本文關(guān)鍵詞：弓形蟲(chóng)P30-P24聯(lián)合DNA疫苗的免疫保護(hù)作用及安全性評(píng)價(jià)　出處：《福建醫(yī)科大學(xué)》2007年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 弓形蟲(chóng) p30基因 p24基因 DNA疫苗 免疫保護(hù) 基因芯片

【摘要】： 疫苗是防治弓形蟲(chóng)病的有效手段之一,弓形蟲(chóng)疫苗研制經(jīng)歷了全蟲(chóng)疫苗、蟲(chóng)體特異組分疫苗、基因工程疫苗、DNA疫苗4個(gè)發(fā)展階段。弓形蟲(chóng)DNA疫苗較傳統(tǒng)疫苗雖有諸多優(yōu)勢(shì),但尚處于研究的起步階段,單價(jià)DNA疫苗可誘導(dǎo)機(jī)體對(duì)弓形蟲(chóng)感染產(chǎn)生細(xì)胞免疫和體液免疫,但免疫效果并不十分理想,聯(lián)合多價(jià)DNA疫苗將是弓形蟲(chóng)疫苗今后的發(fā)展趨勢(shì)。此外,弓形蟲(chóng)DNA疫苗的免疫保護(hù)機(jī)制和安全性還不十分清楚,進(jìn)一步探討疫苗免疫機(jī)制及其對(duì)真核細(xì)胞正�；虮磉_(dá)調(diào)控的影響,將有助于研制高效、安全、實(shí)用的弓形蟲(chóng)DNA疫苗。 一、弓形蟲(chóng)p30、p24 DNA疫苗pVAX1-p30、pVAX1-p24的構(gòu)建及免疫功能研究 本研究利用PCR技術(shù)擴(kuò)增出P30和P24基因片段,分別克隆入pVAX1載體,對(duì)重組質(zhì)粒pVAX1-p30、pVAX1-p24進(jìn)行PCR、雙酶切和測(cè)序鑒定;并將重組質(zhì)粒pVAX1-p30、pVAX1-p24和空載體pVAX1轉(zhuǎn)染入小鼠巨噬細(xì)胞(RAW264.7),建立體外真核細(xì)胞瞬時(shí)表達(dá)系統(tǒng),應(yīng)用免疫細(xì)胞化學(xué)染色(SP法)檢測(cè)表達(dá)產(chǎn)物p30、p24抗原蛋白;最后將pVAX1-p30、pVAX1-p24DNA疫苗經(jīng)肌內(nèi)注射免疫BALB/c小鼠,建立弓形蟲(chóng)攻擊感染小鼠模型,觀察小鼠的生存時(shí)間,檢測(cè)血清抗弓形蟲(chóng)特異性IgG抗體,ELISA法測(cè)定小鼠IFN-γ、IL-2、IL-4細(xì)胞因子水平,MTT法測(cè)定免疫鼠NK細(xì)胞殺傷率,流式細(xì)胞技術(shù)測(cè)定CD4+、CD8+T細(xì)胞亞群。 結(jié)果顯示弓形蟲(chóng)DNA疫苗pVAX1-p30和pVAX1-p240構(gòu)建成功,并能夠在小鼠細(xì)胞巨噬細(xì)胞中表達(dá)p30、p24抗原蛋白。在弓形蟲(chóng)攻擊感染小鼠模型研究中發(fā)現(xiàn),pVAX1-p30+pVAX1-p24聯(lián)合免疫組BALB/c小鼠平均存活時(shí)間達(dá)到(7.60±0.74)d,與對(duì)照組(4.50±0.22)d相比明顯延長(zhǎng)(Log Rank Statistic,P0.05);血清中抗弓形蟲(chóng)特異性IgG的OD值(0.856±0.083)高于對(duì)照組(0.529±0.073)(F=17.412,P0.05);血清IFN-γ水平(853.77±66.74)pg/ml、IL-2水平(192.24±19.90)pg/ml高于對(duì)照組(598.74±67.50、89.44±10.66)pg/ml(F=20.528,F=56.393 , P 0.05) ; pVAX1-p24+pVAX1-p30組脾細(xì)胞CD4+細(xì)胞比例(37.42±4.84)%,與對(duì)照組(48.59±1.85)%間差別具有統(tǒng)計(jì)學(xué)意義(F=16.736,P0.05),CD8+細(xì)胞比例(34.94±5.30)%上升,與對(duì)照組(26.91±2.12)%差別具有統(tǒng)計(jì)學(xué)意義(F=6.896,P0.05);CD4+/CD8+淋巴細(xì)胞比值(1.09±0.19)與對(duì)照組(1.81±0.14)差別具有統(tǒng)計(jì)學(xué)意義(F=27.678,PO.05),且低于pVAX1-p24、pVAX1-p30組(1.66±0.13,1.56±0.22);NK細(xì)胞殺傷率(64.15±7.71)%,高于對(duì)照組(46.81±3.96)%及pVAX1-p24、pVAX1-p30組[(52.03±6.96)%、(55.95±7.37)%(]F=9.816, PO.05)。結(jié)果提示,弓形蟲(chóng)DNA疫苗pVAX1-p30、pVAX1-p24的聯(lián)合應(yīng)用對(duì)弓形蟲(chóng)感染小鼠具有免疫功效,且免疫效果優(yōu)于pVAX1-p24、pVAX1-p30單獨(dú)使用,可明顯提高小鼠的細(xì)胞免疫及體液免疫水平。 二、弓形蟲(chóng)p30、p24雙基因DNA疫苗pBudCE-p24-p30的構(gòu)建及其對(duì)小鼠巨噬細(xì)胞基因表達(dá)譜的影響 本研究將p30、p24兩條基因片段克隆并定位于真核表達(dá)載體pBudCE4.1上,構(gòu)建pBudCE-p24、pBudCE-p30以及pBudCE-p24-p30雙基因重組質(zhì)粒DNA。將重組質(zhì)粒pBudCE-p24、pBudCE-p30以及pBudCE-p24-p30和空載體pBudCE4.1轉(zhuǎn)染入小鼠巨噬細(xì)胞,建立體外真核細(xì)胞瞬時(shí)表達(dá)系統(tǒng),應(yīng)用免疫細(xì)胞化學(xué)染色(SP法)檢測(cè)表達(dá)產(chǎn)物p30、p24抗原蛋白。并利用Affymetrix基因芯片研究p24及p30,特別是p24及p30協(xié)同作用對(duì)真核細(xì)胞正�；虮磉_(dá)調(diào)控的影響,對(duì)異常表達(dá)的基因進(jìn)行檢測(cè)分析,并以RT-PCR技術(shù)對(duì)表達(dá)譜基因芯片結(jié)果進(jìn)行驗(yàn)證。 結(jié)果顯示重組質(zhì)粒pBudCE-p24-p30、pBudCE-p24、pBudCE -p30構(gòu)建成功,并能夠在小鼠細(xì)胞巨噬細(xì)胞中表達(dá)p30、p24抗原蛋白。Affymetrix表達(dá)譜基因芯片檢測(cè)發(fā)現(xiàn),雙基因DNA疫苗pBudCE-p24-p30對(duì)小鼠巨噬細(xì)胞正�；虮磉_(dá)調(diào)控的影響不顯著,pBudCE-p24-p30轉(zhuǎn)染后小鼠巨噬細(xì)胞共有16個(gè)基因表達(dá)出現(xiàn)上調(diào)或下調(diào),上調(diào)的基因有Pdlim4、Pcdh7、CAMK4、Nudcd2等9個(gè)基因,下調(diào)的基因有E2F5、Rpn1、Sh3bgrl等7個(gè)基因。其中與基因轉(zhuǎn)錄有關(guān)的基因6個(gè),與信號(hào)轉(zhuǎn)導(dǎo)有關(guān)的基因6個(gè),部分功能未明的基因4個(gè)。RT-PCR檢測(cè)結(jié)果與表達(dá)譜基因芯片檢測(cè)結(jié)果完全吻合。
[Abstract]:The vaccine is one of the effective methods for preventing and treating toxoplasmosis . The vaccine of toxoplasmosis has many advantages , such as whole worm vaccine , worm - specific component vaccine , genetic engineering vaccine and DNA vaccine . Study on the Construction and Immune Function of One , Toxoplasma gondii ' s p30 , DNA Vaccine of Toxoplasma gondii ( p30 ) and DNA Vaccine ( VAX1 - p30 ) In this study , we amplified the p30 and P24 gene fragments by PCR , and cloned into mouse peritoneal macrophages ( RAW264.7 ) by PCR , double enzyme digestion and sequencing . The recombinant plasmids were then injected into mouse macrophages ( RAW264.7 ) , and the expression products were detected by immunohistochemical staining ( SP method ) . The ratio of CD4 + / CD8 + lymphocytes ( F = 16.736 , P 0.05 ) was higher in BALB / c mice than in the control group ( 8.74 鹵 67.50 , 89.44 鹵 10.66 ) pg / ml ( F = 20.528 , P 0.05 ) . The results showed that the combined application of the DNA vaccine of Toxoplasma gondii DNA vaccine and the DNA vaccine of Toxoplasma gondii has the immune function to the mice infected with toxoplasmosis , and the immune effect is better than that of the single use of the recombinant plasmid pAX1 - p30 and p30 alone , which can obviously improve the cellular immunity and humoral immunity level of the mice . Construction of p30 , p30 and pBudCE - p30 of Toxoplasma gondii and its effect on mouse macrophage gene expression profile In this study , the recombinant plasmid pBudCE4.1 , pBudCE - p30 and pBudCE4.1 were cloned into mouse macrophages to construct the eukaryotic expression vector pBudCE4.1 . The recombinant plasmid pBudCE - p30 and pBudCE4.1 were transfected into mouse macrophages to establish the eukaryotic cell transient expression system . The results showed that the recombinant plasmid pBudCE - p30 , pBudCE1 , pBudCE - p30 was constructed successfully , and the expression of p30 and pBudCE - p30 in mouse peritoneal macrophages was not significant .

【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前2條

1 蘇敏;順鉑誘導(dǎo)小鼠胚胎干細(xì)胞毒性相關(guān)基因的篩選及其初步分析[D];天津醫(yī)科大學(xué);2012年

2 黃國(guó)明;牛瑟氏泰勒蟲(chóng)p23、p33重組蛋白亞單位疫苗聯(lián)合免疫效果評(píng)價(jià)[D];延邊大學(xué);2013年

，

本文編號(hào)：1398749