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

发布时间：2018-05-17 19:18

  本文选题：铜绿假单胞菌 + 重组Bb-OprF疫苗　； 参考：《重庆医科大学》2012年博士论文

【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。
[Abstract]:objective
RBb-OprF vaccine of Pseudomonas aeruginosa was constructed and identified; the expression efficiency of recombinant plasmids and the immunogenicity of recombinant protein OprF were analyzed and detected. After immunization of mice with 4 different inoculation routes, the amount of Pa in the lung tissue of the experimental animal (BALB/c mice) was counted with PAO1 strain, and the changes of serum IgG and its subclasses, IgA and IgE were observed. By detecting the proliferation of spleen cells, the number of CD4~+ and CD8~+T subgroups, the changes of CK (IFN- gamma, IL-12, TNF- A and IL-10) in the supernatant of spleen cells and the apoptosis of spleen cells, the protective immune mechanism of rBb-OprF vaccine was clarified, and the valuable theoretical data for the research of Pa vaccine were provided.
Method
1, vaccine construction
Using the total RNA of the standard strain PAO1 of Pseudomonas aeruginosa as the template, the sequence of the OprF antigen encoding gene was amplified by RT-PCR, and the oprF27-1032 target gene fragment was amplified by PCR, and the recombinant plasmid pGEX-OprF was constructed in the pGEX-1LambdaT shuttle plasmid in the downstream of the pGEX-1LambdaT shuttle plasmid, and the recombinant plasmid pGEX-OprF was constructed, and the recombinant plasmid was transformed into the receptive coliform pole. The strain BL21 (DE3) was induced by isopropylthiosulfate beta -D- galactoside (IPTG), and the recombinant plasmid pGEX-OprF was introduced into Bb by PCR identification and sequencing analysis sequence. The recombinant Bb-OprF vaccine of Pseudomonas aeruginosa was constructed and its expression was detected at mRNA level. PGEX-oprF in Escherichia coli BL21 (IPTG) was induced by SDS-PAGE. The target protein expressed in 1 ~ 13h was analyzed and immunogenicity of recombinant OprF protein was identified by Western blot.
2, protective immunity mechanism
In order to study the protective effect of Pseudomonas aeruginosa rBb-OprF vaccine on acute Pa pneumonia in BALB/c mice, 56 female and clean BALB/c mice were randomly divided into 7 groups according to different immune pathways, with 8 rats in each group.
Group A: rBb-OprF vaccine was injected subcutaneously (SC), and the 5 * 10~6CFU vaccine was suspended in a 100 MRS L liquid medium and injected subcutaneously 1 times on the back.
Group B: intramuscular injection of rBb-OprF (IM), suspension of 5 * 10~6CFU vaccine in 100 L MRS liquid medium, injecting 1 times to four leg muscles of hind leg of mice.
Group C: rBb-OprF nasal mucous membrane inoculation (IN), 5 x 10~5CFU vaccine was suspended in 100 L MRS liquid medium, nasal mucosa was inoculated 1 times.
Group D: rBb-OprF was orally inoculated (PO), and a 5 * 10~8CFU vaccine was suspended in a 100 MRS L liquid medium and administered orally for 1 times.
Group E: empty load control group (Ca), 5 x 10~6CFU Bb (pGEX-1LambdaT) was suspended in MRS liquid medium of 100 L, and 1 times were injected subcutaneously on the back.
Group F: Bb control group (Bb). The Bb of 5 x 10~6CFU was suspended in MRS liquid medium of 100 L, and was injected subcutaneously 1 times on the back.
Group G: MRS control group (MRS), MRS liquid medium of 100 L, subcutaneous injection for 1 times.
Each group was anaesthetized with 8W, ether anaesthetized, and 109CFU/mL of 50 mu L was injected into the nasal cavity with a curved inoculation needle and injected into the nasal cavity. The serum and spleen cells were collected after the PAO1 strain of nasal cavity infection and 1W, and the serum specific IgG, IgA and IgE antibody levels were detected by the classical ELISA method, and the MTT colorimetric assay was used to detect the serum specificity of the mice in each group of BALB/c. The cell proliferation level of splenocytes or PaAg was measured. The changes of CD++4 and CD8 subgroups in splenocytes were detected by FCM, and the levels of IFN- gamma, IL-12, TNF- alpha and IL-10 in the supernatant were detected by ELISA and the apoptosis rate of splenocytes was detected by AnnexinV-FITC kit.
Result
The OprF encoding gene of 1016bp was amplified by RT-PCR, and the recombinant plasmid of pGEX-OprF was successfully constructed. The vector fragment of 4947bp and the target gene fragment of 1016bp were cut out by double enzyme digestion. The sequence analysis found that the recombinant plasmid was in agreement with the expected result. The oprF gene fragment of 1016bp could be obtained with the rBb extraction plasmid as a template, and the positive rBb was obtained. The total RNA was amplified by RT-PCR. The product was identified by 1.2% agarose gel electrophoresis, and a band of the treaty 1016bp was found. It proved that the exogenous gene oprF could express the molecular weight of mRNA.SDS-PAGE analysis expression product of 61kDa in Bb, which was consistent with the expected result, and the expression of the target protein was 16% of the total body protein of the bacteria; Western blot identification. Results the recombinant protein can be identified by Pa outer membrane antigen in mice, suggesting that the recombinant OprF antigen has the same antigenicity with the natural OprF protein and successfully constructed the rBb-OprF vaccine of Pseudomonas aeruginosa.
After immunization with rBb-OprF vaccine, 8W was detected by 1W infection in the nasal cavity infection of a 50 PAO1 L 10~9CFU/ml floating strain.
(1) the amount of Pa charged bacteria in the lung of the vaccine group: SC group, IM group, IN group and PO group were lower than the MRS control group, SC group and IM group, IN group and PO group were different, there was no statistical difference between the no-load body, Bb and the control group.
(2) the results of serum antibody test by ELISA method: the level of serum IgG, IgG1, IgG2a and IgG2b in the immunization group increased significantly, and the level of IgG3 in the subcutaneous injection group and the oral administration group increased significantly; the level of IgA in the subcutaneous injection group increased, and the IgE level in the immunization group was not significantly changed.
(3) the proliferation of splenic lymphocytes detected by MTT method: the proliferation of spleen T cells in all vaccination groups was obvious, while those in group SC and IM were higher than those in IN group and PO group.
(4) the results of splenic CD4+T cells and CD8+T cells were detected by flow cytometry: the CD+4T and CD+8T cells in the splenocytes of each group increased significantly; the CD4+T cells in the SC group were the highest, the CD+8T cells in the SC group, the IM group and the IN higher than the PO group.
(5) using Annexin V-FITC kits to detect the apoptosis of spleen cells in mice: the incidence of splenic cell apoptosis in the vaccination group was lower than that in the IM group, IN and PO group, respectively. The SC group was lower than that of the IM group.
conclusion
The OprF antigen encoding gene was successfully amplified by RT-PCR, and the rBb-OprF vaccine of Pseudomonas aeruginosa was successfully constructed. The recombinant plasmid pGEX-OprF could be expressed in the Escherichia coli BL21, with high expression efficiency. The recombinant fusion protein had the same antigenicity as that of the natural OprF protein, and the rBb-OprF vaccine could induce the stronger protective immunity of BALB/c mice. Pestilence, subcutaneous and intramuscular injection is the better route of inoculation, and Pseudomonas aeruginosa rBb-OprF vaccine can induce mice to produce a mixed Th1 and Th2 immune response.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392

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