慢病毒介导Occludin过表达影响BVDV感染BALB/c小鼠的研究

doi:10.13560/j.cnki.biotech.bull.1985.2021-1156

摘要/Abstract

摘要：

为研究紧密连接蛋白（occludin,OCLN）是否影响牛病毒性腹泻病毒（bovine viral diarrhea virus,BVDV）在BALB/c小鼠体内的复制。构建慢病毒表达载体pLVML-Myc-bOCLN-linker-GFP-IRES-Puro,连同辅助质粒pSPAX2和pMD2.G共同转染至HEK-293T细胞中,转染48 h后收集OCLN-GFP慢病毒悬液并测定其滴度,同时以pLVML-Myc-Mcs-linker-GFP-IRES-Puro载体包装的慢病毒作为阴性对照（GFP）;将4-5周龄BALB/c小鼠随机分为A（0 d）、B（4 d）、C（8 d）、D（10 d）、E（15 d）五组,每组6只,分别注射5×10⁷ IU OCLN-GFP和GFP慢病毒悬液,连续注射两次,间隔48 h,第二次注射后96 h时以1.68×10⁵ TCID₅₀/只BVDV攻毒BALB/c小鼠,分别于攻毒后0、4、8、10、15 d时,解剖小鼠并采集肝脏、脾脏、肺脏、肾脏、小肠等组织,使用实时荧光定量PCR（real-time fluorescent quantitative PCR,qRT-PCR）和Western blot检测OCLN过表达情况,qRT-PCR检测不同组织中BVDV病毒载量;制作组织病理切片观察各组织病变情况。成功包装OCLN-GFP和GFP慢病毒;慢病毒感染BALB/c小鼠96 h后,OCLN-GFP慢病毒感染后OCLN mRNA和蛋白表达水平显著增加;与GFP慢病毒感染的对照组相比,OCLN-GFP慢病毒感染实验组各组织脏器中BVDV病毒载量从攻毒4 d后出现显著性增加;同时与对照组相比,BVDV攻毒后4 d,实验组小鼠肺脏及肝脏器官最早出现明显病变;BVDV攻毒8 d后,肝脏、脾脏、肺脏等器官病变程度较对照组更加严重。研究表明OCLN过表达能显著性增加BVDV在BALB/c小鼠体内的复制,BVDV感染造成的病理变化更为严重,为阐明OCLN介导BVDV复制等分子机制提供重要依据。

关键词: 紧密连接蛋白Occludin, 慢病毒, 牛病毒性腹泻病毒, 病理切片

Abstract:

This work aims to study whether the overexpression of tight junction protein（occludin,OCLN）affects the replication of bovine viral diarrhea virus（BVDV）in BALB/c mice. After lentivirus expression vector pLVML-Myc-bOCLN-linker-GFP-IRES-Puro was constructed,it was co-transfected into HEK-293T cells together with the helper plasmids pSPAX2 and pMD2.G. At 48 h post-transfection,OCLN-GFP lentivirus suspension was collected and its titer was measured. Meanwhile,the lentivirus generated with pLVML-Myc-Mcs-linker-GFP-IRES-Puro vector（GFP）was served as negative control. BALB/c mice aged 4-5 weeks were randomly divided into five groups：A（0 d）,B（4 d）,C（8 d）,D（10 d）and E（15 d）,with 6 mice in each group. Then 5× 10⁷ IU OCLN-GFP and GFP lentivirus suspension were injected twice continuously at an interval of 48 h. At 96 h after the second injection,BALB/c mice were challenged with 1.68 × 10⁵ TCID₅₀ BVDV. At 0,4,8,10 and 15 d after the challenge,mice were dissected and tissues including liver,spleen,lung,kidney and small intestine were collected. The overexpression of OCLN was detected by real-time fluorescent quantitative PCR（qRT-PCR）and Western blot,and the viral loads of BVDV in different tissues were detected by qRT-PCR. Histopathological sections were made to observe the pathological changes of each tissue. As results,OCLN-GFP and GFP lentivirus were successfully generated. After 96 h of lentivirus infection in BALB/c mice,the expressions of OCLN mRNA and protein increased significantly after OCLN-GFP lentivirus infection. Compared with the control group infected with GFP lentivirus,the BVDV viral load in tissues and organs of OCLN-GFP lentivirus infection experimental group increased significantly from 4 d after challenge. Meanwhile,compared with the control group,at 4 d after BVDV challenge,the lung and liver organs of the experimental group appeared obvious lesions at the earliest time;after 8 d of BVDV challenge,the pathological changes of liver,spleen and lung were more serious than those in the control group. In conclusion,the results shows that OCLN overexpression maysignificantly increase the replication of BVDV in BALB/c mice,and the pathological changes caused by BVDV infection are more serious,which provides an important basis for clarifying the molecular mechanism of OCLN mediated BVDV replication.

Key words: tight junction protein Occludin, lentivirus, bovine viral diarrhea virus, pathological section

王万顺, 付强, 魏玉荣, 胡新艳, 陈俊贞, 李泽宇, 史慧君. 慢病毒介导Occludin过表达影响BVDV感染BALB/c小鼠的研究[J]. 生物技术通报, 2022, 38(6): 291-298.

WANG Wan-shun, FU Qiang, WEI Yu-rong, HU Xin-yan, CHEN Jun-zhen, LI Ze-yu, SHI Hui-jun. Effects of Lentivirus-mediated Occludin Overexpression on BVDV Infection in BALB/c Mice[J]. Biotechnology Bulletin, 2022, 38(6): 291-298.

图/表 6

图1 慢病毒质粒图谱和载体鉴定 A：pLVML-Myc-bOCLN-linker-GFP-IRES-Puro慢病毒质粒图谱;B：pLVML-Myc-Mcs-linker-GFP-IRES-Puro慢病毒质粒图谱;C：PCR扩增（M：DNA Marker 2000;1：阴性对照;2-4：单克隆菌落）;D：OCLN基因序列

Fig. 1 Lentivirus plasmid map and vector identification A：pLVML-Myc-bOCLN-linker-GFP-IRES-Puro lentiviral plasmid map. B：pLVML-Myc-Mcs-linker-GFP-IRES-Puro lentiviral plasmid map. C：PCR amplification（M：DNA marker 2000;1：negative control;2-4：monoclonal colonies）. D：OCLN gene sequence

图2 慢病毒感染HEK-293T细胞后绿色荧光表达情况检测

Fig. 2 Detection of green fluorescence expression in the HEK-293T cells infected with lentivirus

图3 qRT-PCR检测各组织中OCLN mRNA表达量

Fig. 3 Detection of OCLN mRNA expressions in various tissues by qRT-PCR *P<0.05,**P<0.01

图4 Western blot检测各组织中OCLN蛋白表达水平

Fig. 4 Expressions of OCLN protein in various tissues by Western blot

图5 qRT-PCR检测各组织中BVDV载量

Fig. 5 BVDV loading in various tissue by qRT-PCR ***P<0.001

图6 病理切片检测BALB/c小鼠各组织病变情况

Fig. 6 Detection of pathological changes of various tissues in BALB/c mice by pathological section

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