IFN-λ1在嗜甲醇酵母中的表达、纯化和活性测定以及其受体信号通路的研究
发布时间：2018-08-04 10:30
【摘要】：IFN-λs是最新发现的一类具有抗病毒活性的干扰素样细胞因子，包括IFN-λ1(IL-29)、IFN-λ2(IL-28A)和IFN-λ3(IL-28B)。IFN-λs在基因结构上与IL-10家族十分相似，而在氨基酸组成和功能方面与Ⅰ型IFN更为接近，在IL-10家族和Ⅰ型IFN之间建立了进化上的联系。IFN-λs，尤其是IFN-λ1，具有开发成广谱抗病毒药物的潜在价值，为此开展了该干扰素基因的克隆、表达、生物活性检测和有关信号蛋白相互作用的研究。 本论文中通过RT-PCR从人外周血淋巴细胞中克隆了IFN-λ1 cDNA，其序列与GenBank的报道有一个碱基的差异，但氨基酸序列完全一致。为了大量获得IFN-λ1蛋白，IFN-λ1 cDNA先后被转化到大肠杆菌和酵母表达体系中。在大肠杆菌中，IFN-λ1的表达水平极低，这可能与密码子偏爱性或IFN-λ1对大肠杆菌的毒性有关。随后IFN-λ1 cDNA以串联多拷贝的形式转化到甲醇营养酵母Pichia pastoris中，并通过α因子前导肽分泌到胞外，表达水平约为28mg／L。理论上，重组IFN-λ1(rhIFN-λ1)前体蛋白将受到酵母KEX2蛋白酶的切割，释放出与天然IFN-λ1一级结构完全相同的重组蛋白。但Western Blotting和氨基酸测序发现rhIFN-λ1受到不明蛋白酶的加工，，产生了具有不同N末端的三种蛋白，其中两种蛋白N端带有残留的α因子前导肽序列，第三种蛋白N端缺失了13个氨基酸残基。这可能是因为IFN-λ1的N末端氨基酸Pro属于强刚性氨基酸，抑制了KEX2在其识别序列DKR羧基端的剪切。用FPLC SP Sepharose Fast Flow层析柱纯化了rhIFN-λ1，回收率大于70％。纯化的蛋白能够有效上调Hela细胞内磷酸化STAT1(pY-STAT1)的水平，表明IFN-λ1的N端缺失或冗余不会对激活STAT1造成太大的影响。 IFN-λs受体隶属于Ⅱ型细胞因子受体家族(CRF2)，由两个亚基构成，即CRF2-12和CRF2-4。其中CRF2-12是IFN-λs受体特有的亚基，CRF2-4最早是作为IL-10受体(IL-10R)的小亚基被发现的，故又称为IL-10R2，同时CRF2-4还是IL-22R和IL-26R的共有亚基。通过氨基酸序列分析发现，CRF2-12胞内区含有一个TRAF6结合位点，并有众多的激酶位点。为了验证CRF2-12与TRAF6的相互作用，我们构建了原核表达载体pGEX-6P-TRAF6和真核表达载体pCMV-Myc-CRF2-12、pBudCE4-TRAF6，并从体内
[Abstract]:IFN- 位 s is a newly discovered class of interferon-like cytokines with antiviral activity, including IFN- 位 1 (IL-29), IFN- 位 2 (IL-28A) and IFN- 位 3 (IL-28B). IFN- 位 s is very similar to IL-10 family in gene structure, but closer to type I IFN in amino acid composition and function. The evolutionary association between IL-10 family and type I IFN. IFN- 位 s, especially IFN- 位 1, has the potential value of developing a broad-spectrum antiviral drug. Therefore, the cloning and expression of the interferon gene have been carried out. Detection of biological activity and study of signal protein interaction. IFN- 位 1 cDNA was cloned from human peripheral blood lymphocytes by RT-PCR. The sequence of IFN- 位 1 cDNAwas different from that reported by GenBank, but the amino acid sequence was identical. In order to obtain IFN- 位 1 protein, IFN- 位 1 cDNA was transformed into Escherichia coli and yeast expression system. The expression level of IFN- 位 1 in Escherichia coli is very low, which may be related to the codon preference or the toxicity of IFN- 位 1 to Escherichia coli. Then IFN- 位 1 cDNA was transformed into Pichia pastoris in series and multiple copies, and secreted to extracellular by 伪 -factor prepeptide, the expression level was about 28mg / L. In theory, the recombinant IFN- 位 1 (rhIFN- 位 1) precursor protein will be cleavage by yeast KEX2 protease and release the same recombinant protein as the natural IFN- 位 1 primary structure. However, Western Blotting and amino acid sequencing showed that rhIFN- 位 1 was processed by unknown protease, resulting in three proteins with different N-terminal, two of which contained residual 伪 -factor prepeptide sequence. The N terminal of the third protein contains 13 amino acid residues. This may be due to the fact that the N-terminal amino acid Pro of IFN- 位 1 belongs to a strongly rigid amino acid, which inhibits the shear of KEX2 at the carboxyl terminal of its recognized sequence DKR. RhIFN- 位 1 was purified by FPLC SP Sepharose Fast Flow chromatography, and the recovery was more than 70%. The purified protein can effectively up-regulate the level of phosphorylated STAT1 (pY-STAT1) in Hela cells. The results indicate that the deletion or redundancy of IFN- 位 1 does not have much effect on the activation of STAT1. IFN- 位 s receptor belongs to the type 鈪
本文编号：2163649
[Abstract]:IFN- 位 s is a newly discovered class of interferon-like cytokines with antiviral activity, including IFN- 位 1 (IL-29), IFN- 位 2 (IL-28A) and IFN- 位 3 (IL-28B). IFN- 位 s is very similar to IL-10 family in gene structure, but closer to type I IFN in amino acid composition and function. The evolutionary association between IL-10 family and type I IFN. IFN- 位 s, especially IFN- 位 1, has the potential value of developing a broad-spectrum antiviral drug. Therefore, the cloning and expression of the interferon gene have been carried out. Detection of biological activity and study of signal protein interaction. IFN- 位 1 cDNA was cloned from human peripheral blood lymphocytes by RT-PCR. The sequence of IFN- 位 1 cDNAwas different from that reported by GenBank, but the amino acid sequence was identical. In order to obtain IFN- 位 1 protein, IFN- 位 1 cDNA was transformed into Escherichia coli and yeast expression system. The expression level of IFN- 位 1 in Escherichia coli is very low, which may be related to the codon preference or the toxicity of IFN- 位 1 to Escherichia coli. Then IFN- 位 1 cDNA was transformed into Pichia pastoris in series and multiple copies, and secreted to extracellular by 伪 -factor prepeptide, the expression level was about 28mg / L. In theory, the recombinant IFN- 位 1 (rhIFN- 位 1) precursor protein will be cleavage by yeast KEX2 protease and release the same recombinant protein as the natural IFN- 位 1 primary structure. However, Western Blotting and amino acid sequencing showed that rhIFN- 位 1 was processed by unknown protease, resulting in three proteins with different N-terminal, two of which contained residual 伪 -factor prepeptide sequence. The N terminal of the third protein contains 13 amino acid residues. This may be due to the fact that the N-terminal amino acid Pro of IFN- 位 1 belongs to a strongly rigid amino acid, which inhibits the shear of KEX2 at the carboxyl terminal of its recognized sequence DKR. RhIFN- 位 1 was purified by FPLC SP Sepharose Fast Flow chromatography, and the recovery was more than 70%. The purified protein can effectively up-regulate the level of phosphorylated STAT1 (pY-STAT1) in Hela cells. The results indicate that the deletion or redundancy of IFN- 位 1 does not have much effect on the activation of STAT1. IFN- 位 s receptor belongs to the type 鈪
本文编号：2163649
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