【作者】 金标；

【导师】 陈洪岩；

【作者基本信息】 东北农业大学 ， 动物学， 2007， 硕士

【摘要】 干扰素-γ（Interferon gamma，INF-γ）又称Ⅱ型干扰素，是一种由活化的NK细胞和CD4⁺、CD8⁺等T淋巴细胞产生的细胞因子。无论在基因水平还是在蛋白质水平它都与Ⅰ型干扰素（如INF-α、INF-β）不同。它显示出低特异性的抗病毒活性，而具有更明显的免疫调节活性，所以也称为免疫干扰素；而白细胞介素-2（interleukin-2，IL-2）是最早发现的具有广泛生物活性的细胞因子，它能刺激T、B细胞的生长增殖，激活T细胞分泌IFN-γ、CSF等，增强Tc（cytotoxic T cell）、NK、巨噬细胞的细胞毒活性，增强抗原提呈作用，是机体免疫调节的重要细胞因子。我们根据GenBank上发表的山羊（Caprine）干扰素-γ和白细胞介素-2基因序列，设计了两套引物。应用RT-PCR方法从山羊外周血淋巴细胞中分别克隆了二者，将其克隆到pMD18-T载体中，经酶切鉴定、PCR鉴定及DNA测序分析，表明扩增片段分别为山羊1FN-γ和IL-2基因。测序结果显示克隆的IFN-γ基因全长501个核苷酸，编码166个氨基酸，该基因与GenBank发表的山羊干扰素-γ基因序列（U34232）比较，核苷酸／氨基酸序列同源性为99.4％。经SignalP 3.0分析表明，前23个氨基酸为信号肽序列；IL-2基因开放阅读框架共有468bp，编码一条155个氨基酸的多肽，与GenBank发表的山羊白细胞介素-2基因序列（AF535145）比较核苷酸／氨基酸同源性为100.0％，与应琦华等的山羊IL-2序列的同源性为99.1％，前20个氨基酸为该蛋白的信号肽序列。应用DNA重组技术，将IL-2基因和去除信号肽的IFN-γ基因分别插入到原核表达载体pET-30a（+）的BamHⅠ和HindⅢ位点上，构建原核表达质粒pET30a-CapIL-2和pET30a-CapIFN-γ，转化大肠杆菌BL21（DE3），经IPTG诱导后进行SDS-PAGE电泳，结果表明目的基因在大肠杆菌中以包涵体形式融合表达。分别用ProBond^TM蛋白纯化试剂盒纯化，用所获得的重组蛋白纯化产物经三次背部皮下多点注射新西兰白兔，制备了兔抗山羊IFN-γ和兔抗山羊IL-2多克隆血清。经Western Blot鉴定，多克隆抗血清可与纯化的山羊重组蛋白发生特异性反应，说明重组蛋白具有抗原活性，为我们进一步研究它们的免疫治疗作用和免疫佐剂作用奠定了基础。更多还原

【Abstract】 Cloning and Prokaryotic Expression of CaprineInterferon-gamma,Interleukin-2 and Preparation of Their AntiserumInterferon gamma(IFN-γ), also termed typeⅡor immune IFN, is a cytokine producedprincipally by natural killer cells and certain subpopulations of T lymphocytes, predominantly type1 CD4~+and CD8~+ lymphocytes,which is not related to the typeⅠIFN(the IFN-αand IFN-β) at boththe genetic and the protein levels. It has a lower specific antiviral activity, but presents moreimmunomodulatory properties.lnterleukin-2 (IL-2) is the first of a series of lymphocytotrophichormones to be recognized and completely characterized, it is a important cytokine inimmunomodulatory which can active the proliferation of T and B cells, induce expression ofIFN-γ、CSF in T cells, enhance the cytotoxic active of Tc(cytotoxic T cell),NK and macrophage,resulte in an enhance in function of antigen presenting. Twe pairs of oligonucletide primers weredesigned to amplify the genes coding for the caprine interferon-gamma and Interleukin-2,respectively. The primers were chosen by the analysis of caprine relative sequences available inGenBank. The genes were amplified from peripheral blood mononuclear cells by the reversetranscription-polymerase chain reaction (RT-PCR), the PCR products were cloned into pMD18-Tvector and identified, respectively. The cDNA open reading frame (ORF) of IFN-γis 501bp,encoding a putative 166 amino acid (aa) protein (19.327KD), the DNA sepuence homology of thiscaprine IFN-γand the corresponding caprine(U34232) cytokine is 99.4％, SignalP 3.0 analysisdemonstrated that the first 23 amino terminal aa sequence compose a hydrophobic signal sequence.The IL-2 gene is 468bp, encoding a putative 155 amino acid (aa) protein, the DNA sepuencehomology of this caprine IL-2 and the corresponding caprine(AF535145) cytokine is 100.0％, thegene homology to Qihua Ying’s is 99.1％, the first 20 amino terminal aa sequence compose ahydrophobic signal sequence. The caprine IL-2 gene and the IFN-γgene without the signal peptidesequence were subcloned into (5’) Bam H I and (3’) HindⅢrestriction sites of pET30a (+)plasmid, the recombinant plasmid were transformed into E. coli BL21(DE3) and induced withIPTG, they were demonstrated by SDS-PAGE that the genes were expressed as inclusion bodies.The proteins were purified and used to prepare the rabbit antiserum against caprine IFN-γor IL-2,the results showed that the antibody could react with the purified proteins in Western blotting.These work laid a foundation and prepared experimental material for the future studies on thebioactivity 0fcaprine IFN-γor IL-2.更多还原