本文選題：銅綠假單胞菌 + 重組Bb-OprF疫苗　； 參考：《重慶醫(yī)科大學(xué)》2012年博士論文

【摘要】：目的 構(gòu)建并鑒定銅綠假單胞菌rBb-OprF疫苗；分析和檢測重組質(zhì)粒的表達效率和重組蛋白OprF的免疫原性；采用4種不同接種途徑疫苗免疫小鼠后，用PAO1菌株攻擊，計數(shù)實驗動物（BALB/c小鼠）肺組織Pa菌荷量，觀察血清IgG及其亞類、IgA和IgE的變化；通過檢測小鼠脾細胞增殖情況、CD4~+和CD8~+T亞群數(shù)量、脾細胞培養(yǎng)上清液中CK (IFN-γ、IL-12、TNF-α和IL-10)的變化和脾細胞凋亡情況，從而初步闡明rBb-OprF疫苗的保護性免疫機制，為Pa疫苗的研制提供有價值的理論資料。 方法 1、疫苗構(gòu)建 以銅綠假單胞菌標準株P(guān)AO1的總RNA為模板，通過RT-PCR擴增獲得OprF抗原編碼基因序列，用PCR擴增oprF27-1032目的基因片段，定向克隆入pGEX-1LambdaT穿梭質(zhì)粒中GST標簽下游的MCS，構(gòu)建重組表達質(zhì)粒pGEX-OprF；將重組質(zhì)粒pGEX-OprF轉(zhuǎn)化感受態(tài)大腸桿菌BL21（DE3），經(jīng)異丙基硫代-β-D-半乳糖苷(IPTG)誘導(dǎo)表達；用PCR鑒定和測序分析序列正確后用電穿孔法將重組質(zhì)粒pGEX-OprF導(dǎo)入Bb，構(gòu)建銅綠假單胞菌重組Bb-OprF疫苗，在mRNA水平檢測其表達情況。用SDS-PAGE對pGEX-oprF在大腸桿菌BL21（DE3）中經(jīng)IPTG誘導(dǎo)1～13h表達的目的蛋白進行分析，并用Western blot對重組OprF蛋白進行免疫原性鑒定。 2、保護性免疫機制 為了研究銅綠假單胞菌rBb-OprF疫苗對BALB/c小鼠急性Pa肺炎的保護作用，設(shè)計將56只雌性、清潔級的BALB/c小鼠按不同免疫途徑隨機分為7組，每組8只。 A組: rBb-OprF疫苗皮下注射(SC)，將5×10~6CFU疫苗懸浮于100μL的MRS液體培養(yǎng)基，背部皮下注射1次； B組：rBb-OprF肌肉注射(IM)，將5×10~6CFU的疫苗懸浮于100μL的MRS液體培養(yǎng)基，對小鼠后腿股四頭肌注射1次； C組：rBb-OprF鼻腔粘膜接種(IN)，將5×10~5CFU的疫苗懸浮于100μL的MRS液體培養(yǎng)基，鼻腔黏膜接種1次； D組：rBb-OprF口服灌胃接種(PO)，將5×10~8CFU的疫苗懸浮于100μL的MRS液體培養(yǎng)基，，口服灌胃1次； E組：空載體對照組(Ca)，將5×10~6CFU的Bb(pGEX-1LambdaT)懸浮于100μL的MRS液體培養(yǎng)基，背部皮下注射1次； F組：Bb對照組(Bb)，將5×10~6CFU的Bb懸浮于100μL的MRS液體培養(yǎng)基，背部皮下注射1次； G組：MRS對照組(MRS)，100μL的MRS液體培養(yǎng)基，背部皮下注射1次。 各組按上述接種BALB/c小鼠后8w，乙醚麻醉，用彎曲接種針吸50μL的109CFU/mL的浮游PAO1菌株，從鼻腔注入。在PAO1株鼻腔感染攻擊后1w，收集血清和脾細胞，用經(jīng)典的ELISA法檢測各組小鼠血清特異性的IgG及其亞類、IgA和IgE抗體水平；用MTT比色法檢測脾細胞原液或PaAg刺激后細胞增殖水平變化；利用FCM檢測脾細胞CD++4和CD8亞群變化；用ELISA法檢測脾細胞原液或PaAg刺激后培養(yǎng)上清液中IFN-γ、IL-12、TNF-α和IL-10的水平以及用AnnexinV-FITC試劑盒檢測脾細胞凋亡率。 結(jié)果 采用RT-PCR擴增出1016bp的OprF編碼基因；成功構(gòu)建pGEX-OprF重組質(zhì)粒，經(jīng)雙酶切鑒定，切出4947bp的載體片段和1016bp的目的基因片段；序列分析發(fā)現(xiàn)其與預(yù)期結(jié)果一致；以rBb抽提質(zhì)粒為模板進行PCR擴增可得到1016bp的oprF基因片段，以陽性rBb的總RNA為模板進行RT-PCR擴增，產(chǎn)物經(jīng)1.2%的瓊脂糖凝膠電泳鑒定，可見一條約1016bp的條帶，證明外源基因oprF能夠在Bb中表達mRNA。SDS-PAGE分析表達產(chǎn)物分子質(zhì)量約為61kDa，與預(yù)期結(jié)果一致，表達的目的蛋白質(zhì)占菌體總蛋白的16%；Western blot鑒定結(jié)果重組蛋白能被Pa外膜粗抗原免疫小鼠血清識別，提示重組OprF抗原與天然OprF蛋白具有相同的抗原性，成功構(gòu)建銅綠假單胞菌rBb-OprF疫苗。 rBb-OprF疫苗免疫后8w，用50μL的10~9CFU/ml的浮游PAO1菌株鼻腔內(nèi)感染攻擊后1w檢測： （1）疫苗組肺Pa荷菌量：SC組、IM組、IN組和PO組肺組織Pa菌荷量均低于MRS對照組，SC組與IM組、IN組和PO組間存在差異；空載體、Bb和MRS對照組間差異無統(tǒng)計學(xué)意義。 （2）ELISA法檢測血清抗體結(jié)果：疫苗免疫組血清IgG、IgG1、IgG2a和IgG2b水平顯著增加；皮下注射組和口服灌胃組IgG3水平顯著增加；皮下注射組IgA水平增加；疫苗免疫組IgE水平無明顯變化。 （3）MTT法檢測的小鼠脾淋巴細胞增殖結(jié)果:所有疫苗接種組脾T細胞增殖明顯，SC組和IM組高于IN組和PO組。 （4）流式細胞儀檢測脾CD4+T細胞和CD8+T細胞結(jié)果：各疫苗組脾細胞CD+4T細胞和CD+8T細胞顯著增加；CD4+T細胞在SC組最高，CD+8T細胞在SC組、IM組和IN高于PO組。 （5）應(yīng)用Annexin V-FITC kits檢測小鼠脾細胞凋亡結(jié)果：疫苗接種組脾細胞凋亡發(fā)生率降低，SC組低于IM組、IN和PO組。 結(jié)論 通過RT-PCR成功擴增出OprF抗原編碼基因；成功構(gòu)建銅綠假單胞菌rBb-OprF疫苗；重組質(zhì)粒pGEX-OprF能在大腸桿菌BL21中表達，表達效率較高，重組融合蛋白具有與天然OprF蛋白相同的抗原性；rBb-OprF疫苗能誘導(dǎo)BALB/c小鼠產(chǎn)生較強的保護性免疫反應(yīng)，皮下和肌肉注射接種是較好的接種途徑；銅綠假單胞菌rBb-OprF疫苗可誘導(dǎo)小鼠產(chǎn)生混合型的Th1和Th2免疫應(yīng)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R392

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