本文關(guān)鍵詞：重組人干擾素λ3的制備及聚乙二醇修飾　出處：《北京協(xié)和醫(yī)學(xué)院》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 重組人干擾素λ3 原核表達(dá) 純化 mPEG-丁醛 抗病毒活性

【摘要】：目的：人干擾素λ(Human interferon lambdas, IFN-λs)是一類新發(fā)現(xiàn)的干擾素,在調(diào)節(jié)人粘膜／上皮組織的抗病毒反應(yīng)和保護(hù)胃腸道上皮細(xì)胞中發(fā)揮著極其重要的作用。然而,有關(guān)IFN-λs的生物學(xué)特性和其在整個(gè)免疫系統(tǒng)中的作用機(jī)制尚須進(jìn)一步的研究。本研究主要以人干擾素λ3為研究對象,利用大腸桿菌表達(dá)體系表達(dá)重組人干擾素λ3,并進(jìn)行純化,對重組蛋白進(jìn)行聚乙二醇定點(diǎn)修飾,以期獲得具有生物學(xué)活性的聚乙二醇化重組人干擾素λ3。 方法：化學(xué)合成法合成密碼子優(yōu)化的rtIFN-λ3基因,并與pThioHisA載體連接構(gòu)建原核表達(dá)質(zhì)粒,轉(zhuǎn)化大腸桿菌Top10中誘導(dǎo)表達(dá)；表達(dá)產(chǎn)物經(jīng)透析復(fù)性、層析純化后用EK酶切下硫氧還蛋白。對進(jìn)一步層析純化分離得到的rhIFN-λ3蛋白進(jìn)行單甲氧基聚乙二醇-丁醛(mPEG-ButyrALD)修飾,并對修飾產(chǎn)物進(jìn)行初步分離純化和活性檢測。 結(jié)果：通過限制性內(nèi)切酶消化、PCR擴(kuò)增和測序確定獲得了重組表達(dá)質(zhì)粒pThioHisA-rhIFN-λ3。該重組質(zhì)粒在大腸桿菌中主要以包涵體形式表達(dá)重組蛋白,經(jīng)透析復(fù)性、EK酶切及離子交換層析后獲得分子量為19kDa的rhIFN-λ3,純度達(dá)90%。mPEG-丁醛對rhIFN-λ3蛋白的最適修飾條件為rhIFN-λ3蛋白和10kDamPEG-丁醛按1：10摩爾比混合,于pH6.5的磷酸鹽緩沖液中在室溫反應(yīng)8h。反應(yīng)混合物經(jīng)陽離子交換柱層析純化后獲得的聚乙二醇單點(diǎn)修飾產(chǎn)物(PEG-rhIFN-λ3)純度達(dá)86%以上。rhIFN-λ3在WISH細(xì)胞中對VSV的半數(shù)效應(yīng)濃度(EC50)為8.43ng/ml,PEG-rhIFN-λ3的EC50為49.19ng/ml,PEG修飾后保留了17.14%的體外活性。 結(jié)論：成功實(shí)現(xiàn)rhIFN-λ3在大腸桿菌表達(dá)體系中的表達(dá)；表達(dá)產(chǎn)物經(jīng)復(fù)性后具有體外抗病毒活性：制備得到活性保留率約為17.14%的聚乙二醇化rhIFN-λ3,為其進(jìn)一步功能性研究奠定了基礎(chǔ)。修飾后rhIFN-λ3的免疫原性及抗原性、體內(nèi)生物學(xué)活性及穩(wěn)定性等有待進(jìn)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.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 王一欣;孫雅煊;;聚乙二醇干擾素的研究和應(yīng)用[J];甘肅科技;2006年06期

2 鄧培媛;裴振峨;朱素君;;干擾素不良反應(yīng)綜述[J];臨床藥物治療雜志;2003年04期

3 寧云山,李妍,王小寧;包含體蛋白質(zhì)的復(fù)性研究進(jìn)展[J];生物技術(shù)通訊;2001年03期

4 姜忠義,高蓉,王艷強(qiáng),孫彥;蛋白質(zhì)和多肽藥物聚乙二醇化的問題與對策[J];藥學(xué)學(xué)報(bào);2002年05期

，

本文編號(hào)：1389226