Application of KIH Structure in Recombinant Expression of Human Interleukin-35 in vitro
10.13865/j.cnki.cjbmb.2024.04.1051
- VernacularTitle:KIH结构在人白介素-35体外重组表达中的应用
- Author:
Kai-Yue ZHANG
1
,
2
;
Zong-Yan LI
;
Rui-Qing CAO
;
Lin-Lin MENG
;
Xiang-Wei HU
;
Yu-Chao GU
;
Jian-Gang WANG
Author Information
1. 中国海洋大学海洋药物教育部重点实验室,中国海洋大学医药学院,山东青岛 266100
2. 康立泰生物医药(青岛)有限公司,山东青岛 266000
- Keywords:
human interleukin-35(IL-35);
Knobs in to Holes(KIH);
fusion protein;
polymer
- From:
Chinese Journal of Biochemistry and Molecular Biology
2024;40(6):857-866
- CountryChina
- Language:Chinese
-
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.
