【摘要】：目的構(gòu)建卡氏肺孢子菌真核表達(dá)質(zhì)粒pcDNA3.1(+)-582,并在真核細(xì)胞表達(dá)系統(tǒng)中進(jìn)行表達(dá)。用構(gòu)建的質(zhì)粒免疫小鼠后檢測(cè)其體液免疫及細(xì)胞免疫反應(yīng)。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(hù)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(kuò)增出p55基因片段,將其連接至pGEM-T載體進(jìn)行篩選和鑒定,通過(guò)酶切和連接,將此片段連接至真核表達(dá)載體pcDNA3.1(+)上,構(gòu)建真核表達(dá)質(zhì)粒pcDNA3.1(+)-582;經(jīng)鑒定序列正確后將此表達(dá)質(zhì)粒瞬時(shí)轉(zhuǎn)染COS-7細(xì)胞,分別通過(guò)RT-PCR及Western-blotting鑒定其RNA及蛋白表達(dá)。將此質(zhì)粒免疫BALB/c小鼠后,檢測(cè)抗體滴度,用MTT法測(cè)定脾淋巴細(xì)胞增殖活性;同時(shí)以此質(zhì)粒免疫SD大鼠,用Real-time PCR法檢測(cè)該疫苗的免疫保護(hù)效果。 結(jié)果經(jīng)酶切鑒定、PCR和測(cè)序分析結(jié)果表明,真核表達(dá)質(zhì)粒pcDNA3.1(+)-582構(gòu)建成功,此質(zhì)粒免疫小鼠后能誘導(dǎo)產(chǎn)生特異性抗體,小鼠的T淋巴細(xì)胞增殖活性增強(qiáng)。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(dǎo)BALB/c小鼠產(chǎn)生一定的細(xì)胞免疫和體液免疫,對(duì)大鼠肺孢子菌感染具有一定的免疫保護(hù)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(xué)位授予單位】：南通大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392.1

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