霍乱弧菌tcpA基因的克隆表达及其免疫保护作用研究
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摘要
霍乱弧菌(Vibrio cholerae,VC)是水产动物中常见的、危害最大的病原菌之一,不仅可引起鱼类严重的脱黏病和细菌性肠炎,而且还可通过水产品感染人类,引起人类烈性肠道传染病。目前控制该病的主要手段是使用抗菌药物,但是由于该菌感染的普遍性、耐药菌株的不断增加,在很大程度上限制了抗菌药物的疗效。因此,该病免疫防治的研究具有重要意义。鉴于霍乱弧菌通过毒素共调菌毛(toxin-coregulated pilus,Tcp)黏附定植于小肠黏膜是其致病的前提,阻断霍乱弧菌的黏附定植即可防止感染的发生。对此,本研究选择暴露于细菌表面并与霍乱弧菌黏附定植密切相关的黏附素TcpA为目标,通过基因工程技术获得重组蛋白GST-TcpA,研究其免疫保护作用,以确定重组TcpA蛋白在霍乱弧菌基因工程亚单位疫苗研制中的应用价值。
首先,应用PCR方法从霍乱弧菌Y1菌株基因组DNA中扩增tcpA结构基因并克隆至pMD18-T载体进行序列测定与分析。序列分析显示tcpA结构基因由675个核苷酸组成,开放阅读框完整无中断,编码由224个氨基酸组成的相对分子量约为21 kD的TcpA蛋白。tcpA结构基因在霍乱弧菌Y1菌株与Genbank中登录的20个霍乱弧菌参考株之间均具有较高的同源性,彼此间的核苷酸序列和推导的氨基酸序列同源性分别介于71.8%-99.7%和57.7%-99.3%之间。这些研究结果为tcpA基因的亚克隆和表达奠定了基础。
其次,将tcpA基因亚克隆至表达载体pGEX-4T-1并在大肠杆菌BL21(DE3)中表达。根据pGEX-4T-1表达载体阅读框的要求,设计1对包含BamHⅠ和XhoⅠ酶切位点的特异引物,以重组克隆质粒pMD-18T-tcpA为模板,扩增tcpA结构基因。PCR产物经BamHⅠ/XhoⅠ双酶切后,亚克隆到表达载体pGEX-4T-1中,并转化至大肠杆菌BL21(DE3)。pGEX-4T-1-tcpA/BL21重组菌经IPTG诱导后,可以包涵体形式高效表达分子量约47.0 kD的重组蛋白GST-TcpA,并经提纯后获得大量的该重组蛋白,从而为进一步研究其免疫保护作用奠定了基础。
最后,分别采用Western blot、细胞黏附抑制试验和免疫保护性试验测定重组蛋白GST-TcpA的抗原性和体内外免疫保护作用。pGEX-4T-1-tcpA/BL21重组菌的诱导表达产物经SDS-PAGE分离后,转移至PVDF膜,以霍乱弧菌菌毛蛋白抗血清为一抗进行Western-blot分析,结果显示重组蛋白GST-TcpA可与菌毛蛋白抗血清发生特异性免疫反应,证实重组TcpA蛋白保持了天然Tcp蛋白的抗原性。以霍乱弧菌菌体、霍乱弧菌菌毛蛋白、重组蛋白GST-TcpA和GST蛋白分别免疫小鼠制备抗血清,检测这四种抗血清在细胞黏附抑制试验中的作用。结果显示菌体抗血清可完全抑制细菌对HEp-2细胞的黏附,菌毛抗血清和重组蛋白抗血清可明显降低细菌对HEp-2细胞的黏附,而GST抗血清无黏附抑制作用。表明重组蛋白抗血清在体外能明显抑制霍乱弧菌Y1菌株对HEp-2细胞的黏附作用,除Tcp外,霍乱弧菌Y1菌株尚表达其它黏附素。实验小鼠分别经纯化重组蛋白GST-TcpA和GST蛋白四次免疫后,用Y1株菌液进行攻击,结果显示重组蛋白免疫组小鼠的免疫保护率为81.25%,而GST免疫组小鼠和生理盐水对照组小鼠全部死亡。说明重组TcpA蛋白对霍乱弧菌感染具有较好的免疫保护作用。
综上所述,本研究在成功构建重组表达质粒pGEX-4T-1-tcpA并获得重组蛋白GST-TcpA的基础上,探明了重组TcpA蛋白仍保留了天然Tcp蛋白的抗原性和免疫保护作用,在霍乱弧菌基因工程疫苗的研制中具有潜在的应用价值。
Vibrio Cholerae is one of the most common and damaging pathogen in aquaculture, which not only leads to serious mucous sloughing disease and bacterial enteritis of fish but also causes human's potent intestinal infectious diseases Cholerae by aquatic products.At present,the primary means to control the disease is to use antibiotics,but the effects of its are limited by the infection of V.cholerae universally and the increase of drug resistant strains continuously,which result in abundant death of aquatic animals suffered from those. So,studying on immunoprophylaxis and immunotherapy of the disease are important. Since the first elementary step of initialing infection is to adhere to the intestinal mucosa by toxin-coregulated pilus(TCP) of V.cholerae,blocking adhesion and colonization of V.cholerae to the intestinal mucosa might prevent V.cholerae infenction.In this study TcpA protein of V.cholerae is taken as a target protein.In order to determine the applied value of recombinant protein TcpA in the developing genetic engineering subunit vaccine of V.cholerae,tcpA gene from V.cholerae strain Y1 was cloned and expressed in prokaryotic by genetic engineering technolongy,and then antigenicity and immuneprotection of the recombinant protein GST-TcpA were detected.
Firstly,tcpA gene was amplified from strain Y1 isolated in Anhui by PCR method and cloned into pMD18-T vector,then sequenced and analyzed by BioXM software.The results revealed that tcpA complete gene contained an open reading frame(ORF) about 675 bp in length without interruption,encoding 224 amino acids with a approximately 21 kD molecular weight.There are 71.8%-99.7%nucleotide sequences homology and 3.4%-87.6%amino acid sequences homology between strain Y1 and some referent strains of V.cholerae registered in GenBank.These results laid a foundation for subcloning and prokaryotic expression of tcpA gene.
Secondly,tcpA gene was subcloned into expressive plasmid pGEX-4T-1 and expressed in E.coli BL21(DE3).A pair of specific primers containing BamHⅠand XhoⅠrestriction enzyme sites were designed.tcpA gene was amplified by PCR method from pMD-18T-tcpA.The purified PCR product was digested by BamHⅠand XhoⅠ,and then was inserted into pGEX-4T-1 and transformed into E.coli BL21(DE3).The recombinant protein GST-TcpA with 47 kD molecular weight was hyperexpressed in the form of inclusion bodies after pGEX-4T-1-tcpA/ BL21 was induced by IPTG.A large number of the recombinant protein GST-TcpA was extracted by conventional protein separate technique at same time.These results laid a foundation for the study on antigenicity and immuneprotection of the recombinant protein GST-TcpA.
Finally,antigenicity and immuneprotection in vitro and in vivo of the recombinant protein GST-TcpA were investigated by western blot,inhibition of cell adhesion and immuneprotection trial respectively.After the Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),the expessive recombinant protein GST-TcpA induced from pGEX-4T-1-tcpA/BL21 was shifted to PVDF film and detected by western blot with antipilus of strain Y1 serum.Western blot result showed anti-serum could react specifically with the recombinant protein,which indicates that the recombinant TcpA has the same antigenicity as natural one's.Anti-serum were prepared in experimental mice immunized with V.cholerae,pili protein of V.cholerae,recombinant protein GST-TcpA and GST protein respectively,and their adhesion inhibition effect was determined by Hep-2 cell infection model.The results showed that anti-V.cholerae serum could inhibit bacteria adhering to Hep-2 cells completely,anti-pili serum and anti-GST-TcpA serum could reduce the capacity of bacteria adhering to Hep-2 cells markedly,but the anti-GST serum showed no adhesion inhibition effect.These indicates that anti-GST-TcpA serum has a powerful anti-adhesion effect on V.cholerae in vitro and V.cholerae can expess other adhesins besides TcpA.After being immunized four times with recombinant protein GST-TcpA or GST protein,the immunized mice were challenged of V.cholerae strain Y1 with 10~8 CFU/mL dose.The mice immunized with recombinant protein GST-TcpA displayed 81.25%immuneprotection rate,but the mice immunized with GST protein and unimmunized mice wholly died.These suggested recombinant protein could induce an immmune response to provide better protection against V.cholerae infection.
The results showed that recombinant protein TcpA remain antigenicity and immuneprotection in vitro and in vivo on the basis of successfully constructing recombinant expressive plasmids pGEX-4T-tcpA and obtaining recombinant protein GST-TcpA,and thus recombinant protein TcpA has the potential to be used for developing of genetic engineering subunit vaccine of V.cholerae.
首先,应用PCR方法从霍乱弧菌Y1菌株基因组DNA中扩增tcpA结构基因并克隆至pMD18-T载体进行序列测定与分析。序列分析显示tcpA结构基因由675个核苷酸组成,开放阅读框完整无中断,编码由224个氨基酸组成的相对分子量约为21 kD的TcpA蛋白。tcpA结构基因在霍乱弧菌Y1菌株与Genbank中登录的20个霍乱弧菌参考株之间均具有较高的同源性,彼此间的核苷酸序列和推导的氨基酸序列同源性分别介于71.8%-99.7%和57.7%-99.3%之间。这些研究结果为tcpA基因的亚克隆和表达奠定了基础。
其次,将tcpA基因亚克隆至表达载体pGEX-4T-1并在大肠杆菌BL21(DE3)中表达。根据pGEX-4T-1表达载体阅读框的要求,设计1对包含BamHⅠ和XhoⅠ酶切位点的特异引物,以重组克隆质粒pMD-18T-tcpA为模板,扩增tcpA结构基因。PCR产物经BamHⅠ/XhoⅠ双酶切后,亚克隆到表达载体pGEX-4T-1中,并转化至大肠杆菌BL21(DE3)。pGEX-4T-1-tcpA/BL21重组菌经IPTG诱导后,可以包涵体形式高效表达分子量约47.0 kD的重组蛋白GST-TcpA,并经提纯后获得大量的该重组蛋白,从而为进一步研究其免疫保护作用奠定了基础。
最后,分别采用Western blot、细胞黏附抑制试验和免疫保护性试验测定重组蛋白GST-TcpA的抗原性和体内外免疫保护作用。pGEX-4T-1-tcpA/BL21重组菌的诱导表达产物经SDS-PAGE分离后,转移至PVDF膜,以霍乱弧菌菌毛蛋白抗血清为一抗进行Western-blot分析,结果显示重组蛋白GST-TcpA可与菌毛蛋白抗血清发生特异性免疫反应,证实重组TcpA蛋白保持了天然Tcp蛋白的抗原性。以霍乱弧菌菌体、霍乱弧菌菌毛蛋白、重组蛋白GST-TcpA和GST蛋白分别免疫小鼠制备抗血清,检测这四种抗血清在细胞黏附抑制试验中的作用。结果显示菌体抗血清可完全抑制细菌对HEp-2细胞的黏附,菌毛抗血清和重组蛋白抗血清可明显降低细菌对HEp-2细胞的黏附,而GST抗血清无黏附抑制作用。表明重组蛋白抗血清在体外能明显抑制霍乱弧菌Y1菌株对HEp-2细胞的黏附作用,除Tcp外,霍乱弧菌Y1菌株尚表达其它黏附素。实验小鼠分别经纯化重组蛋白GST-TcpA和GST蛋白四次免疫后,用Y1株菌液进行攻击,结果显示重组蛋白免疫组小鼠的免疫保护率为81.25%,而GST免疫组小鼠和生理盐水对照组小鼠全部死亡。说明重组TcpA蛋白对霍乱弧菌感染具有较好的免疫保护作用。
综上所述,本研究在成功构建重组表达质粒pGEX-4T-1-tcpA并获得重组蛋白GST-TcpA的基础上,探明了重组TcpA蛋白仍保留了天然Tcp蛋白的抗原性和免疫保护作用,在霍乱弧菌基因工程疫苗的研制中具有潜在的应用价值。
Vibrio Cholerae is one of the most common and damaging pathogen in aquaculture, which not only leads to serious mucous sloughing disease and bacterial enteritis of fish but also causes human's potent intestinal infectious diseases Cholerae by aquatic products.At present,the primary means to control the disease is to use antibiotics,but the effects of its are limited by the infection of V.cholerae universally and the increase of drug resistant strains continuously,which result in abundant death of aquatic animals suffered from those. So,studying on immunoprophylaxis and immunotherapy of the disease are important. Since the first elementary step of initialing infection is to adhere to the intestinal mucosa by toxin-coregulated pilus(TCP) of V.cholerae,blocking adhesion and colonization of V.cholerae to the intestinal mucosa might prevent V.cholerae infenction.In this study TcpA protein of V.cholerae is taken as a target protein.In order to determine the applied value of recombinant protein TcpA in the developing genetic engineering subunit vaccine of V.cholerae,tcpA gene from V.cholerae strain Y1 was cloned and expressed in prokaryotic by genetic engineering technolongy,and then antigenicity and immuneprotection of the recombinant protein GST-TcpA were detected.
Firstly,tcpA gene was amplified from strain Y1 isolated in Anhui by PCR method and cloned into pMD18-T vector,then sequenced and analyzed by BioXM software.The results revealed that tcpA complete gene contained an open reading frame(ORF) about 675 bp in length without interruption,encoding 224 amino acids with a approximately 21 kD molecular weight.There are 71.8%-99.7%nucleotide sequences homology and 3.4%-87.6%amino acid sequences homology between strain Y1 and some referent strains of V.cholerae registered in GenBank.These results laid a foundation for subcloning and prokaryotic expression of tcpA gene.
Secondly,tcpA gene was subcloned into expressive plasmid pGEX-4T-1 and expressed in E.coli BL21(DE3).A pair of specific primers containing BamHⅠand XhoⅠrestriction enzyme sites were designed.tcpA gene was amplified by PCR method from pMD-18T-tcpA.The purified PCR product was digested by BamHⅠand XhoⅠ,and then was inserted into pGEX-4T-1 and transformed into E.coli BL21(DE3).The recombinant protein GST-TcpA with 47 kD molecular weight was hyperexpressed in the form of inclusion bodies after pGEX-4T-1-tcpA/ BL21 was induced by IPTG.A large number of the recombinant protein GST-TcpA was extracted by conventional protein separate technique at same time.These results laid a foundation for the study on antigenicity and immuneprotection of the recombinant protein GST-TcpA.
Finally,antigenicity and immuneprotection in vitro and in vivo of the recombinant protein GST-TcpA were investigated by western blot,inhibition of cell adhesion and immuneprotection trial respectively.After the Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),the expessive recombinant protein GST-TcpA induced from pGEX-4T-1-tcpA/BL21 was shifted to PVDF film and detected by western blot with antipilus of strain Y1 serum.Western blot result showed anti-serum could react specifically with the recombinant protein,which indicates that the recombinant TcpA has the same antigenicity as natural one's.Anti-serum were prepared in experimental mice immunized with V.cholerae,pili protein of V.cholerae,recombinant protein GST-TcpA and GST protein respectively,and their adhesion inhibition effect was determined by Hep-2 cell infection model.The results showed that anti-V.cholerae serum could inhibit bacteria adhering to Hep-2 cells completely,anti-pili serum and anti-GST-TcpA serum could reduce the capacity of bacteria adhering to Hep-2 cells markedly,but the anti-GST serum showed no adhesion inhibition effect.These indicates that anti-GST-TcpA serum has a powerful anti-adhesion effect on V.cholerae in vitro and V.cholerae can expess other adhesins besides TcpA.After being immunized four times with recombinant protein GST-TcpA or GST protein,the immunized mice were challenged of V.cholerae strain Y1 with 10~8 CFU/mL dose.The mice immunized with recombinant protein GST-TcpA displayed 81.25%immuneprotection rate,but the mice immunized with GST protein and unimmunized mice wholly died.These suggested recombinant protein could induce an immmune response to provide better protection against V.cholerae infection.
The results showed that recombinant protein TcpA remain antigenicity and immuneprotection in vitro and in vivo on the basis of successfully constructing recombinant expressive plasmids pGEX-4T-tcpA and obtaining recombinant protein GST-TcpA,and thus recombinant protein TcpA has the potential to be used for developing of genetic engineering subunit vaccine of V.cholerae.
引文
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50. Beck NA, Krukonis ES, DiRita VJ. TcpH influences virulence gene expression in Vibrio cholerae by inhibiting degradation of the transcription activator TcpP[J]. J Bacteriol, 2004, 186(24): 8309-8316
51. Harkey CW, Everiss KD, Peterson KM. The Vibrio cholerae toxin-coregulated pilus gene tcpI encodes a homology of methyl-accepting chemotaxis proteins[J]. Infect Immun, 1994, 62(7): 2669-2678
52. Ogierman MA, Voss E, Meaney C, et al. Comparison of the promoter proximal regions of the tcp gene cluster in classical and El Tor strains of Vibrio choterae O1[J]. Gene, 1996,170(1): 9-16
53. Hulbert RR and Taylor RK. Mechanism of ToxT-dependent transcriptional activation at the Vibrio cholerae tcpA promoter[J]. J Bacteriol, 2002,184(20): 5533-5544
54. Withey JH, DiRita VJ. The toxbox: specific DNA sequence requirements for activation of Vibrio cholerae virulence genes by ToxT[J]. Mol Microbiol, 2006, 59(6): 1779-1989
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56. Miller VL, Taylor RK, Mekalanos JJ. Cholera toxin transcriptional activator toxR is a transmem-brane DNA binding protein[J]. Cell, 1987,48(2): 271-279
57. Dirita VJ, Mekalanos JJ . Periplasmic interaction between two membrane regulatory proteins, ToxR and ToxS, results in signal transduction and transcriptional activation[J]. Cell, 1991,64(1): 29-37
58. Lee AY, Park SG, Jang M, et al. Proteomic analysis of pathogenic bacterium Vibrio vulnificus[J]. Proteomics, 2006,6(4): 1283-1289
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64.胡守奎,李槿年,余为一.蟹源致病性拟态弧菌的黏附及侵袭特性[J].水产学报,2004,28(5):541-546
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72.贾文祥,杜艳,刘莉等.霍乱弧菌O139菌毛提取及其疫苗的试制[J].中华微生物学和免疫学杂志,1999,19(6):519-522
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76.陈清,王雅贤,俞守义.环境对产毒大肠杆菌定居因子表达的调控[J].中华微生物学和免疫学杂志,2001,21(1):6-9
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