抗TSLP全人源单链抗体的体外亲和力成熟
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摘要
目的:对抗TSLP-scFv全人源单链抗体进行单点突变,以提高其亲和力。方法:本研究前期筛选了特异性好的抗TSLP-scFv,本研究利用Discovery Studio系统模拟TSLP和抗TSLP-scFv三维结构,进行分子对接,对结合表位氨基酸进行随机突变,选取可显著提高其亲和力的氨基酸突变点,根据突变位点设计引物,采用重叠延伸PCR对氨基酸位点进行突变。将突变后的scFv基因与表达载体pLZ16连接,转化E.coli DH5αF',表达后筛选亲和力提高的抗TSLP-scFv。结果:DS系统筛选出可以提高scFv亲和力的5个单点突变氨基酸位点,PCR扩增出突变后的抗TSLP-scFv DNA片段大小为1000bp左右。将测序正确的菌株进行表达,通过ELISA筛选出与抗原结合力提升的抗TSLP-scFv;通过生物大分子相互作用技术,测定单抗的亲和力,突变后的scFv M4亲和力较突变前提高了10倍左右。结论:成功提升了抗TSLP-scFv全人源单链抗体的亲和力。
Objective: To single amino acid mutation of the full human scFvs against TSLP to enhance its affinity. Methods:The specific scFvs against TSLP was screened in our previous study and here the three-dimensional structures of TSLP and anti-TSLP scFvs were simulated by Discovery Studio system,then the molecular docking was made. The amino acids of binding epitope were randomly mutated and the mutated amino acids were selected which could remarkably improve the affinity of scFvs. The primers were designed based on the sequence of mutation amino acids and the scFv sequences were mutated by the overlapping extension PCR. The DNA of mutated scFvs was ligated with the expression vector p LZ16 and transformed into E. coli DH5αF'. Then the scFvs were expressed and the scFvs with improved affinity were selected by ELISA and BIAcore. Results: The five scFvs with single amino acid mutation were screened out by DS system,which could elevate the affinity of scFvs. The mutated anti-TSLP-scFvs were amplified by PCR,which size was about 1 000 bp. The mutated scFvs with correct sequence were expressed,and the mutated scFvs with improved affinity were detected by ELISA and BIAcore. The affinity of selected mutated scFv( M4) has been about 10 times higher than the scFv nonmutation. Conclusion: The affinity of anti-TSLP-scFv has been improved successfully.
Objective: To single amino acid mutation of the full human scFvs against TSLP to enhance its affinity. Methods:The specific scFvs against TSLP was screened in our previous study and here the three-dimensional structures of TSLP and anti-TSLP scFvs were simulated by Discovery Studio system,then the molecular docking was made. The amino acids of binding epitope were randomly mutated and the mutated amino acids were selected which could remarkably improve the affinity of scFvs. The primers were designed based on the sequence of mutation amino acids and the scFv sequences were mutated by the overlapping extension PCR. The DNA of mutated scFvs was ligated with the expression vector p LZ16 and transformed into E. coli DH5αF'. Then the scFvs were expressed and the scFvs with improved affinity were selected by ELISA and BIAcore. Results: The five scFvs with single amino acid mutation were screened out by DS system,which could elevate the affinity of scFvs. The mutated anti-TSLP-scFvs were amplified by PCR,which size was about 1 000 bp. The mutated scFvs with correct sequence were expressed,and the mutated scFvs with improved affinity were detected by ELISA and BIAcore. The affinity of selected mutated scFv( M4) has been about 10 times higher than the scFv nonmutation. Conclusion: The affinity of anti-TSLP-scFv has been improved successfully.
引文
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[9]Li H,Zhao H,Yu J,et al.Increased prevalence of regulatory T cells in the lung cancer microenvironment:a role of thymic stromal lymphopoietin[J].Cancer Immunol Imunother,2011,60(11):1587-1596.
[10]Takahashi N,Sugaya M,Suga H,et al.Thymic stromal chemokine TSLP acts through Th2 cytokine production to induce Cutaneous T-cell lymphoma[J].Cancer Res,2016,76(21):6241-6252.
[11]Lo Kuan E.Thymic stromal lymphopoietin and cancer[J].J Immunol,2014,193(9):4283-4288.
[12]Zhang Y.Effects of TSLP on obstetrical and gynecological diseases[J].Am Reprod Immunol(New York,N.Y.:1989),2017,77(1):doi:10.1111/aji.12.612.
[13]Semlali A,Jacques E,Koussih L,et al.Thymicstromallymphopoietin-induced human asthmatic airway epithelial cell proliferation through an IL-13-dependent pathway[J].J Allergy Clin Immunol,2010,125(4):844-850.
[14]Tatsuno K,Fujiyama T,Yamaguchi H,et al.TSLP directly interacts with skin-homing Th2 cells highly expressing its receptor to enhance IL-4 production in atopic dermatitis[J].J Invest Dermatol,2015,135(12):3017-3024.
[15]Akasaki S,Matsushita K,Kato Y,et al.Murine allergic rhinitis and nasal Th2 activation are mediated via TSLP-and IL-33-signaling pathways[J].Int Immunol,2016,28(2):65-76.
[16]Mitchell PD.Epithelial derived cytokines in Asthma[J].Chest,2017,151(6):1338-1344.
[17]孙延鹏,卢祖能,王云甫.胸腺基质淋巴细胞生成素与自身免疫性疾病的研究进展[J].医学研究杂志,2013,42(1):183-186.
[18]Park JH,Jeong DY,Peyrin-Biroulet L,et al.Insight into the role of TSLP in inflammatory bowel diseases[J].Autoimmun Rev,2017,16(1):55-63.
[19]孙延鹏,卢祖能,孙强,等.重症肌无力胸腺基质淋巴细胞生成素表达与CD4+CD25+Foxp3+Treg细胞表型的研究[J].中国神经免疫学和神经病学杂志,2012,19(6):439-443.
[20]Baatjes AJ,Smith SG,Dua B,et al.Treatment with anti-OX40L or anti-TSLP does not alter the frequency of T regulatory cells in allergic asthmatics[J].Allergy,2015,70(11):1505-1508.
[21]朱建光,袁青,黄黎,等.抗TSLP全人源单链抗体筛选与初步鉴定[J].中国免疫学杂志,2014,30(12):1662-1665,1669.
[2]Friend SL,Hosier S,Nelson A,et al.Athymic stromal cell line supports in vitro development of surface Ig M+B cells and produces a novel growth factor affecting B and T lineage cells[J].Exp Hematol,1994,22(3):321-328.
[3]Harada M,Hirota T,Jodo AI,et al.Thymic stromal lymphopoietin gene promoter polymorphisms are associated with susceptibility to bronchial asthma[J].Am J Respirat cell Mol Biol,2011,44(6):787-793.
[4]Watson B.Thymic stromal lymphopoietin:a central regulator of allergic asthma[J].Expert Opinion Thera Targets,2014,18(7):771-785.
[5]Soumelis V,Reche PA,Kanzler H,et al.Human epithelial cells trigger dendritic cell mediated allergic inflammation by producing TSLP[J].Nat Immunol,2002,3(7):673-680.
[6]Al-Shami A,Spolski R,Kelly J,et al.A role for TSLP in the development of inflammation in an asthma model[J].J Exp Med,2005,202(6):829-839.
[7]Rochman Y,Spolski R.New insights into the regulation of T cells by gamma(c)family cytokines[J].Nat Rev Immunol,2009,9(7):480-490.
[8]Erdmann RB,Gartner JG,Leonard WJ.Lack of functional TSLPreceptors mitigates Th2 polarization and the establishment and growth of 4T1 primary breast tumours but has different effects on tumour quantities in the lung and brain[J].Scand J Immunol,2013,78(5):408-418.
[9]Li H,Zhao H,Yu J,et al.Increased prevalence of regulatory T cells in the lung cancer microenvironment:a role of thymic stromal lymphopoietin[J].Cancer Immunol Imunother,2011,60(11):1587-1596.
[10]Takahashi N,Sugaya M,Suga H,et al.Thymic stromal chemokine TSLP acts through Th2 cytokine production to induce Cutaneous T-cell lymphoma[J].Cancer Res,2016,76(21):6241-6252.
[11]Lo Kuan E.Thymic stromal lymphopoietin and cancer[J].J Immunol,2014,193(9):4283-4288.
[12]Zhang Y.Effects of TSLP on obstetrical and gynecological diseases[J].Am Reprod Immunol(New York,N.Y.:1989),2017,77(1):doi:10.1111/aji.12.612.
[13]Semlali A,Jacques E,Koussih L,et al.Thymicstromallymphopoietin-induced human asthmatic airway epithelial cell proliferation through an IL-13-dependent pathway[J].J Allergy Clin Immunol,2010,125(4):844-850.
[14]Tatsuno K,Fujiyama T,Yamaguchi H,et al.TSLP directly interacts with skin-homing Th2 cells highly expressing its receptor to enhance IL-4 production in atopic dermatitis[J].J Invest Dermatol,2015,135(12):3017-3024.
[15]Akasaki S,Matsushita K,Kato Y,et al.Murine allergic rhinitis and nasal Th2 activation are mediated via TSLP-and IL-33-signaling pathways[J].Int Immunol,2016,28(2):65-76.
[16]Mitchell PD.Epithelial derived cytokines in Asthma[J].Chest,2017,151(6):1338-1344.
[17]孙延鹏,卢祖能,王云甫.胸腺基质淋巴细胞生成素与自身免疫性疾病的研究进展[J].医学研究杂志,2013,42(1):183-186.
[18]Park JH,Jeong DY,Peyrin-Biroulet L,et al.Insight into the role of TSLP in inflammatory bowel diseases[J].Autoimmun Rev,2017,16(1):55-63.
[19]孙延鹏,卢祖能,孙强,等.重症肌无力胸腺基质淋巴细胞生成素表达与CD4+CD25+Foxp3+Treg细胞表型的研究[J].中国神经免疫学和神经病学杂志,2012,19(6):439-443.
[20]Baatjes AJ,Smith SG,Dua B,et al.Treatment with anti-OX40L or anti-TSLP does not alter the frequency of T regulatory cells in allergic asthmatics[J].Allergy,2015,70(11):1505-1508.
[21]朱建光,袁青,黄黎,等.抗TSLP全人源单链抗体筛选与初步鉴定[J].中国免疫学杂志,2014,30(12):1662-1665,1669.