低血清培养对PRRSV NX-1株在Marc-145细胞中增殖的影响  

Effects of low serum level on proliferation of PRRSV NX-1 in Marc-145 cells

作　　者：管旭科 邓吉平 郭松扬念 董尚坤 张佳颖 袁安文[1] 杨青[1] GUAN Xu-ke;DENG Ji-ping;GUO Song-yang-nian;DONG Shang-kun;ZHANG Jia-ying;YUAN An-wen;YANG Qing(College of Veterinary Medicine,Hunan Agricultural University,Changsha 410128,China)

机构地区：[1]湖南农业大学动物医学院,湖南长沙410128

出　　处：《中国兽医科学》2023年第10期1307-1313,共7页Chinese Veterinary Science

基　　金：湖南省技术攻关“揭榜挂帅”项目(2021NK1030);国家自然科学基金项目(31972761)。

摘　　要：本研究旨在探索低浓度血清培养条件对猪繁殖与呼吸综合征病毒(PRRSV)NX-1株在宿主细胞Marc-145中增殖的影响。首先,用感染复数(MOI)为0.2的PRRSV NX-1株接种Marc-145细胞1.5 h后,分别用含0、1%和2%胎牛血清(FBS)的维持液培养72 h,显微镜下观察细胞形态变化,通过CCK-8法检测细胞活力;采用间接免疫荧光法(IFA)和荧光定量PCR法分别检测病毒感染后病原在细胞中的表达定位及其ORF7基因的表达;最后,用Reed-Muench法检测病毒滴度。结果显示,感染后第72小时,Marc-145细胞出现明显的细胞病变(CPE),2%FBS培养时比1%FBS的CPE更明显;细胞活力检测结果显示,感染后第24和48小时,添加FBS时显著增强细胞的活力,感染后第72小时,添加2%FBS组的细胞活力显著低于其他2组;在添加1%或2%FBS培养时,IFA显示在细胞胞质中抗原阳性信号较强,而在无FBS培养细胞中信号弱。当维持液中添加FBS培养72 h时,病毒ORF7基因在宿主细胞中的表达显著高于无血清组,且2%FBS组的表达量显著较高,其相应病毒液的滴度也显著高于其他2组。上述结果表明,PRRSV NX-1株感染Marc-145细胞后,在含2%血清的培养基中病毒的增殖能力和毒株感染力较强。This study aimed to investigate the effect of low serum level on proliferation of PRRSV NX-1 stain in Marc-145 cells.Firstly,Marc-145 cells were inoculated with 0.2 multiple infection index(MOI)PRRSV NX-1 for 1.5 h,then cultured in maintenance culture medium containing 0,1%and 2%fetal bovine serum(FBS)for 72 h.Cell morphology was observed under a microscope,and cell viability was detected by CCK-8 assay.Indirect immunofluorescence assay(IFA)and real-time quantitative PCR were used to detect the localization and expression of pathogen and its ORF7 gene expression in host cells post infection.Finally,the virus titer was tested using the Reed Munch method.The results showed that obvious cytopathic effect(CPE)was observed after the cells infected with PRRSV NX-1 for 72 h,which was more obvious in the 2%FBS group when compared to the 1%FBS or Mock group.CCK-8 assay showed that FBS supplementation significantly increased cell viability at 24 h and 48 h post infection,which was lower in the 2%FBS group than those of the other two groups at 72 h.Strong positive signal of the antigen was observed in cells cultured with 1%or 2%FBS by IFA assay,and weak fluorescence signal was observed in the FBS-free cultured cells.In addition,FBS supplementation significantly increased the expression of the viral ORF7 gene in cells after 72 h of infection,and the corresponding viral titers also showed a similar increase.The present findings suggest that the PRRSV NX-1 strain exhibits a strong proliferative ability and infectivity when host cells were cultured in 2%serum medium.

关 键 词：猪繁殖与呼吸综合征病毒 血清 细胞增殖 ORF7基因 

分 类 号：S852.659.6[农业科学—基础兽医学]