本文關(guān)鍵詞： 卡氏肺孢子菌 pVAX-p55-v0 pVAX-p55-v3 構(gòu)建 表達(dá) 免疫應(yīng)答　出處：《重慶醫(yī)科大學(xué)》2011年博士論文　論文類型：學(xué)位論文

【摘要】：目的: 本研究擬采用分子生物學(xué)和分子免疫學(xué)的方法構(gòu)建卡氏肺孢子菌(Pneumocystis carinii, PC) p55-v3 DNA疫苗,同時(shí)將p55-v0抗原作為陽性對照,免疫SD大鼠后,對p55-v3 DNA疫苗預(yù)防PC的作用進(jìn)行評定,并進(jìn)一步對其免疫作用機(jī)理進(jìn)行探討,為闡明PC與宿主相互作用的分子機(jī)制及新型疫苗的研制提供基礎(chǔ),進(jìn)而為PCP的防治提供一種新的方法和手段。 方法: 1.建立PCP動物模型并制備PC抗血清。 2.提取PC感染鼠肺組織總RNA,運(yùn)用RT-PCR擴(kuò)增p55-v3和p55-v0抗原基因。 3.運(yùn)用分子克隆的方法構(gòu)建p55-v3和p55-v0的真核表達(dá)載體。 4.運(yùn)用脂質(zhì)體2000將鑒定正確的真核表達(dá)載體轉(zhuǎn)染COS-7細(xì)胞,通過RT-PCR和Western-blot分別在mRNA及蛋白水平對所轉(zhuǎn)染細(xì)胞兩種抗原蛋白的表達(dá)進(jìn)行檢測。 5.動物實(shí)驗(yàn):分別將p55-v3、p55-v0 DNA疫苗免疫SD大鼠(以pVAX1空載體及PBS作為對照)后,按常規(guī)方法構(gòu)建PCP模型,于第6周處死大鼠,通過一般情況、肺重/體重、肺印片包囊計(jì)數(shù)、病理切片及體液免疫、細(xì)胞免疫的檢測,觀察p55-v3和p55-v0 DNA疫苗對大鼠的免疫保護(hù)作用并進(jìn)行比較,從而對p55-v3的免疫保護(hù)機(jī)制及效應(yīng)進(jìn)行評價(jià)。 結(jié)果: 1.模型鼠肺印片(GMS染色),可見大量被染成棕黑色的PC包囊。免疫組化證實(shí)血清中抗PC抗體陽性。 2.以總RNA為模板進(jìn)行RT-PCR后,1 %瓊脂糖凝膠電泳分析,在1200 bp、1000 bp左右處見一特異性條帶,分別與p55-v0、p55-v3抗原基因大小相符。 3.將擴(kuò)增產(chǎn)物與T載體連接,測序正確后構(gòu)建重組載體pVAX-p55-v0,pVAX-p55-v3。酶切鑒定表明p55-v3、p55-v0抗原基因片段已成功克隆入pVAX1載體。 4.將重組真核表達(dá)載體轉(zhuǎn)染COS-7細(xì)胞后提取總RNA,以其為模板進(jìn)行RT-PCR,1 %瓊脂糖凝膠電泳觀察可見重組質(zhì)粒轉(zhuǎn)染組有明顯的特異性條帶,分別位于1200 bp及1000 bp左右,其大小與p55-v0及p55-v3基因片段一致,而空質(zhì)粒轉(zhuǎn)染組僅見內(nèi)參(GAPDH)條帶,未見特異性條帶;Western-blot分析發(fā)現(xiàn)重組質(zhì)粒轉(zhuǎn)染組均可見約55 kDa大小的特異性條帶,提示在COS-7細(xì)胞中重組質(zhì)粒從mRNA及蛋白水平均有表達(dá)。 5.構(gòu)建的DNA疫苗免疫SD大鼠后發(fā)現(xiàn)pVAX-p55-v0及pVAX-p55-v3免疫組肺重/體重、包囊計(jì)數(shù)較PBS及pVAX1空載體組明顯減少,而pVAX-p55-v0與pVAX-p55-v3免疫組之間無顯著性差異。病理切片觀察發(fā)現(xiàn)PBS及pVAX1空載體組(HE染色)肺泡間隔增寬,間質(zhì)水腫明顯,GMS染色下可見肺泡壁及間質(zhì)中大量被染成棕黑色的PC包囊,而pVAX-p55-v0及pVAX-p55-v3免疫組明顯減輕,且pVAX-p55-v0與pVAX-p55-v3之間無顯著性差異。與對照組相比,免疫組血清IgG顯著增高,脾淋巴細(xì)胞顯著增殖,血清IFN-γ,IL-2增高明顯,pVAX-p55-v0及pVAX-p55-v3免疫組之間無明顯差異。各組大鼠血清IL-4水平無顯著性差異, 結(jié)論: 1.成功構(gòu)建PCP模型并制備PC抗血清。 2.成功擴(kuò)增p55-v0及p55-v3基因。 3.成功構(gòu)建重組真核表達(dá)載體pVAX-p55-v0及pVAX-p55-v3。 4.重組真核表達(dá)載體pVAX-p55-v0及pVAX-p55-v3體外轉(zhuǎn)染COS-7細(xì)胞,RT-PCR及Western-blot鑒定證實(shí)在mRNA及蛋白水平均有表達(dá)。 5.重組DNA疫苗pVAX-p55-v3可誘導(dǎo)大鼠產(chǎn)生部分保護(hù)性免疫應(yīng)答,其免疫保護(hù)效應(yīng)與p55-v0 DNA疫苗無顯著性差異。
[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.

【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】：2011
【分類號】：R519;R346

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