摘要
白细胞介素35(interleukin-35,IL-35)是非常重要的免疫抑制性细胞因子,被证实在多种疾病的免疫应答中发挥作用.本研究通过克隆人IL-35基因编码序列,构建单亚基表达载体pXC17.4-p35和pcDNA3.1(+)-EBI3,共转染CHO-K1细胞体外表达IL-35,经检测未发现p35亚基与EBI3亚基的结合.Knobs-into-Holes(KIH)可解决异源抗体重链错配的问题,因此,在原始序列的基础上融合KIH结构构建表达载体pXC17.4-p35-Fch和pcDNA3.1(+)-EBI3-Fck来表达KIH-IL-35重组融合蛋白质;同时交换2个亚基的表达载体来验证不同表达载体对KIH-IL-35表达量的影响.经过多种蛋白质检测方法的分析,显示KIH-IL-35结构的正确表达率明显提高.大量表达后对KIH-IL-35进行亲和纯化,采用ELISA法检测纯化后的KIH-IL-35与糖蛋白130(gp130)分子的结合活性,结果表明,KIH-IL-35与gp130的结合呈浓度依赖性.采用细胞活性检测方法检测KIH-IL-35与M1细胞的间接活性,结果表明,M1细胞的抑制率与KIH-IL-35的浓度呈剂量依赖性增长.此外,本研究成功建立了 一种利用激活的人外周血单核细胞(peripheral blood mononuclear cells,PBMCs)对IL-35进行活性检测的方法,结果显示,激活的PBMCs与KIH-IL-35的浓度呈剂量依赖性增长.总之,本研究利用KIH-IL-35模型,提高了重组人IL-35的正确表达率,并验证其在体外的高活性,为IL-35的研究及类似异源二聚体细胞因子的重组表达提供新思路.
Abstract
Interleukin-35(IL-35)is an important immunosuppressive cytokine that has been shown to play a role in the immune response of various diseases.In this study,we cloned the coding sequence of human IL-35 gene,constructed single subunit expression vectors pXC17.4-p35 and pcDNA3.1(+)-EBI3,and co-transfected CHO-K1 cells to express IL-35 in vitro.No binding was found between subunits of p35 and EBI3.Knobs-into-Holes(KIH)can solve the problem of heavy chain mismatch of heterolo-gous antibodies.Therefore,expression vectors pXC17.4-p35-Fch and pcDNA3.1(+)-EBI3-Fck were constructed by fusing KIH structures on the basis of the original sequences to express the recombinant fu-sion protein of KIH-IL-35.The expression vectors of two subunits were exchanged at the same time to verify the influence of different vectors on the expression level of KIH-IL-35.The analysis of various pro-tein detection methods showed that the correct expression rate of KIH-IL-35 structure was significantly im-proved.Affinity purification of KIH-IL-35 was performed after large amount of expression,and the bind-ing activity of KIH-IL-35 to glycoprotein 130(gp130)was detected by ELISA.The results showed that the binding of KIH-IL-35 to gp130 was concentration dependent.The indirect activity of KIH-IL-35 and M1 cells was detected by cell activity assay.Further results showed that the inhibition rate of M1 cells in-creased in a dose-dependent manner with the concentration of KIH-IL-35.In addition,a method for de-termining IL-35 activity by activated human peripheral blood mononuclear cells was successfully estab-lished.Activated PBMCs increased in a dose-dependent manner with KIH-IL-35 concentration.In sum-mary,this study utilized the KIH-IL-35 model to enhance the expression of recombinant human IL-35 and validated its high activity in vitro,providing new ideas for the study of IL-35 and the recombinant expres-sion of similar heterodimeric cytokines.
基金项目
山东省重点研发计划2023(2023CXGC010510)
青岛市关键技术攻关及产业化示范类项目(23-1-4-xxgg-14-nsh)
NETL
NSTL
万方数据