可溶性重组人IL23R-CHR在大肠埃希菌中的表达、纯化及活性研究
Expression, purification and functional assay of rhIL23R-CHR
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摘要: 利用人白细胞cDNA文库体外扩增获得hIL23R-CHR目的基因，克隆至原核表达载体pET22b/pET28a/pET32a，转化BL21(DE3)宿主菌，并对工程菌的表达方式(直接表达、可溶性标签融合表达、分泌表达及包涵体表达)及表达量进行比较，确立pET 32a为表达载体，经IPTG诱导表达后通过镍亲和柱分离纯化融合蛋白Trx-IL23R-CHR，肠激酶4 ℃酶切Trx-IL23R-CHR分子24 h，上镍柱进行二次纯化，获得纯度达90%以上的rhIL23R-CHR分子。亲和力实验证实:原核重组表达的hIL23R-CHR与hIL23在物质的量比为1∶1时可以有效结合，小脑HE病理切片提示rhIL23R-CHR分子对实验性变态反应性脑脊髓炎(EAE)模型鼠有一定的保护作用。
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关键词:
- IL-23 /
- hIL23R-CHR /
- 原核表达 /
- 分离纯化
Abstract: The IL23R-CHR gene was cloned from human leukocyte cDNA library and inserted into pMD18T vector. The recombinant plasmid was confirmed by DNA sequencing. Followed, the IL23R-CHR was subcloned into pET22b/pET28a/pET32a vector, and then the recombinant expression plasmids were transformed into BL21(DE3). In this study, we optimized the expression condition by comparing direct expression between soluble fusion protein expression, secretion expression and inclusion body expression. Finally, hIL23R-CHR was expressed and purified from Trx-hIL23R-CHR fusion protein with a purity of 90%. Affinity assay also demonstrated that purified hIL23R-CHR was able to bind to human IL-23. Histological examination revealed that hIL23R-CHR can protect the central nervous system of EAE mice.-
Keywords:
- IL23 /
- hIL23R-CHR /
- expression /
- purification
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[1] Zhou L,Ivanov II,Spolski R,et al.IL-6 programs T(H)-17 cell differentiation by promoting sequential engagement of the IL-21 and IL-23 pathways[J].Nat Immunol,2007,8(9):967-74. [2] Chen Y, Claire LL, Brent M, et al. Anti-IL-23 therapy inhibits multiple inflammatory pathways and ameliorates autoimmune encephalomyelitis[J].J Clin Invest,2006,116(5):1 317-1 326. [3] Miossec P,Kolls JK.Targeting IL-17 and TH17 cells in chronic inflammation[J].Nat Rev Drug Discov,2012,11(10):763-776. [4] Bettelli E,Korn T,Oukka M,et al.Induction and effector functions of T(H)17 cells[J].Nature,2008,453(7 198):1 051-1 057. [5] Ciofani M,Madar A,Galan C,et al.A validated regulatory network for Th17 cell specification[J].Cell,2012,151(2):289-303. [6] Griffiths CE,Strober BE,van de Kerkhof P,et al.Comparison of ustekinumab and etanercept for moderate-to-severe psoriasis[J].N Engl J Med,2010,362(2):118-128. [7] Sandborn WJ,Gasink C,Gao LL,et al.Ustekinumab induction and maintenance therapy in refractory Crohn′s disease[J].N Engl J Med,2012,367(16):1 519-1 528.
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