago N,Miura Y,et al.The importance of serum creatine phosphokinase level in the early diagnosis,and as a prognostic factor,of Vibrio vulnificus infection[J].Br J Dermatol,2001,145(2):280-284.
10.Chang JJ,Sheen IS,Peng SM,et al.Vibrio vulnificus infection-report of 8 cases and review of cases[J].Changgeng Yi Xue Za Zhi,1994,17(4):339-346.
11.Hlady WG,Mullen RC,Hopkin RS.Vibrio vulnificus from raw oysters.Leading cause of re- ported deaths from foodbome illness in Florida[J].J Fla Med Assoc,1993,80(8):536-538.
12. Lerstloompleephunt N, Tantawichien T, Sitprija V. Renal failure in Vibrio vulnificus infecti- on[J]. Ren Fail, 2000,22(3):337-343.
13. AU A, Mehra MR, Stapleton DD, et al. Vibrio vulnificus sepsis in solid organ transplantati- on: a medical nemesis [J]. J Heart Lung Transplant, 1995,14 (3):598-600.
14. Hlady Wg, Klontz Kc. The epidemiology of Vibrio infections in Florida, 1981-1993.[J]. In- fect Dis, 1996,173(5): 1176-1183.
15. Lowenstein CJ, Dinerman JL, Snyder SH. Nitric oxide: A physiologic messenger[J]. Ann Intern Med, 1994,120,227-237.
16. Xie Q, Nathan C. (1994) The high-output nitric oxidepathway: Role and regulation. [J]. Leu- kocyte Biol,1994,56, 576-582.
17. Moncada S, Palmer RM,Higgs EA.Nitric oxide:Physiology, pathophysiology, and pharmacy- ology. Pharmacol.Rev.199 43, 109-142.
18. Kang MK Jhee FC.et al. Induction of Nitric Oxide Synthase Expression by Vibrio vulnificus Cytolysin[J]. Bioch Bioph Res Comm,2002,290,1090-1095.
19. Cohen J. Immunol.1993,14,126-130.
20. Kwon KB, Yang JY.et al. Vibrio vulnificus Cytolysin Induces Superoxide Anion-initiated Apoptotic Signaling Pathway in Human ECV304 Cells[J].Biol Chem,2001.27(50):47518 -47523.
21. Rho HW, Choi MJ. Cytotoxic mechanism of vibrio vulnificus cytolysin in CPAE cells[J]. Life Sci.2002,70:1923-1934.
22. Lee YR, Park KH. A Calcium-Calmodulin Antagonist Blocks Experimental Vibrio vulnificus Cytolysin-Induced Lethality in an Experimental Mouse Model[J].Infe Immu. 2004, 72(10): 6157-6159.
23. Kim HR, Rho HW, Jeong MH, et al.Hemolytic mechanism of cytolysin produced from V. vulnificus[J]. Life Sci 1993;53:571-578.
24. Kim BS, Kim JS. Cholesterol induce oligomerization of Vibrio vulnificus cytolysin specifica- lly[J].Eexp Mole Med,2002,34(3): 239-242.
25. Shao CP, Hor LI. Metalloprotease is not essential for Vibrio vulnificus virulence in mice[J].Infect Immun,2000,68(6):3569-3573.
26.Banner CR,Remove JS,Fryer L,et al.A rapid method for labeling rabbit immunoglobulin with fluorescent for use in detection of fish pathogens[J].Bull eur Assoc Fish Patyol,1992,2:35-37.
27.Baxa DV,Dawai K,Kusuda R,et al.Detection of Flexibacter mantims by fluorescnt antibo- dy technique in experimentino tally infected black sea bream fry[J].Fish pathology,1998,23(3):29-32.
28.张晓华,徐怀恕.中国对虾弧菌病的间接荧光抗体诊断技术[J].海洋湖沼,1997,28(6):604-610.
29.叶林,俞开康.皱纹盘鲍幼鲍溃烂病病原菌的ELISA检测法[J].中国水产科学,1998,5(2):118-122.
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