【摘要】：IFN-λs是最新發(fā)現(xiàn)的一類具有抗病毒活性的干擾素樣細(xì)胞因子，包括IFN-λ1(IL-29)、IFN-λ2(IL-28A)和IFN-λ3(IL-28B)。IFN-λs在基因結(jié)構(gòu)上與IL-10家族十分相似，而在氨基酸組成和功能方面與Ⅰ型IFN更為接近，在IL-10家族和Ⅰ型IFN之間建立了進(jìn)化上的聯(lián)系。IFN-λs，尤其是IFN-λ1，具有開發(fā)成廣譜抗病毒藥物的潛在價值，為此開展了該干擾素基因的克隆、表達(dá)、生物活性檢測和有關(guān)信號蛋白相互作用的研究。 本論文中通過RT-PCR從人外周血淋巴細(xì)胞中克隆了IFN-λ1 cDNA，其序列與GenBank的報道有一個堿基的差異，但氨基酸序列完全一致。為了大量獲得IFN-λ1蛋白，IFN-λ1 cDNA先后被轉(zhuǎn)化到大腸桿菌和酵母表達(dá)體系中。在大腸桿菌中，IFN-λ1的表達(dá)水平極低，這可能與密碼子偏愛性或IFN-λ1對大腸桿菌的毒性有關(guān)。隨后IFN-λ1 cDNA以串聯(lián)多拷貝的形式轉(zhuǎn)化到甲醇營養(yǎng)酵母Pichia pastoris中，并通過α因子前導(dǎo)肽分泌到胞外，表達(dá)水平約為28mg／L。理論上，重組IFN-λ1(rhIFN-λ1)前體蛋白將受到酵母KEX2蛋白酶的切割，釋放出與天然IFN-λ1一級結(jié)構(gòu)完全相同的重組蛋白。但Western Blotting和氨基酸測序發(fā)現(xiàn)rhIFN-λ1受到不明蛋白酶的加工，，產(chǎn)生了具有不同N末端的三種蛋白，其中兩種蛋白N端帶有殘留的α因子前導(dǎo)肽序列，第三種蛋白N端缺失了13個氨基酸殘基。這可能是因為IFN-λ1的N末端氨基酸Pro屬于強(qiáng)剛性氨基酸，抑制了KEX2在其識別序列DKR羧基端的剪切。用FPLC SP Sepharose Fast Flow層析柱純化了rhIFN-λ1，回收率大于70％。純化的蛋白能夠有效上調(diào)Hela細(xì)胞內(nèi)磷酸化STAT1(pY-STAT1)的水平，表明IFN-λ1的N端缺失或冗余不會對激活STAT1造成太大的影響。 IFN-λs受體隸屬于Ⅱ型細(xì)胞因子受體家族(CRF2)，由兩個亞基構(gòu)成，即CRF2-12和CRF2-4。其中CRF2-12是IFN-λs受體特有的亞基，CRF2-4最早是作為IL-10受體(IL-10R)的小亞基被發(fā)現(xiàn)的，故又稱為IL-10R2，同時CRF2-4還是IL-22R和IL-26R的共有亞基。通過氨基酸序列分析發(fā)現(xiàn)，CRF2-12胞內(nèi)區(qū)含有一個TRAF6結(jié)合位點(diǎn)，并有眾多的激酶位點(diǎn)。為了驗證CRF2-12與TRAF6的相互作用，我們構(gòu)建了原核表達(dá)載體pGEX-6P-TRAF6和真核表達(dá)載體pCMV-Myc-CRF2-12、pBudCE4-TRAF6，并從體內(nèi)
[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 偽 -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 偽 -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

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