重组人干扰素λ3的制备及聚乙二醇修饰

发布时间：2018-01-06 19:29

本文关键词：重组人干扰素λ3的制备及聚乙二醇修饰　出处：《北京协和医学院》2012年硕士论文　论文类型：学位论文

【摘要】：目的：人干扰素λ(Human interferon lambdas, IFN-λs)是一类新发现的干扰素,在调节人粘膜／上皮组织的抗病毒反应和保护胃肠道上皮细胞中发挥着极其重要的作用。然而,有关IFN-λs的生物学特性和其在整个免疫系统中的作用机制尚须进一步的研究。本研究主要以人干扰素λ3为研究对象,利用大肠杆菌表达体系表达重组人干扰素λ3,并进行纯化,对重组蛋白进行聚乙二醇定点修饰,以期获得具有生物学活性的聚乙二醇化重组人干扰素λ3。方法：化学合成法合成密码子优化的rtIFN-λ3基因,并与pThioHisA载体连接构建原核表达质粒,转化大肠杆菌Top10中诱导表达；表达产物经透析复性、层析纯化后用EK酶切下硫氧还蛋白。对进一步层析纯化分离得到的rhIFN-λ3蛋白进行单甲氧基聚乙二醇-丁醛(mPEG-ButyrALD)修饰,并对修饰产物进行初步分离纯化和活性检测。结果：通过限制性内切酶消化、PCR扩增和测序确定获得了重组表达质粒pThioHisA-rhIFN-λ3。该重组质粒在大肠杆菌中主要以包涵体形式表达重组蛋白,经透析复性、EK酶切及离子交换层析后获得分子量为19kDa的rhIFN-λ3,纯度达90%。mPEG-丁醛对rhIFN-λ3蛋白的最适修饰条件为rhIFN-λ3蛋白和10kDamPEG-丁醛按1：10摩尔比混合,于pH6.5的磷酸盐缓冲液中在室温反应8h。反应混合物经阳离子交换柱层析纯化后获得的聚乙二醇单点修饰产物(PEG-rhIFN-λ3)纯度达86%以上。rhIFN-λ3在WISH细胞中对VSV的半数效应浓度(EC50)为8.43ng/ml,PEG-rhIFN-λ3的EC50为49.19ng/ml,PEG修饰后保留了17.14%的体外活性。结论：成功实现rhIFN-λ3在大肠杆菌表达体系中的表达；表达产物经复性后具有体外抗病毒活性：制备得到活性保留率约为17.14%的聚乙二醇化rhIFN-λ3,为其进一步功能性研究奠定了基础。修饰后rhIFN-λ3的免疫原性及抗原性、体内生物学活性及稳定性等有待进一步研究。
[Abstract]:Objective: human interferon lambda (Human interferon Lambdas, IFN- s) is a newly identified interferon, play an extremely important role in gastrointestinal epithelial cells and regulating human mucosal protection antiviral response / epithelial tissue. However, the biological characteristics of the IFN- lambda s and its role in the whole immune system the mechanism is to be further studied. This research focuses on human interferon lambda 3 as the research object, using the Escherichia coli expression system of recombinant human interferon lambda 3, and was purified for site-specific PEGylation of recombinant protein, pegylated recombinant human interferon lambda in order to obtain biologically active 3.
Methods: rtIFN- lambda synthesis codon optimization of the chemical synthesis of the 3 gene, and constructed the prokaryotic expression plasmid and pThioHisA vector induced expression in Escherichia coli Top10; the expression product was dialyzed and purified by EK enzyme digestion by thioredoxin. Further purification of the isolated rhIFN- lambda 3 protein single methoxy polyethylene glycol aldehyde (mPEG-ButyrALD) modification, and the modified products were preliminary separation purification and activity detection.
Results: by restriction endonuclease digestion, PCR amplification and sequencing the recombinant expression plasmid pThioHisA-rhIFN- lambda 3. the recombinant plasmid in Escherichia coli is mainly expressed in a form of inclusion body of recombinant protein, after renaturation, EK enzyme digestion and ion exchange chromatography to obtain the molecular weight of 19kDa rhIFN- lambda 3, the purity of 90%.mPEG- aldehyde of the 3 protein rhIFN- lambda optimum conditions for modified rhIFN- lambda 3 protein and 10kDamPEG- aldehyde mixed according to the molar ratio of 1:10, phosphate buffer in pH6.5 reaction at room temperature 8h. reaction mixture by cation exchange column chromatography to obtain polyethylene glycol single point modified product (PEG-rhIFN- lambda 3) reached a purity of more than 86%.RhIFN- in 3. WISH cells in half effect on the concentration of VSV (EC50) 8.43ng/ml, PEG-rhIFN- 3 EC50 lambda 49.19ng/ml, the modified PEG retains 17.14% activity in vitro.
Conclusion: the successful implementation of rhIFN- lambda 3 in Escherichia coli expression system; expression product after renaturation with antiviral activity in vitro: prepared activity retention rate is about 17.14% of the pegylated rhIFN- lambda 3, is the foundation for further functional studies. The modified rhIFN- lambda 3 immunogenicity and antigenicity that should be further studied in vivo biological activity and stability.

【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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