铜绿假单胞菌重组Bb-oprE疫苗构建及其免疫机制的初步研究
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摘要
目的
构建并鉴定铜绿假单胞菌rBb-OprF疫苗；分析和检测重组质粒的表达效率和重组蛋白OprF的免疫原性；采用4种不同接种途径疫苗免疫小鼠后，用PAO1菌株攻击，计数实验动物（BALB/c小鼠）肺组织Pa菌荷量，观察血清IgG及其亚类、IgA和IgE的变化；通过检测小鼠脾细胞增殖情况、CD4~+和CD8~+T亚群数量、脾细胞培养上清液中CK (IFN-γ、IL-12、TNF-α和IL-10)的变化和脾细胞凋亡情况，从而初步阐明rBb-OprF疫苗的保护性免疫机制，为Pa疫苗的研制提供有价值的理论资料。
方法
1、疫苗构建
以铜绿假单胞菌标准株PAO1的总RNA为模板，通过RT-PCR扩增获得OprF抗原编码基因序列，用PCR扩增oprF27-1032目的基因片段，定向克隆入pGEX-1LambdaT穿梭质粒中GST标签下游的MCS，构建重组表达质粒pGEX-OprF；将重组质粒pGEX-OprF转化感受态大肠杆菌BL21（DE3），经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达；用PCR鉴定和测序分析序列正确后用电穿孔法将重组质粒pGEX-OprF导入Bb，构建铜绿假单胞菌重组Bb-OprF疫苗，在mRNA水平检测其表达情况。用SDS-PAGE对pGEX-oprF在大肠杆菌BL21（DE3）中经IPTG诱导1～13h表达的目的蛋白进行分析，并用Western blot对重组OprF蛋白进行免疫原性鉴定。
2、保护性免疫机制
为了研究铜绿假单胞菌rBb-OprF疫苗对BALB/c小鼠急性Pa肺炎的保护作用，设计将56只雌性、清洁级的BALB/c小鼠按不同免疫途径随机分为7组，每组8只。
A组: rBb-OprF疫苗皮下注射(SC)，将5×10~6CFU疫苗悬浮于100μL的MRS液体培养基，背部皮下注射1次；
B组：rBb-OprF肌肉注射(IM)，将5×10~6CFU的疫苗悬浮于100μL的MRS液体培养基，对小鼠后腿股四头肌注射1次；
C组：rBb-OprF鼻腔粘膜接种(IN)，将5×10~5CFU的疫苗悬浮于100μL的MRS液体培养基，鼻腔黏膜接种1次；
D组：rBb-OprF口服灌胃接种(PO)，将5×10~8CFU的疫苗悬浮于100μL的MRS液体培养基，口服灌胃1次；
E组：空载体对照组(Ca)，将5×10~6CFU的Bb(pGEX-1LambdaT)悬浮于100μL的MRS液体培养基，背部皮下注射1次；
F组：Bb对照组(Bb)，将5×10~6CFU的Bb悬浮于100μL的MRS液体培养基，背部皮下注射1次；
G组：MRS对照组(MRS)，100μL的MRS液体培养基，背部皮下注射1次。
各组按上述接种BALB/c小鼠后8w，乙醚麻醉，用弯曲接种针吸50μL的109CFU/mL的浮游PAO1菌株，从鼻腔注入。在PAO1株鼻腔感染攻击后1w，收集血清和脾细胞，用经典的ELISA法检测各组小鼠血清特异性的IgG及其亚类、IgA和IgE抗体水平；用MTT比色法检测脾细胞原液或PaAg刺激后细胞增殖水平变化；利用FCM检测脾细胞CD++4和CD8亚群变化；用ELISA法检测脾细胞原液或PaAg刺激后培养上清液中IFN-γ、IL-12、TNF-α和IL-10的水平以及用AnnexinV-FITC试剂盒检测脾细胞凋亡率。
结果
采用RT-PCR扩增出1016bp的OprF编码基因；成功构建pGEX-OprF重组质粒，经双酶切鉴定，切出4947bp的载体片段和1016bp的目的基因片段；序列分析发现其与预期结果一致；以rBb抽提质粒为模板进行PCR扩增可得到1016bp的oprF基因片段，以阳性rBb的总RNA为模板进行RT-PCR扩增，产物经1.2%的琼脂糖凝胶电泳鉴定，可见一条约1016bp的条带，证明外源基因oprF能够在Bb中表达mRNA。SDS-PAGE分析表达产物分子质量约为61kDa，与预期结果一致，表达的目的蛋白质占菌体总蛋白的16%；Western blot鉴定结果重组蛋白能被Pa外膜粗抗原免疫小鼠血清识别，提示重组OprF抗原与天然OprF蛋白具有相同的抗原性，成功构建铜绿假单胞菌rBb-OprF疫苗。
rBb-OprF疫苗免疫后8w，用50μL的10~9CFU/ml的浮游PAO1菌株鼻腔内感染攻击后1w检测：
（1）疫苗组肺Pa荷菌量：SC组、IM组、IN组和PO组肺组织Pa菌荷量均低于MRS对照组，SC组与IM组、IN组和PO组间存在差异；空载体、Bb和MRS对照组间差异无统计学意义。
（2）ELISA法检测血清抗体结果：疫苗免疫组血清IgG、IgG1、IgG2a和IgG2b水平显著增加；皮下注射组和口服灌胃组IgG3水平显著增加；皮下注射组IgA水平增加；疫苗免疫组IgE水平无明显变化。
（3）MTT法检测的小鼠脾淋巴细胞增殖结果:所有疫苗接种组脾T细胞增殖明显，SC组和IM组高于IN组和PO组。
（4）流式细胞仪检测脾CD4+T细胞和CD8+T细胞结果：各疫苗组脾细胞CD+4T细胞和CD+8T细胞显著增加；CD4+T细胞在SC组最高，CD+8T细胞在SC组、IM组和IN高于PO组。
（5）应用Annexin V-FITC kits检测小鼠脾细胞凋亡结果：疫苗接种组脾细胞凋亡发生率降低，SC组低于IM组、IN和PO组。
结论
通过RT-PCR成功扩增出OprF抗原编码基因；成功构建铜绿假单胞菌rBb-OprF疫苗；重组质粒pGEX-OprF能在大肠杆菌BL21中表达，表达效率较高，重组融合蛋白具有与天然OprF蛋白相同的抗原性；rBb-OprF疫苗能诱导BALB/c小鼠产生较强的保护性免疫反应，皮下和肌肉注射接种是较好的接种途径；铜绿假单胞菌rBb-OprF疫苗可诱导小鼠产生混合型的Th1和Th2免疫应答。
Objective
To construct and identify recombinant Bifidobacteria(rBb)-OprFvaccine of Pseudomonas aeruginosa，and then to decompose the expressionof recombinated plasmid pGEX-OprF induced by IPTG in BL21(DE3).BALB/c mice were immunized with the vaccine subcutaneously,intramuscularly, intranasallyand orally，thenthe mice were challenged with109CFU PAO1strain8th week after vaccination and sacrificed on9th weekafter infection. The number of viable bacteria in lung homogenate wascounted. Sera were collected to determine the levels of IgA, IgE, IgG andtheir subclasses by ELISA. The proliferation of splenocytes wassignificantly increased by methyltetrazolium(MTT) assay. Number of CD4+and CD8+subsets in4immunization groups were more than control by flowcytometry(FCM). The level of IFN-γ, IL-12, TNF-αand IL-10in the culturesupernatants were markedly elevated by ELISA. If so, the protectiveimmunologic mechanism of BALB/c mice induced the recombinantBb-OprF vaccine in acute pneumonia of PAO1strain could be preliminarilyexplained and a kind of helpful vaccine to control Pseudomonas aeruginosa pneumonia would be provided.
Methods
OprF coding coding gene was respectively amplified by RT-PCR fromthe total RNA of PAO1strain. oprF27-1032DNA fragment was cloned intoE.coli-Bifidobacterium shuttle expressing plasmid pGEX-1LambdT toconstruct pGEX-OprF. The recombinant plasmid was introduced into E.coliBL21(DE3) and the oprF gene was expressed in the presence ofisopropyl-β-D-thiogalactopyranosid(IPTG). The recombinant plasmidpGEX-OprF was transformed into Bifidobacterium bifidum(Bb) byelectroporation. The recombinant OprF protein was analyzed and identifiedby sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE)and Western blot.
To investigate protective immunity mechanisms induced in BALB/cmice by inoculation with the recombinant Bb vaccine, and subsequentlychallenged with PAO1strain. Fifty-six female BALB/c mice aged12-14weeks were randomly divided into seven groups, eight in each group. GroupA: rBb-OprF was subcutaneously injected per capita5×10~6CFU. Group B:rBb-OprF was intramuscularly injected per capita5×10~6CFU. Group C:rBb-OprF was intranasally injected per capita5×10~6CFU. Group D:rBb-OprF was orally inoculated per capita5×10~8CFU. Group E:Bb(pGEX-1LambdaT) was subcutaneously injected per capita5×10~6CFU.Group F: Bb was subcutaneously injected per capita5×10~6CFU. Group G: 100μLMRS served subcutaneously as control. All the mice were challengedwith5.0×106CFU PAO1of Pseudomonas aeruginosa strain8th week aftervaccination and sacrificed on9th week after infection. The number of viablebacteria in lung homogenate was counted. Sera were collected to determinethe levels of IgA, IgE, IgG and their subclasses by ELISA. The proliferationof splenocytes was examined by methyltetrazolium(MTT) assay. Thepercent of CD4+and CD8+and apoptosis of splenocytes were detected byflow cytometry. The level of IFN-γ, IL-12, TNF-αand IL-10in the culturesupernatants were markedly elevated by ELISA. The percent of apoptosis ofsplenocytes were detected byAnnexin V-FITC kits.
Results
The OprF coding gene was successfully amplified by PCR, thepGEX-OprF plasmid was extracted from the positive clone selected andtestified by an agarose electroporation after restriction endonucleasedigestion were the same as expected (1016bp oprF gene and4947bppGEX-1LambdT). The oprF gene fragment was amplified by PCR fromthe template of pGEX-OprF extracted from rBb vaccine and sequenceanalysis. The oprF gene fragment was amplified by PCR from the templateof the total of rBb. A relative molecular weight of expressed recombinantprotein at approximately61kDa was determined by SDS-PAGE. Theamount of the expressed protein comprised16%of the total bacterialproteins. The fusion protein could be recognized by the sera of mice produced against the crude outer membrane protein.
BALB/c mice were vaccinated with the rBb-OprF vaccinesubcutaneously, intramuscularly, intranasally and orally. Then the mice werechallenged with5.0×10~6CFU PAO1strain on8thweek after vaccination andkilled9thweek after infection. The bacteria count of the4immunizationgroups decreased evidently as compared with control. The levels of IgG,IgG1, IgG2a and IgG2b increased markedly while those of IgE wasinvariability, IgG3increased in subcutaneous and oral groups, IgAincreasedin subcutaneous group. The proliferation of splenocytes was significantlyincreased by MTT assay. Number of CD4~+and CD8~+subsets in4immunization groups were more than control by flow cytometry(FCM). Thelevel of IFN-γ, IL-12, TNF-αand IL-10in the culture supernatants weremarkedly elevated by ELISA. Apoptotic rate of all vaccine group wasdeclined.
Conclusion
OprF coding gene was successfully cloned by RT-PCR. Therecombinant Bb-OprF vaccine of Pseudomonas aeruginosa wassuccessfully constructed. BL21(pGEX-OprF) could express recombinantGST-OprF fusion protein and the expression efficiency was lower. Theantigenicity of the recombinant GST-OprF fusion protein was the same asnative OprF. The recombinant Bb-OprF vaccine could induce moreeffectively protective immune resposes in BALB/c mice. subcutaneous inoculation and intramuscular vaccination are two better immune routes.Mixed Th1/Th2type immune responses may be producted in micevaccinated with the recombinant Bb-OprF vaccine of Pseudomonasaeruginosa.
构建并鉴定铜绿假单胞菌rBb-OprF疫苗；分析和检测重组质粒的表达效率和重组蛋白OprF的免疫原性；采用4种不同接种途径疫苗免疫小鼠后，用PAO1菌株攻击，计数实验动物（BALB/c小鼠）肺组织Pa菌荷量，观察血清IgG及其亚类、IgA和IgE的变化；通过检测小鼠脾细胞增殖情况、CD4~+和CD8~+T亚群数量、脾细胞培养上清液中CK (IFN-γ、IL-12、TNF-α和IL-10)的变化和脾细胞凋亡情况，从而初步阐明rBb-OprF疫苗的保护性免疫机制，为Pa疫苗的研制提供有价值的理论资料。
方法
1、疫苗构建
以铜绿假单胞菌标准株PAO1的总RNA为模板，通过RT-PCR扩增获得OprF抗原编码基因序列，用PCR扩增oprF27-1032目的基因片段，定向克隆入pGEX-1LambdaT穿梭质粒中GST标签下游的MCS，构建重组表达质粒pGEX-OprF；将重组质粒pGEX-OprF转化感受态大肠杆菌BL21（DE3），经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达；用PCR鉴定和测序分析序列正确后用电穿孔法将重组质粒pGEX-OprF导入Bb，构建铜绿假单胞菌重组Bb-OprF疫苗，在mRNA水平检测其表达情况。用SDS-PAGE对pGEX-oprF在大肠杆菌BL21（DE3）中经IPTG诱导1～13h表达的目的蛋白进行分析，并用Western blot对重组OprF蛋白进行免疫原性鉴定。
2、保护性免疫机制
为了研究铜绿假单胞菌rBb-OprF疫苗对BALB/c小鼠急性Pa肺炎的保护作用，设计将56只雌性、清洁级的BALB/c小鼠按不同免疫途径随机分为7组，每组8只。
A组: rBb-OprF疫苗皮下注射(SC)，将5×10~6CFU疫苗悬浮于100μL的MRS液体培养基，背部皮下注射1次；
B组：rBb-OprF肌肉注射(IM)，将5×10~6CFU的疫苗悬浮于100μL的MRS液体培养基，对小鼠后腿股四头肌注射1次；
C组：rBb-OprF鼻腔粘膜接种(IN)，将5×10~5CFU的疫苗悬浮于100μL的MRS液体培养基，鼻腔黏膜接种1次；
D组：rBb-OprF口服灌胃接种(PO)，将5×10~8CFU的疫苗悬浮于100μL的MRS液体培养基，口服灌胃1次；
E组：空载体对照组(Ca)，将5×10~6CFU的Bb(pGEX-1LambdaT)悬浮于100μL的MRS液体培养基，背部皮下注射1次；
F组：Bb对照组(Bb)，将5×10~6CFU的Bb悬浮于100μL的MRS液体培养基，背部皮下注射1次；
G组：MRS对照组(MRS)，100μL的MRS液体培养基，背部皮下注射1次。
各组按上述接种BALB/c小鼠后8w，乙醚麻醉，用弯曲接种针吸50μL的109CFU/mL的浮游PAO1菌株，从鼻腔注入。在PAO1株鼻腔感染攻击后1w，收集血清和脾细胞，用经典的ELISA法检测各组小鼠血清特异性的IgG及其亚类、IgA和IgE抗体水平；用MTT比色法检测脾细胞原液或PaAg刺激后细胞增殖水平变化；利用FCM检测脾细胞CD++4和CD8亚群变化；用ELISA法检测脾细胞原液或PaAg刺激后培养上清液中IFN-γ、IL-12、TNF-α和IL-10的水平以及用AnnexinV-FITC试剂盒检测脾细胞凋亡率。
结果
采用RT-PCR扩增出1016bp的OprF编码基因；成功构建pGEX-OprF重组质粒，经双酶切鉴定，切出4947bp的载体片段和1016bp的目的基因片段；序列分析发现其与预期结果一致；以rBb抽提质粒为模板进行PCR扩增可得到1016bp的oprF基因片段，以阳性rBb的总RNA为模板进行RT-PCR扩增，产物经1.2%的琼脂糖凝胶电泳鉴定，可见一条约1016bp的条带，证明外源基因oprF能够在Bb中表达mRNA。SDS-PAGE分析表达产物分子质量约为61kDa，与预期结果一致，表达的目的蛋白质占菌体总蛋白的16%；Western blot鉴定结果重组蛋白能被Pa外膜粗抗原免疫小鼠血清识别，提示重组OprF抗原与天然OprF蛋白具有相同的抗原性，成功构建铜绿假单胞菌rBb-OprF疫苗。
rBb-OprF疫苗免疫后8w，用50μL的10~9CFU/ml的浮游PAO1菌株鼻腔内感染攻击后1w检测：
（1）疫苗组肺Pa荷菌量：SC组、IM组、IN组和PO组肺组织Pa菌荷量均低于MRS对照组，SC组与IM组、IN组和PO组间存在差异；空载体、Bb和MRS对照组间差异无统计学意义。
（2）ELISA法检测血清抗体结果：疫苗免疫组血清IgG、IgG1、IgG2a和IgG2b水平显著增加；皮下注射组和口服灌胃组IgG3水平显著增加；皮下注射组IgA水平增加；疫苗免疫组IgE水平无明显变化。
（3）MTT法检测的小鼠脾淋巴细胞增殖结果:所有疫苗接种组脾T细胞增殖明显，SC组和IM组高于IN组和PO组。
（4）流式细胞仪检测脾CD4+T细胞和CD8+T细胞结果：各疫苗组脾细胞CD+4T细胞和CD+8T细胞显著增加；CD4+T细胞在SC组最高，CD+8T细胞在SC组、IM组和IN高于PO组。
（5）应用Annexin V-FITC kits检测小鼠脾细胞凋亡结果：疫苗接种组脾细胞凋亡发生率降低，SC组低于IM组、IN和PO组。
结论
通过RT-PCR成功扩增出OprF抗原编码基因；成功构建铜绿假单胞菌rBb-OprF疫苗；重组质粒pGEX-OprF能在大肠杆菌BL21中表达，表达效率较高，重组融合蛋白具有与天然OprF蛋白相同的抗原性；rBb-OprF疫苗能诱导BALB/c小鼠产生较强的保护性免疫反应，皮下和肌肉注射接种是较好的接种途径；铜绿假单胞菌rBb-OprF疫苗可诱导小鼠产生混合型的Th1和Th2免疫应答。
Objective
To construct and identify recombinant Bifidobacteria(rBb)-OprFvaccine of Pseudomonas aeruginosa，and then to decompose the expressionof recombinated plasmid pGEX-OprF induced by IPTG in BL21(DE3).BALB/c mice were immunized with the vaccine subcutaneously,intramuscularly, intranasallyand orally，thenthe mice were challenged with109CFU PAO1strain8th week after vaccination and sacrificed on9th weekafter infection. The number of viable bacteria in lung homogenate wascounted. Sera were collected to determine the levels of IgA, IgE, IgG andtheir subclasses by ELISA. The proliferation of splenocytes wassignificantly increased by methyltetrazolium(MTT) assay. Number of CD4+and CD8+subsets in4immunization groups were more than control by flowcytometry(FCM). The level of IFN-γ, IL-12, TNF-αand IL-10in the culturesupernatants were markedly elevated by ELISA. If so, the protectiveimmunologic mechanism of BALB/c mice induced the recombinantBb-OprF vaccine in acute pneumonia of PAO1strain could be preliminarilyexplained and a kind of helpful vaccine to control Pseudomonas aeruginosa pneumonia would be provided.
Methods
OprF coding coding gene was respectively amplified by RT-PCR fromthe total RNA of PAO1strain. oprF27-1032DNA fragment was cloned intoE.coli-Bifidobacterium shuttle expressing plasmid pGEX-1LambdT toconstruct pGEX-OprF. The recombinant plasmid was introduced into E.coliBL21(DE3) and the oprF gene was expressed in the presence ofisopropyl-β-D-thiogalactopyranosid(IPTG). The recombinant plasmidpGEX-OprF was transformed into Bifidobacterium bifidum(Bb) byelectroporation. The recombinant OprF protein was analyzed and identifiedby sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE)and Western blot.
To investigate protective immunity mechanisms induced in BALB/cmice by inoculation with the recombinant Bb vaccine, and subsequentlychallenged with PAO1strain. Fifty-six female BALB/c mice aged12-14weeks were randomly divided into seven groups, eight in each group. GroupA: rBb-OprF was subcutaneously injected per capita5×10~6CFU. Group B:rBb-OprF was intramuscularly injected per capita5×10~6CFU. Group C:rBb-OprF was intranasally injected per capita5×10~6CFU. Group D:rBb-OprF was orally inoculated per capita5×10~8CFU. Group E:Bb(pGEX-1LambdaT) was subcutaneously injected per capita5×10~6CFU.Group F: Bb was subcutaneously injected per capita5×10~6CFU. Group G: 100μLMRS served subcutaneously as control. All the mice were challengedwith5.0×106CFU PAO1of Pseudomonas aeruginosa strain8th week aftervaccination and sacrificed on9th week after infection. The number of viablebacteria in lung homogenate was counted. Sera were collected to determinethe levels of IgA, IgE, IgG and their subclasses by ELISA. The proliferationof splenocytes was examined by methyltetrazolium(MTT) assay. Thepercent of CD4+and CD8+and apoptosis of splenocytes were detected byflow cytometry. The level of IFN-γ, IL-12, TNF-αand IL-10in the culturesupernatants were markedly elevated by ELISA. The percent of apoptosis ofsplenocytes were detected byAnnexin V-FITC kits.
Results
The OprF coding gene was successfully amplified by PCR, thepGEX-OprF plasmid was extracted from the positive clone selected andtestified by an agarose electroporation after restriction endonucleasedigestion were the same as expected (1016bp oprF gene and4947bppGEX-1LambdT). The oprF gene fragment was amplified by PCR fromthe template of pGEX-OprF extracted from rBb vaccine and sequenceanalysis. The oprF gene fragment was amplified by PCR from the templateof the total of rBb. A relative molecular weight of expressed recombinantprotein at approximately61kDa was determined by SDS-PAGE. Theamount of the expressed protein comprised16%of the total bacterialproteins. The fusion protein could be recognized by the sera of mice produced against the crude outer membrane protein.
BALB/c mice were vaccinated with the rBb-OprF vaccinesubcutaneously, intramuscularly, intranasally and orally. Then the mice werechallenged with5.0×10~6CFU PAO1strain on8thweek after vaccination andkilled9thweek after infection. The bacteria count of the4immunizationgroups decreased evidently as compared with control. The levels of IgG,IgG1, IgG2a and IgG2b increased markedly while those of IgE wasinvariability, IgG3increased in subcutaneous and oral groups, IgAincreasedin subcutaneous group. The proliferation of splenocytes was significantlyincreased by MTT assay. Number of CD4~+and CD8~+subsets in4immunization groups were more than control by flow cytometry(FCM). Thelevel of IFN-γ, IL-12, TNF-αand IL-10in the culture supernatants weremarkedly elevated by ELISA. Apoptotic rate of all vaccine group wasdeclined.
Conclusion
OprF coding gene was successfully cloned by RT-PCR. Therecombinant Bb-OprF vaccine of Pseudomonas aeruginosa wassuccessfully constructed. BL21(pGEX-OprF) could express recombinantGST-OprF fusion protein and the expression efficiency was lower. Theantigenicity of the recombinant GST-OprF fusion protein was the same asnative OprF. The recombinant Bb-OprF vaccine could induce moreeffectively protective immune resposes in BALB/c mice. subcutaneous inoculation and intramuscular vaccination are two better immune routes.Mixed Th1/Th2type immune responses may be producted in micevaccinated with the recombinant Bb-OprF vaccine of Pseudomonasaeruginosa.
引文
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