卡氏肺孢子菌p55基因片段DNA疫苗免疫保护作用的研究

发布时间：2019-03-14 19:48

【摘要】：目的构建卡氏肺孢子菌真核表达质粒pcDNA3.1(+)-582,并在真核细胞表达系统中进行表达。用构建的质粒免疫小鼠后检测其体液免疫及细胞免疫反应。用该质粒免疫SD大鼠,观察其抗肺孢子菌肺炎的免疫保护作用。方法利用PCR技术从提取的卡氏肺孢子菌基因组DNA中扩增出p55基因片段,将其连接至pGEM-T载体进行筛选和鉴定,通过酶切和连接,将此片段连接至真核表达载体pcDNA3.1(+)上,构建真核表达质粒pcDNA3.1(+)-582;经鉴定序列正确后将此表达质粒瞬时转染COS-7细胞,分别通过RT-PCR及Western-blotting鉴定其RNA及蛋白表达。将此质粒免疫BALB/c小鼠后,检测抗体滴度,用MTT法测定脾淋巴细胞增殖活性;同时以此质粒免疫SD大鼠,用Real-time PCR法检测该疫苗的免疫保护效果。结果经酶切鉴定、PCR和测序分析结果表明,真核表达质粒pcDNA3.1(+)-582构建成功,此质粒免疫小鼠后能诱导产生特异性抗体,小鼠的T淋巴细胞增殖活性增强。免疫大鼠后DNA疫苗组大鼠肺组织菌体含量与空质粒组相比有明显降低(P0.05)。结论成功构建了卡氏肺孢子菌DNA疫苗,此疫苗能诱导BALB/c小鼠产生一定的细胞免疫和体液免疫,对大鼠肺孢子菌感染具有一定的免疫保护效果。
[Abstract]:Aim to construct eukaryotic expression plasmid pcDNA3.1 ()-582 and express it in eukaryotic expression system. The humoral and cellular immune responses were detected after immunization with the constructed plasmid. This plasmid was used to immunize SD rats and observe its immunoprotection against Pneumocystis pneumoniae (Pneumocystis pneumoniae). Methods the fragment of P55 gene was amplified from genomic DNA of Pneumosporium carinii by PCR technique. The fragment was ligated to pGEM-T vector for screening and identification. The fragment was ligated into eukaryotic expression vector pcDNA3.1 () to construct eukaryotic expression plasmid pcDNA3.1 ()-582. The expression plasmid was transiently transfected into COS-7 cells. The expression of RNA and protein were identified by RT-PCR and Western-blotting, respectively. After the BALB/c mice were immunized with this plasmid, the antibody titers were detected and the proliferation activity of splenic lymphocytes was determined by MTT method. Meanwhile, the SD rats were immunized with this plasmid and the immune protective effect of the vaccine was detected by Real-time PCR method. Results Eukaryotic expression plasmid pcDNA3.1 ()-582 was successfully constructed by restriction endonuclease digestion, PCR and sequencing analysis. The recombinant plasmid could induce the production of specific antibody and enhance the proliferation of T lymphocytes in mice. After immunization, the bacterial content in lung tissue of DNA vaccine group was significantly lower than that of empty plasmid group (P0.05). Conclusion Pneumocystis carinii DNA vaccine has been successfully constructed. This vaccine can induce cellular immunity and humoral immunity in BALB/c mice, and has a certain immune protective effect on Pneumocystis carinii infection in rats.
【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.1

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