马MHC-I-B_(2)蛋白对马疱疹病毒1型胞内复制影响的研究  

Study on the effect of equine MHC-I-B_(2) protein on intracellula replication of equine herpes virus type 1

作　　者：郑学功 刘建华[1] 孙佳琪 车传忠 祖力亚江·阿不都热合曼 冉多良[1] ZHENG Xue-gong;LIU Jian-hua;SUN Jia-qi;CHE Chuan-zhong;ZULYAJAN Abdurahman;RAN Duo-liang(College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,China)

机构地区：[1]新疆农业大学动物医学学院,新疆乌鲁木齐830052

出　　处：《中国预防兽医学报》2021年第12期1263-1268,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：基于EHV-1新疆株马传染性鼻肺炎灭活疫苗研发(2017A01002-2-4)。

摘　　要：为探明马MHC-I-B_(2)蛋白对马疱疹病毒1型(EHV-1)胞内复制的影响,本研究合成马MHC-I-B_(2)基因,并将其克隆至慢病毒表达载体,构建重组质粒pLVML-HA-MHC-IRES-Puro。利用慢病毒载体系统将pSPAX2、pMAD.2G、pLVML-HA-MHC-IRES-Puro共转染HEK-293T细胞,同时共转染pLenti-GFP-N2,pSPAX2和pMAD.2G质粒作为对照组,待对照组细胞出现绿色荧光时收获各组感染后的BHK-21细胞获得相应慢病毒。将上述慢病毒感染BHK-21细胞并经嘌呤霉素筛选后将获得表达马MHC-I-B_(2)的细胞并传20代,分别取第5、10、15、20代细胞,通过PCR检测马MHC-I-B_(2)的重链基因序列,分析构建细胞系的遗传稳定性,并利用western blot检测第20代细胞中马MHC-I-B_(2)重链基因的表达。结果显示,正确构建了稳定过表达马MHC-I-B_(2)蛋白的BHK-21细胞系(BHK-21-Equus MHC-I-B_(2))。采用EHV-1(MOI 0.1)感染BHK-21-Equus MHC-I-B2与其亲本细胞BHK-21,检测病毒滴度,结果显示BHK-21-Equus MHC-I-B_(2)细胞病变效应(CPE)更明显,病毒滴度比其亲本细胞提高了3.2~4.4倍,可见马MHC-I-B_(2)蛋白可显著提高EHV-1的增殖水平。本研究建立了稳定过表达马MHC-I-B_(2)蛋白的BHK-21细胞系,并首次证实马MHC-I-B_(2)蛋白对EHV-1的增殖具有促进作用,为今后EHV-1的感染机制研究奠定物质基础。In order to investigate the effect of equine MHC-I B_(2) protein on the intracellular replication of equid herpesvirus-1(EHV-1),the equine MHC-I-B_(2) gene was synthesized and cloned it into the lentiviral expression vector to construct recombinant plasmid pLVML-HA-MHC-IRES-Puro.HEK-293T cells were co-transfected with lentiviral vector system pSPAX2,pMAD.2G and pLVML-HA-MHC-IRES-PURO,and the green fluorescent protein of control cells showed that the pseudovirus was successfully packaged and infected BHK-21 cells.After screening with puromycin,PCR and western blot were used to detect the protein of the horse MHC-I B_(2) heavy chain gene.The results showed that BHK-21 cell line(BHK-21-Equus MHC-I-B_(2))overexpressing equine MHC-I-B_(2) was successfully constructed.EHV-1(MOI 0.1)infected BHK-21-Equus MHC-I-B_(2) and its parent cells.The results showed that the cytopathic effect(CPE)was more obvious after BHK-21-Equus MHC-I-B_(2) was infected with EHV-1,and the virus titer was increased by 3.2 to 4.4 times compared with its parent cells.The study successfully established BHK-21 cell line with stable expression of equine MHC-I B_(2) protein,which proved for the first time that equine MHC-B_(2) protein can promote the replication of EHV-1,providing material basis for future studies on the mechanism of EHV-1 infection.

关 键 词：马疱疹病毒1型 马MHCⅠ类分子 细胞 

分 类 号：S855.3[农业科学—临床兽医学]