卡氏肺孢子菌p55-v3DNA疫苗的构建及其对大鼠免疫保护作用的研究

发布时间：2018-01-29 09:07

本文关键词： 卡氏肺孢子菌 pVAX-p55-v0 pVAX-p55-v3 构建表达免疫应答　出处：《重庆医科大学》2011年博士论文　论文类型：学位论文

【摘要】：目的: 本研究拟采用分子生物学和分子免疫学的方法构建卡氏肺孢子菌(Pneumocystis carinii, PC) p55-v3 DNA疫苗,同时将p55-v0抗原作为阳性对照,免疫SD大鼠后,对p55-v3 DNA疫苗预防PC的作用进行评定,并进一步对其免疫作用机理进行探讨,为阐明PC与宿主相互作用的分子机制及新型疫苗的研制提供基础,进而为PCP的防治提供一种新的方法和手段。方法: 1.建立PCP动物模型并制备PC抗血清。 2.提取PC感染鼠肺组织总RNA,运用RT-PCR扩增p55-v3和p55-v0抗原基因。 3.运用分子克隆的方法构建p55-v3和p55-v0的真核表达载体。 4.运用脂质体2000将鉴定正确的真核表达载体转染COS-7细胞,通过RT-PCR和Western-blot分别在mRNA及蛋白水平对所转染细胞两种抗原蛋白的表达进行检测。 5.动物实验:分别将p55-v3、p55-v0 DNA疫苗免疫SD大鼠(以pVAX1空载体及PBS作为对照)后,按常规方法构建PCP模型,于第6周处死大鼠,通过一般情况、肺重/体重、肺印片包囊计数、病理切片及体液免疫、细胞免疫的检测,观察p55-v3和p55-v0 DNA疫苗对大鼠的免疫保护作用并进行比较,从而对p55-v3的免疫保护机制及效应进行评价。结果: 1.模型鼠肺印片(GMS染色),可见大量被染成棕黑色的PC包囊。免疫组化证实血清中抗PC抗体阳性。 2.以总RNA为模板进行RT-PCR后,1 %琼脂糖凝胶电泳分析,在1200 bp、1000 bp左右处见一特异性条带,分别与p55-v0、p55-v3抗原基因大小相符。 3.将扩增产物与T载体连接,测序正确后构建重组载体pVAX-p55-v0,pVAX-p55-v3。酶切鉴定表明p55-v3、p55-v0抗原基因片段已成功克隆入pVAX1载体。 4.将重组真核表达载体转染COS-7细胞后提取总RNA,以其为模板进行RT-PCR,1 %琼脂糖凝胶电泳观察可见重组质粒转染组有明显的特异性条带,分别位于1200 bp及1000 bp左右,其大小与p55-v0及p55-v3基因片段一致,而空质粒转染组仅见内参(GAPDH)条带,未见特异性条带;Western-blot分析发现重组质粒转染组均可见约55 kDa大小的特异性条带,提示在COS-7细胞中重组质粒从mRNA及蛋白水平均有表达。 5.构建的DNA疫苗免疫SD大鼠后发现pVAX-p55-v0及pVAX-p55-v3免疫组肺重/体重、包囊计数较PBS及pVAX1空载体组明显减少,而pVAX-p55-v0与pVAX-p55-v3免疫组之间无显著性差异。病理切片观察发现PBS及pVAX1空载体组(HE染色)肺泡间隔增宽,间质水肿明显,GMS染色下可见肺泡壁及间质中大量被染成棕黑色的PC包囊,而pVAX-p55-v0及pVAX-p55-v3免疫组明显减轻,且pVAX-p55-v0与pVAX-p55-v3之间无显著性差异。与对照组相比,免疫组血清IgG显著增高,脾淋巴细胞显著增殖,血清IFN-γ,IL-2增高明显,pVAX-p55-v0及pVAX-p55-v3免疫组之间无明显差异。各组大鼠血清IL-4水平无显著性差异, 结论: 1.成功构建PCP模型并制备PC抗血清。 2.成功扩增p55-v0及p55-v3基因。 3.成功构建重组真核表达载体pVAX-p55-v0及pVAX-p55-v3。 4.重组真核表达载体pVAX-p55-v0及pVAX-p55-v3体外转染COS-7细胞,RT-PCR及Western-blot鉴定证实在mRNA及蛋白水平均有表达。 5.重组DNA疫苗pVAX-p55-v3可诱导大鼠产生部分保护性免疫应答,其免疫保护效应与p55-v0 DNA疫苗无显著性差异。
[Abstract]:Objective:
This research adopts the methods of molecular biology and molecular immunology of Pneumocystis carinii (Pneumocystis carinii PC) p55-v3 DNA vaccine, the p55-v0 antigen was used as positive control, immune SD rats, to evaluate the effect of p55-v3 DNA vaccine against PC, and further explore the mechanism of immune function. To provide a basis for the development of PC and elucidate the molecular mechanism of host interaction and novel vaccines, provide a new method and means for the prevention and treatment of PCP.
Method:
1. the animal model of PCP was established and the antiserum of PC was prepared.
2. the total RNA of lung tissue of PC infected rats was extracted and the p55-v3 and p55-v0 antigen genes were amplified by RT-PCR.
3. the eukaryotic expression vector of p55-v3 and p55-v0 was constructed by molecular cloning.
4., we used liposome 2000 to identify the correct eukaryotic expression vector to transfect COS-7 cells, and detected the expression of two antigen proteins at mRNA and protein level respectively by RT-PCR and Western-blot.
5. animal experiment: respectively, p55-v3, p55-v0 DNA vaccine SD rats (pVAX1 vector and PBS as control), PCP model is constructed according to the conventional method, in sixth weeks the rats were killed by the general situation, the lung weight / body weight, lung imprint cyst count, pathological sections and humoral immune detection. Cell immunity, p55-v3 and p55-v0 were compared to observe DNA vaccine on rats and immune protective effect, so as to evaluate the immune protection mechanism and effect of p55-v3.
Result:
1. model rat lung prints (GMS staining) showed a large number of brown black PC capsules. Immunohistochemistry confirmed that the anti PC antibody was positive in the serum.
2. after total RNA as template for RT-PCR, after 1% agarose gel electrophoresis, a specific band appeared at 1200 BP, 1000 BP, which was consistent with the size of p55-v0 and p55-v3 antigen genes.
3., the amplified products were connected to T vector. After sequencing, the recombinant vector pVAX-p55-v0 was constructed. PVAX-p55-v3. digestion showed that p55-v3 and p55-v0 gene fragments were successfully cloned into pVAX1 vector.
4. the recombinant eukaryotic expression vector was transfected into COS-7 cells after the extraction of total RNA as the template for RT-PCR, 1% were confirmed by agarose gel electrophoresis of recombinant plasmid transfection group has obvious specific bands are located at 1200 BP and 1000 BP, and its size is p55-v0 and the p55-v3 fragment, and empty plasmid only the reference group (GAPDH) bands had no specific bands; Western-blot analysis showed that the recombinant plasmids were found in about 55 of the size of the kDa specific bands, suggesting that the recombinant plasmid expression from mRNA and protein level were significantly in COS-7 cells.
DNA vaccine SD rats 5. construction found after pVAX-p55-v0 and the pVAX-p55-v3 group the lung weight / body weight, cyst count compared to PBS and pVAX1 empty vector group were significantly reduced, but there is no significant difference between pVAX-p55-v0 and pVAX-p55-v3 immune group. Pathological observation showed that PBS and pVAX1 empty vector group (HE staining) of alveolar septum. Interstitial edema, GMS staining of PC cysts and interstitial alveolar wall in a large number of dyed dark brown, while pVAX-p55-v0 and pVAX-p55-v3 expression were significantly reduced, and between pVAX-p55-v0 and pVAX-p55-v3. No significant difference compared with the control group, immune serum IgG significantly increased spleen lymphocyte proliferation significantly, serum IFN- gamma IL-2, obviously, there is no significant difference between pVAX-p55-v0 and pVAX-p55-v3 immune group. No significant difference in the serum IL-4 level in rats,
Conclusion:
1. the PCP model was successfully constructed and the antiserum of PC was prepared.
2. the p55-v0 and p55-v3 genes were amplified successfully.
3. the successful construction of recombinant eukaryotic expression vector pVAX-p55-v0 and pVAX-p55-v3.
4. the recombinant eukaryotic expression vector pVAX-p55-v0 and pVAX-p55-v3 were transfected into COS-7 cells in vitro. The RT-PCR and Western-blot identification proved that the level of mRNA and protein were expressed.
5. the recombinant DNA vaccine pVAX-p55-v3 could induce partial protective immune response in rats, and there was no significant difference between the immune protective effect and the p55-v0 DNA vaccine.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R519;R346

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