【作者】 陈降华；

【导师】 李文平； 周庆丰；

【作者基本信息】 湖南农业大学 ， 临床兽医学， 2010， 硕士

【摘要】 本试验对三种细胞因子猪白细胞介素18(porcine interleukin 18, pIL18)、猪粒细胞-巨噬细胞集落刺激因子(granulocytemacrophage colony-stimulating factor, GM-CSF)、猪干扰素y (Interferon gamma, IFNy)的免疫增强作用进行了生物学研究。利用重组PCR方法分别将它们与圆环病毒结构蛋白编码基因(PCV2ORF2)嵌合,将测序正确的嵌合基因亚克隆至穿梭载体pShuttle-CMV,电转化含腺病毒基因组(Adeasy-1)的大肠杆菌细胞BJ5183-Ad-1,成功获得重组腺病毒DNA。重组腺病毒DNA经纯化后转染AD-293细胞,获得了表达融合蛋白的重组腺病毒。对表达的融合蛋白进行免疫特性分析,试验分三部分,如下所述：试验Ⅰ：采用重叠延伸PCR方法,通过一基因接头(linker)将PCV2ORF2基因和前三种细胞因子成熟肽基因分别拼接成嵌合基因,并将其克隆至pMD18-T Simple载体上,成功构建了目的基因克隆载体。经酶切和核苷酸测序鉴定,结果显示：获得的嵌合基因pIL18-ORF2、pGMCSF-ORF2、pIFNy-ORF2序列全长分别为1338bp、1194bp、1259bp,片段均与GeneBank公布的相应序列一致,并符合原阅读框。试验Ⅱ：将测序正确的三个嵌合基因亚克隆至穿梭载体pShuttle-CMV,电转化含腺病毒基因组(Adeasy-1)的大肠杆菌细胞BJ5183-Ad-1,成功获得了重组腺病毒DNA。将纯化后的重组腺病毒DNA转染AD293细胞,经过病毒基因组的PCR鉴定、转录水平的RT-PCR鉴定以及蛋白表达水平的免疫荧光分析,初步表明获得了表达融合蛋白的重组腺病毒rAd-LF2、rAd-GF2、rAd-NF2。试验Ⅲ：本研究通过小鼠试验比较了PCV2ORF2重组腺病毒与pIL18-ORF2、pGMCSF-ORF2、pIFNy-ORF2嵌合基因共表达重组腺病毒的免疫特性。试验结果表明：重组腺病毒rAd-LF2、rAd-GF2、rAd-NF2和rAd-Cap均可诱导小鼠产生体液免疫应答,并可产生很好的免疫保护反应,且rAd-LF2、rAd-GF2、rAd-NF2的免疫效果均较rAd-Cap更好,其中rAd-NF2免疫增强效果非常明显,说明细胞因子可以一定程度的增强免疫效果。本实验结果显示：在成功构建嵌合基因克隆载体的前提下,利用腺病毒表达载体成功获得了表达正确的重组腺病毒,通过小鼠免疫试验可以看出：pIL18、pGM-CSF.pIFNy的免疫佐剂作用确实,和单一ORF2重组腺病毒表达产物相比,嵌合基因重组腺病毒表达的融合蛋白免疫原性和反应原性都更好。更多还原

【Abstract】 In this test, we have studied the immune enhancement of three kinds of cytokines on porcine interleukin-18 (porcine interleukin 18, pIL18), porcine granulocyte macrophage colony-stimulating factor (granulocytemacrophage colony-stimulating factor, GM-CSF), porcine interferon-γ(Interferon gamma, IFNγ). Recombinant adenovirus DNA were constructed based on the following methods:establishing chimeric fragment of the above genes and circovirus structural protein coding gene(PCV2ORF2) with recombinent PCR methods,respectively.And the sequence of the chimeric gene was then subcloned into a shuttle vector of pShuttle-CMV, electro-transformation with adenovirus genome (Adeasy-1) of Escherichia coli BJ5183-Ad-1.After purification, recombinant DNA were transfected into AinD-293 cells and expression of fusion protein of recombinant adenovirus were obtained.The test was divided into three parts to analyse the immuno characterization of the expressed fusion protein:Test 1:Constrcuting chimeric genes of PCV2ORF2 and the above three cytokines’mature peptide gene with a genetic connection(linker) by overlapping extension PCR method,respectively.The chimeric genes were then cloned into pMD18-T Simple vector.Enzyme digesion and nucleotide sequencing results showed that the obtained chimeric gene fragments of pIL18-ORF2,pGMCSF-ORF2, pIFNγ-ORF2 were 1338bp,1194bp,1259bp,respectively,which were accordance with relevant sequences in Genebank and the original reading frame.Test 2:The three chimeric genes were sequenced and subcloned into shuttle vector of pShuttle-CMV.The recombinant vectors were transformed into Escherichia coli BJ5183-Ad-1 by electroporation.Purifying the recombinant adenovirus DNA and transfected them into AD293 cell.PCR identification,RT-PCR indentification and immunofluorescence of protein expression results showed that the three recombinant vectors which expressed fusion proteins were constructed succesfuly.Test 3:Comparing the immuno characterization between PCV2ORF2 recombinent adenovirus and the above chimeric genes’ co-expression proteins through mice experiments.The results indicated that rAd-LF2、rAd-GF2、rAd-NF2 and rAd-Cap could induce humoral immune response in mice and produce good immun protection,moreover,the immune effects of rAd-LF2、rAd-GF2 and rAd-NF2 were better than rAd-Cap,among which rAd-NF2 showed a significant effect of immuno enhancement.Our data proved that cytokines could enhance immuno effects to a certain degree.The studies show that: in the context of successfully constructing the chimeric gene vector, we have obtained the correct expressed recombinant adenovirus under the premise of using adenovirus expression vector. Also through the immune test it can be seen: the immune effect of immune adjuvant, such as pIL18, pGM-CSF, pIFNy, is significant. And compared to a single recombinant ORF2 adenovirus expression product, the mmunogenicity and reactogenicity of fusion protein, which is expressed by chimeric gene recombinant adenovirus, are better.更多还原