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Both tumor necrosis factor and interferon gamma Share and may work synergitically in, the activities of antivirus, antitumor and immunomodulation we fused the interferon-gamma gene and tumor necrosis factor gene in frame, with a deletion of 480 bp including translational terminator and an insertion of an artificial linking sequence in between.The fused gene was expressed in E.coli anda bif unctional hybrid protein was obtained. 肿瘤坏死因子与γ干扰素具有非常相似的生物学活性,并在一些主要的生物活性方面表现有较强的协同作用。本文采用寡核苷酸指导下的定位缺失-插入体外基因突变技术,删除了干扰素与肿瘤坏死因子串联基因之间的非编码区,去徐了γ干扰素的翻译终止信号,插入了一个人工设计的连接肽,使γ干扰素与肿瘤坏死因子基因在不改变读码框架的条件下融合起来,并在大肠杆菌表达系统中表述了兼有γ干扰素和肿瘤坏死因子功能的融合蛋白。 An E.coli DH5α strain harboring human interferon gamma gene was cultured in fermentors.The mean harvest of E.coli in three lots was 14.1g/L,and teh expression yield of the interferon was 1.02×10 9IU/L.The E.coli was homogenized and the inclusion bodies were collected.The interferon was extracted by using 7mol/L guanidine HCl.About 78.4% of bacterial proteins was removed,but the loss of biological activity of the interferon was only 10.4% in this process.The crude interferon was... An E.coli DH5α strain harboring human interferon gamma gene was cultured in fermentors.The mean harvest of E.coli in three lots was 14.1g/L,and teh expression yield of the interferon was 1.02×10 9IU/L.The E.coli was homogenized and the inclusion bodies were collected.The interferon was extracted by using 7mol/L guanidine HCl.About 78.4% of bacterial proteins was removed,but the loss of biological activity of the interferon was only 10.4% in this process.The crude interferon was renatured and the biological activity increased to 405%.The specific activity of renatured interferon also increased significantly.The mean specific activity of three lots was 1.76 ×10 7IU/mg protein.The renatured interfenon was purified by three-step chromatography.The mean recovery rate was 28.13%.The specific activities were 3.22,2.78 and 3.01×10 7IU/mg protein,respectively.The purity of three lots all was >99% determined by SDS-PAGE and >98% determined by HPLC.Other assay items were all qualified. 用带有表达质粒pBV220(含有γ干扰素基因)的大肠杆菌DH5α株进行发酵培养,3批中试的菌产量平均为14.1克/L,γ干扰素的表达量平均为1.02×109IU/L,收集的菌体经高压匀质破菌后收集包涵体,用7mol/L盐酸胍提取干扰素,此过程可去除78.4%菌体蛋白,而干扰素活性仅损失10.41%,粗制干扰素经复性可使干扰素活性提高405%,比活也有明显提高,3批平均为1.76×107IU/mg蛋白,复性γ干扰素用三步柱层析法进行纯化,其其得率平均为28.13%,其比活分别为3.22、2.78和3.01×107IU/mg蛋白。3批中试的纯化γ干扰素纯度在SDS-PAGE测定均>99%,高效液相层析(HPLC)测定>98%,其它检定项目也均合乎规程要求。 Swine interferon-gamma gene(SwIFN-γ2 ) was cloned from Con A-stimulated swine spleen cells total RNA by RT-PCR. SwIFN-γ2 was inserted into pGEM T-Easy vector and identified by endonuclease and DNA sequencing. The results indicated that a site mutation at 462 position in SwIFN-γ2. To construct high level expression plasmid, SwIFN-γ2 was ligated with the expression vector pQE30, then transformed into SG13009(pREP4) E.coli strain, and induced with IPTG. SDS-PAGE and Western blot confirmed that... Swine interferon-gamma gene(SwIFN-γ2 ) was cloned from Con A-stimulated swine spleen cells total RNA by RT-PCR. SwIFN-γ2 was inserted into pGEM T-Easy vector and identified by endonuclease and DNA sequencing. The results indicated that a site mutation at 462 position in SwIFN-γ2. To construct high level expression plasmid, SwIFN-γ2 was ligated with the expression vector pQE30, then transformed into SG13009(pREP4) E.coli strain, and induced with IPTG. SDS-PAGE and Western blot confirmed that confluent protein of 6His-recombinant swine interferon-gamma (6His-rSwIFN-γ2) was expressed efficiently in inclusion bodies in E.coli with the yield accounting for 33.5% of the total proteins the. After purification with Ni-NTA agarose, the purity of 6His-rSwIFN-γ2 was greater than 90%. 6His-rSwIFN-γ2 solubilized in a denatured solution containing 8 mol/L urea, refolded by dialysis, have antiviral activity of 8×103~1.6×104 U/mg. 采用RT-PCR技术从猪脾淋巴细胞总RNA中扩增出猪干扰素γcDNA(SwIFN-γ2)。SwIFN-γ2与已报道的猪干扰素γ序列基本一致,仅在462位核苷酸处发生了点变异。在SwIFN-γ2成熟蛋白基因两端分别合成含SphⅠ和HindⅢ酶切位点的引物,用PCR扩增后亚克隆到pQE30表达载体,转化宿主菌SG13009(pREP4),经IPTG诱导表达了N'端含6个组氨酸的重组猪干扰素γ(6His-rSwIFNγ2)。重组干扰素的表达量占总菌体蛋白的33.5%。采用Ni-NTA金属螯合亲和层析纯化6His-rSwIFNγ2,其纯度达90%以上,经8mol/L尿素溶液变性、透析复性后,其抗滤泡性口炎病毒比活性为8×103~1.6×104 U/mg。
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