弓形虫P30-P24联合DNA疫苗的免疫保护作用及安全性评价

发布时间：2018-01-08 21:26

本文关键词：弓形虫P30-P24联合DNA疫苗的免疫保护作用及安全性评价　出处：《福建医科大学》2007年博士论文　论文类型：学位论文

【摘要】： 疫苗是防治弓形虫病的有效手段之一,弓形虫疫苗研制经历了全虫疫苗、虫体特异组分疫苗、基因工程疫苗、DNA疫苗4个发展阶段。弓形虫DNA疫苗较传统疫苗虽有诸多优势,但尚处于研究的起步阶段,单价DNA疫苗可诱导机体对弓形虫感染产生细胞免疫和体液免疫,但免疫效果并不十分理想,联合多价DNA疫苗将是弓形虫疫苗今后的发展趋势。此外,弓形虫DNA疫苗的免疫保护机制和安全性还不十分清楚,进一步探讨疫苗免疫机制及其对真核细胞正常基因表达调控的影响,将有助于研制高效、安全、实用的弓形虫DNA疫苗。一、弓形虫p30、p24 DNA疫苗pVAX1-p30、pVAX1-p24的构建及免疫功能研究本研究利用PCR技术扩增出P30和P24基因片段,分别克隆入pVAX1载体,对重组质粒pVAX1-p30、pVAX1-p24进行PCR、双酶切和测序鉴定;并将重组质粒pVAX1-p30、pVAX1-p24和空载体pVAX1转染入小鼠巨噬细胞(RAW264.7),建立体外真核细胞瞬时表达系统,应用免疫细胞化学染色(SP法)检测表达产物p30、p24抗原蛋白;最后将pVAX1-p30、pVAX1-p24DNA疫苗经肌内注射免疫BALB/c小鼠,建立弓形虫攻击感染小鼠模型,观察小鼠的生存时间,检测血清抗弓形虫特异性IgG抗体,ELISA法测定小鼠IFN-γ、IL-2、IL-4细胞因子水平,MTT法测定免疫鼠NK细胞杀伤率,流式细胞技术测定CD4+、CD8+T细胞亚群。结果显示弓形虫DNA疫苗pVAX1-p30和pVAX1-p240构建成功,并能够在小鼠细胞巨噬细胞中表达p30、p24抗原蛋白。在弓形虫攻击感染小鼠模型研究中发现,pVAX1-p30+pVAX1-p24联合免疫组BALB/c小鼠平均存活时间达到(7.60±0.74)d,与对照组(4.50±0.22)d相比明显延长(Log Rank Statistic,P0.05);血清中抗弓形虫特异性IgG的OD值(0.856±0.083)高于对照组(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高于对照组(598.74±67.50、89.44±10.66)pg/ml(F=20.528,F=56.393 , P 0.05) ; pVAX1-p24+pVAX1-p30组脾细胞CD4+细胞比例(37.42±4.84)%,与对照组(48.59±1.85)%间差别具有统计学意义(F=16.736,P0.05),CD8+细胞比例(34.94±5.30)%上升,与对照组(26.91±2.12)%差别具有统计学意义(F=6.896,P0.05);CD4+/CD8+淋巴细胞比值(1.09±0.19)与对照组(1.81±0.14)差别具有统计学意义(F=27.678,PO.05),且低于pVAX1-p24、pVAX1-p30组(1.66±0.13,1.56±0.22);NK细胞杀伤率(64.15±7.71)%,高于对照组(46.81±3.96)%及pVAX1-p24、pVAX1-p30组[(52.03±6.96)%、(55.95±7.37)%(]F=9.816, PO.05)。结果提示,弓形虫DNA疫苗pVAX1-p30、pVAX1-p24的联合应用对弓形虫感染小鼠具有免疫功效,且免疫效果优于pVAX1-p24、pVAX1-p30单独使用,可明显提高小鼠的细胞免疫及体液免疫水平。二、弓形虫p30、p24双基因DNA疫苗pBudCE-p24-p30的构建及其对小鼠巨噬细胞基因表达谱的影响本研究将p30、p24两条基因片段克隆并定位于真核表达载体pBudCE4.1上,构建pBudCE-p24、pBudCE-p30以及pBudCE-p24-p30双基因重组质粒DNA。将重组质粒pBudCE-p24、pBudCE-p30以及pBudCE-p24-p30和空载体pBudCE4.1转染入小鼠巨噬细胞,建立体外真核细胞瞬时表达系统,应用免疫细胞化学染色(SP法)检测表达产物p30、p24抗原蛋白。并利用Affymetrix基因芯片研究p24及p30,特别是p24及p30协同作用对真核细胞正常基因表达调控的影响,对异常表达的基因进行检测分析,并以RT-PCR技术对表达谱基因芯片结果进行验证。结果显示重组质粒pBudCE-p24-p30、pBudCE-p24、pBudCE -p30构建成功,并能够在小鼠细胞巨噬细胞中表达p30、p24抗原蛋白。Affymetrix表达谱基因芯片检测发现,双基因DNA疫苗pBudCE-p24-p30对小鼠巨噬细胞正常基因表达调控的影响不显著,pBudCE-p24-p30转染后小鼠巨噬细胞共有16个基因表达出现上调或下调,上调的基因有Pdlim4、Pcdh7、CAMK4、Nudcd2等9个基因,下调的基因有E2F5、Rpn1、Sh3bgrl等7个基因。其中与基因转录有关的基因6个,与信号转导有关的基因6个,部分功能未明的基因4个。RT-PCR检测结果与表达谱基因芯片检测结果完全吻合。
[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 卤 67.50 , 89.44 卤 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 .

【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392

【引证文献】