犬干扰素α2的原核表达及抗病毒活性分析
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摘要
本研究将人工合成的犬干扰素α2成熟区序列插入原核表达载体p ET-28a(+)中,构建重组表达质粒p ET-28a-Ca IFN-α2;然后将该质粒转化至大肠杆菌Rosetta感受态中进行IPTG诱导表达,经SDS-PAGE和Western blot分析鉴定,分子量约为23 k Da的目的蛋白表达,表达的重组蛋白主要以包涵体的形式存在,表达量约占菌体总蛋白的52.5%。包涵体蛋白经变性、复性和纯化处理后,获得的重组Ca IFN-α2纯度为92%;用MDCK/VSV微量细胞病变抑制法检测重组蛋白的抗病毒活性为3.16×106/m L。本研究结果为进一步研制新型犬用干扰素制品奠定了物质基础。
In this study, the mature encoding sequence of canine interferon α 2 subtype was synthesized and inserted into the prokaryotic expression vector p ET28 a(+) to construct the recombinant plasmid p ET-28 a-Ca IFN-α2. The resulting p ET-28 a-Ca IFN-α2 was transformed into E.coli Rosetta competent cells for protein expression with induction of IPTG. The recombinant Ca IFN-α2 was determined to be 23 k Da of molecular mass in SDS-PAGE and Western blot. In addition, the recombinant Ca IFN-α2 mainly existed in a form of inclusion body and accounted for 52.5% of the total bacterial proteins. The recombinant Ca IFN-α2 with the purity of 92% was obtained through a series of denaturation, renaturation and purification treatments. The antiviral activity of the recombinant Ca IFN-α 2 was determined by CPE reduction of vesicular stomatitis virus infection in MDCK cells. The results showed that the antiviral activity of the recombinant Ca IFN-α2 was 3.16×106 U/m L, which laid the foundation for the development of novel canine interferon agent.
In this study, the mature encoding sequence of canine interferon α 2 subtype was synthesized and inserted into the prokaryotic expression vector p ET28 a(+) to construct the recombinant plasmid p ET-28 a-Ca IFN-α2. The resulting p ET-28 a-Ca IFN-α2 was transformed into E.coli Rosetta competent cells for protein expression with induction of IPTG. The recombinant Ca IFN-α2 was determined to be 23 k Da of molecular mass in SDS-PAGE and Western blot. In addition, the recombinant Ca IFN-α2 mainly existed in a form of inclusion body and accounted for 52.5% of the total bacterial proteins. The recombinant Ca IFN-α2 with the purity of 92% was obtained through a series of denaturation, renaturation and purification treatments. The antiviral activity of the recombinant Ca IFN-α 2 was determined by CPE reduction of vesicular stomatitis virus infection in MDCK cells. The results showed that the antiviral activity of the recombinant Ca IFN-α2 was 3.16×106 U/m L, which laid the foundation for the development of novel canine interferon agent.
引文
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[12]Peciak K,Tommasi R,Choi J W,et al.Expression of soluble and active interferon consensus in SUMO fusion expression system in E.coli[J].Protein Expr Purif,2014,99:18-26.
[13]Srivastava P,Bhattacharaya P,Pandey G,et al.Overexpression and purification of recombinant human interferon alpha2b in Escherichia coli[J].Protein Expr Purif,2005,41(2):313-322.
[14]Yang L M,Xue Q H,Sun L,et al.Cloning and characterization of a novel feline IFN-omega[J].J Interferon Cytokine Res,2007,27(2):119-127.
[15]Basu A,Li X,Leong S S.Refolding of proteins from inclusion bodies:rational design and recipes[J].Appl Microbiol Biotechnol,2011,92(2):241-251.
[16]Leong S S,Middelberg A P.The refolding of differentα-fetoprotein variants[J].Protein Sci,2006,15(9):2040-2050.
[2]Samuel C E.Antiviral actions of interferons[J].Clin Microbiol Rev,2001,14(4):778-809.
[3]Malmgaard L.Induction and regulation of IFNs during viral infections[J].J Interferon Cytokine Res,2004,24(8):439-454.
[4]Yang L,Xu L,Li Y,et al.Molecular and functional characterization of canine interferon-epsilon[J].J Interferon Cytokine Res,2013,33(12):760-768.
[5]Taira O,Watanugi I,Hagiwara Y,et al.Cloning and expression of canine interferon-alpha genes in Escherichia coli[J].J Vet Med Sci,2005,67(10):1059-1062.
[6]范翠英,冯利兴,樊金玲,等.重组蛋白表达系统的研究进展[J].生物技术,2012,22(2):76-80.
[7]Bis R L,Stauffer T M,Singh S M,et al.High yield soluble bacterial expression and streamlined purification of recombinant human interferonα-2a[J].Protein Expr Purif,2014,99:138-146.
[8]林德贵.注射用重组犬干扰素α治疗犬瘟热病犬的临床应用[C].北京全国兽医外科学第13次学术研讨会、小动物医学第1次学术研讨会暨奶牛疾病第3次学术讨论会,2006.
[9]苏建东.重组犬α干扰素活性检测及对犬细小病毒病的疗效研究[D].北京:中国农业科学院,2013.
[10]邹勇,宋继萍,吕宏亮,等.狂犬病-干扰素联合制剂的免疫原性及治疗作用[J].中国生物制品学杂志,2002,15(5):287-289.
[11]徐晓娟,王怡飞,王玉,等.犬干扰素α1基因在大肠埃希菌中的表达及其抗病毒活性的测定[J].中国生物制品学杂志,2013,26(8):1084-1087.
[12]Peciak K,Tommasi R,Choi J W,et al.Expression of soluble and active interferon consensus in SUMO fusion expression system in E.coli[J].Protein Expr Purif,2014,99:18-26.
[13]Srivastava P,Bhattacharaya P,Pandey G,et al.Overexpression and purification of recombinant human interferon alpha2b in Escherichia coli[J].Protein Expr Purif,2005,41(2):313-322.
[14]Yang L M,Xue Q H,Sun L,et al.Cloning and characterization of a novel feline IFN-omega[J].J Interferon Cytokine Res,2007,27(2):119-127.
[15]Basu A,Li X,Leong S S.Refolding of proteins from inclusion bodies:rational design and recipes[J].Appl Microbiol Biotechnol,2011,92(2):241-251.
[16]Leong S S,Middelberg A P.The refolding of differentα-fetoprotein variants[J].Protein Sci,2006,15(9):2040-2050.