【摘要】：目的構(gòu)建卡氏肺孢子菌真核表達質(zhì)粒pcDNA3.1(+)-582,并在真核細胞表達系統(tǒng)中進行表達。用構(gòu)建的質(zhì)粒免疫小鼠后檢測其體液免疫及細胞免疫反應。用該質(zhì)粒免疫SD大鼠,觀察其抗肺孢子菌肺炎的免疫保護作用。 方法利用PCR技術(shù)從提取的卡氏肺孢子菌基因組DNA中擴增出p55基因片段,將其連接至pGEM-T載體進行篩選和鑒定,通過酶切和連接,將此片段連接至真核表達載體pcDNA3.1(+)上,構(gòu)建真核表達質(zhì)粒pcDNA3.1(+)-582;經(jīng)鑒定序列正確后將此表達質(zhì)粒瞬時轉(zhuǎn)染COS-7細胞,分別通過RT-PCR及Western-blotting鑒定其RNA及蛋白表達。將此質(zhì)粒免疫BALB/c小鼠后,檢測抗體滴度,用MTT法測定脾淋巴細胞增殖活性;同時以此質(zhì)粒免疫SD大鼠,用Real-time PCR法檢測該疫苗的免疫保護效果。 結(jié)果經(jīng)酶切鑒定、PCR和測序分析結(jié)果表明,真核表達質(zhì)粒pcDNA3.1(+)-582構(gòu)建成功,此質(zhì)粒免疫小鼠后能誘導產(chǎn)生特異性抗體,小鼠的T淋巴細胞增殖活性增強。免疫大鼠后DNA疫苗組大鼠肺組織菌體含量與空質(zhì)粒組相比有明顯降低(P0.05)。 結(jié)論成功構(gòu)建了卡氏肺孢子菌DNA疫苗,此疫苗能誘導BALB/c小鼠產(chǎn)生一定的細胞免疫和體液免疫,對大鼠肺孢子菌感染具有一定的免疫保護效果。
[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.
【學位授予單位】：南通大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392.1

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