摘要
B7-H4分子是B7家族的成员之一。在人和小鼠的研究显示,该分子对T细胞免疫学功能的发挥具有重要调节作用。但是该分子在猪免疫系统中的功能还没有报道。本研究通过PCR技术扩增到了猪的免疫调控分子B7-H4（pB7-H4）的基因片段,并将其克隆到pET-32a（＋）原核表达载体,获得了pET-32a-pB7-H4。用原核表达并纯化的重组pB7-H4蛋白免疫BALB/c小鼠,取其脾淋巴细胞与SP2/0细胞进行融合,通过间接ELISA筛选出了1株抗pB7-H4蛋白的单克隆抗体（MAb）杂交瘤细胞株3C8,MAb亚类鉴定结果显示其抗体亚类为IgG1。流式细胞仪分析结果显示,该MAb能特异性结合细胞系上瞬时表达的pB7-H4。利用该单抗进一步分析pB7-H4在猪外周血T淋巴细胞、巨噬细胞和肺泡巨噬细胞上的表达,结果,pB7-H4在肺泡巨噬细胞和外周血巨噬细胞中高效表达,但是在T淋巴细胞中检测不到明显的表达。上述研究结果表明,该单抗的制备为进一步研究pB7-H4的免疫学功能提供了有效工具。
B7-H4 is one member of B7 family.Many researches in mice and humans show that B7-H4 plays an important regulatory role on the function of T cells.However,there is no report on the function of B7-H4 in porcine immune system so far.In this research,the gene fragment of porcine B7-H4（pB7-H4）was amplified by PCR and cloned into prokaryotic expression vector pET-32 a（＋）to obtain pET-32 a-pB7-H4.SP2/0 myeloma cells were fused with spleen cells from the BALB/c mice immunized with purified recombinant pB7-H4 expressed in E.coli.One hybridoma cell line,named as 3 C8,stably secreting MAb against pB7-H4 protein was identified by indirect ELISA.The MAb is Ig G1 subtype.Flow cytometic analysis showed the MAb can specifically bind with the pB7-H4 temporally expressed on a cell line.It was used to analyze the pB7-H4 expression on the porcine immune cells.The results indicated that pB7-H4 molecule is highly expressed on macrophages in both porcine pulmonary alveoli and peripheral blood mononuclear cell（PBMC）,but almost not on T lymphocytes.In conclusion,the successful production of MAb specific for pB7-H4 provides a useful tool for further research on pB7-H4 immune functions.
作者
郭杰
刘璐
朱可蒙
王宁
辛九庆
刘恒贵
GUO Jie;LIU Lu;ZHU Ke-meng;WANG Ning;XIN Jiu-qing;LIU Heng-gui(State Key Laboratory of Veterinary Biotechnology/Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences ,Harbin 150069, China;College of Life Science and Technology, Harbin Normal University ,Harbin 150069, China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2018年第5期545-551,共7页
Chinese Veterinary Science
基金
中央级公益性科研院所基本科研业务费专项(1610302016005
1610302017014)
关键词
猪B7-H4
原核表达
单克隆抗体
免疫细胞
流式细胞检测
porcine B7-H4
prokaryotic expression
monoclonal antibody
immune cells
flow cytometry analysis
