猪CD40L的可溶性表达及其生物学活性检测  

Soluble expression and biological activity detection of porcine CD40L

作　　者：贺艳艳 郭慧琛[2] 孙世琪[2] 穆素雨 敬晓棋[1] HE Yanyan;GUO Huichen;SUN Shiqi;MU Suyu;JING Xiaoqi(College of Life Sciences,Yulin University,Yulin 719000,China;State Key Laboratory for Animal Disease Control and Prevention/College of Animal Medicine of Lanzhou University/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730000,China)

机构地区：[1]榆林学院生命科学学院,陕西榆林719000 [2]中国农业科学院、兰州兽医研究所、兰州大学动物医学与生物安全学院、动物疫病防控全国重点实验室,甘肃兰州730000

出　　处：《中国兽医科学》2025年第3期389-395,共7页Chinese Veterinary Science

基　　金：国家成都农业科技中心地方财政专项(NASC2024KR06);兰州市人才创业创新项目(2023-RC-3)。

摘　　要：为探讨可溶性猪CD40L(第113—261位氨基酸)对猪外周血单核细胞(PBMC)的活化作用,将构建好的含猪CD40L基因的质粒在大肠杆菌BL21(DE3)中表达,纯化的重组CD40L蛋白偶联FITC后与PBMC进行原位杂交,同时使用荧光显微镜和流式细胞仪分析研究CD40L蛋白刺激B细胞的生物学活性。SDS-PAGE和Western-blot结果显示,表达的重组CD40L蛋白大小约14 kDa,确定最佳表达条件为16℃、D_(600 nm)值为1.1、0.5 mmol/L IPTG诱导12 h;流式细胞仪分析表明,重组CD40L蛋白能与PBMC表面的CD40蛋白分子结合,重组CD40L蛋白可以明显活化B细胞(P<0.001)。结论:CD40L具有促进B细胞活化的生物学活性,CD40L能促进免疫细胞反应,具有免疫调节作用。本研究为CD40L在重要家畜物种和人类疾病动物模型中的作用奠定了基础。In order to investigate the activation of soluble porcine CD40L(113-261 amino acids) on porcine peripheral blood mononuclear cells(PBMC),a plasmid containing porcine CD40L gene was constructed and expressed in Escherichia coli BL21(DE3).The purified recombinant CD40L protein was conjured with FITC and then hybridized with PBMC in situ.The biological activity of B cells stimulated by CD40L protein was studied by fluorescence microscopy and flow cytometry.SDS-PAGE and Western-blot results showed that the size of recombinant CD40L protein was about 14 kDa.The optimal expression conditions were 16 ℃,D_(600 nmvalue) was 1.1,and the concentration of 0.5 mmol/L was induced by IPTG for 12 h.Flow cytometry analysis showed that the recombinant CD40L protein could bind to CD40 protein molecules on the surface of PBMC,and the recombinant CD40L protein could significantly activate B cells(P<0.001).In conclusion,CD40L had the biological activity of promoting B cell activation,and CD40L can promote immune cell reaction,and have the immunomodulatory effect.This study provided a basis for the role of CD40L in important livestock species and animal models of human disease.

关 键 词：猪CD40L 原核表达 生物学活性 

分 类 号：S852.4[农业科学—基础兽医学]