【作者】 沈翼；

【导师】 刘世贵； 高荣；

【作者基本信息】 四川大学 ， 遗传学， 2003， 硕士

【摘要】 为探索构建具有多功能、高活性，可显著增加免疫调节范围、增强免疫效应的新型细胞因子基因，获得融合蛋白，本实验应用PCR技术对猪的IL-6和IL-4基因分别加以改造。去除IL-6的终止密码子，并在两端引入BamHⅠ和SalⅠ酶切位点，去除IL-4的起始密码子，并在两端引入SalⅠ和XhoⅠ酶切位点，同时在二者之间加上Gly-Ser-Ser-Thr柔性连接肽序列，克隆PGR产物，并构建了pGEX-4T-1-IL6／IL4原核表达载体，经BamHⅠ，SalⅠ和XhoⅠ酶切鉴定，证实IL6／IL4融合基因已克隆到原核表达载体pGEX-4T-1，为进一步研究其诱导表达条件及生物学功能奠定了基础。 将已构建的II-6／IL-4融合蛋白表达载体质粒转化大肠杆菌BL21(DE3)，用2×YTA培养基培养，以IPTG诱导融合蛋白表达，并作RT-PCR及SDS-PAGE分析转录及表达情况；融合蛋白的表达产物以，包涵体形式存在，将包涵体经过变性，稀释、透析复性处理后用MTT法测定重组蛋白刺激猪淋巴母细胞增殖反应活性。RT-PCR结果显示，该融合蛋白能在大肠杆菌中正确转录，经SDS-PAGE分析表达蛋白分子量为64KD，与相同浓度的重组猪IL-6和IL-4比较，经复性处理的融合蛋白促进猪淋巴母细胞增殖反应活性显著增强。表明我们所构建的IL-6／IL-4融合蛋白是一种新型高效免疫调节分子，可望进一步研制成新的高效免疫增强制剂。更多还原

【Abstract】 In order to construct a fusion cytokine protein which could have more and stronger bioactivities to enhance the immnuity than the original cytokines, we use the PCR technique to reconstruct the porcine IL-6 and IL-4 gene. The termination code of the IL-6 gene was deleted and the enzyme sites of BamHI and Sail were introduced to the two ends, named as FPIL-6 cDNA; the starting code of the IL-4 gene was cut and the enzyme sites of Sail and Xhol were added to the two flanks, designed as FPIL-4 cDNA. The reconstructed IL-6 and IL-4 gene were connected together via a linker sequence encoding Gly-Ser-Ser-Thr amino acid residues. The PCR amplified products of the IL-6 and IL-4 gene was digested respectively with BamHI and Sail, Sail and Xhol endonucleases, and then cloned into the pGEX-4T-l expression plasmid which was also digested with the sarne endonucleases. The recombinant plasmid was digested with BamH I, Sail and Xhol to identify whether the FPIL-6 and FPIL-4 cDNA fragments were inserted into the plasmid in correct orientation. .The BL21 (DE3) competent cells were transformed with the recombinant pGFPIL-6/IL-4, incubated with 2xYTA culture and induced to express the fusion protein by IPTG. The mRNA and protein expression of the recombinant plasmids were assayed by RT-PCR and SDS-PAGE. The results were found that the mRNA of the fusion gene could be correctly transcribed, and the fusion protein was expressed in the form of inclusion body with 64KD molecular weight revealed by SDS-PAGE analysis. The bioactivities of refolded fusion protein were detected by MTT assay to stimulate the proliferation of ConA blast cells of porcine. The results showed that through the treatment of denature, dilution and dialysis, the inclusion body of fusion protein restored the bioactivity and had marked bioactivity to promote the proliferation of porcine ConA blasts. These suggest that the fusion protein of porcine IL-6 and IL-4 could be employed to develop a novel effective immunoadjuvant to enhance the immunity of animals against infectious diseases.更多还原