牛病毒性腹泻病毒感染对CD46基因修饰树突状细胞表型及其细胞因子分泌的影响

摘要

为研究CD46分子在BVDV感染树突状细胞(DC)介导的免疫应答中的作用,本研究根据牛CD46基因保守序列设计3对短发卡RNA(shRNA)的正义、反义寡核苷酸链,以及3对乱序列作为阴性对照,将其退火后分别克隆至pLL3.7质粒载体中,获得CD46基因shRNA重组质粒后将其转染MDBK细胞.采用荧光定量PCR和western blot技术筛选最佳沉默CD46基因的shRNA重组质粒,将其和表达载体pVAX1-CD46转染新疆褐牛DC (MDDCs)后利用BVDV感染MDDCs,通过荧光定量PCR检测DC表型及其细胞因子mRNA转录水平.结果显示,与未转染细胞组相比,CD46基因过表达的MDDCs中CD40、CD80、CD86和MHC-Ⅱ的mRNA转录水平均明显升高(p＜0.05);而CD46基因沉默的MDDCs中CD40 mRNA转录水平也明显升高(p＜0.05),CD86 mRNA转录水平变化不明显(p＞0.05),CD80和MHC-Ⅱ的mRNA转录水平均明显下降(p＜0.05).与未转染细胞组相比,CD46基因过表达组IL-10和IL-12 mRNA转录水平变化不明显(p＞0.05),而CD46基因沉默的MDDCs中IL-10和IL-12mRNA的表达均明显下降(p＜0.05).本研究从新的角度初步揭示了CD46分子对BVDV感染DC成熟、初始T细胞增殖和分化的影响,为解决DCs对BVDV的免疫抑制作用提供新的思路.%In order to investigate the role of CD46 molecule in immune responses mediated by dendritic cells infected with BVDV,in this study,according to the consensus sequences of bovine CD46 gene,three short hairpin oligonucleotides and the complementary strands were designed.The sense and antisense oligonucleotides were annealed,and cloned into the expression vector pLL3.7.The resultant plasmids,which express short hairpin RNA (shRNA) against bovine CD46 gene,were transfected into MDBK cells,and then real-time PCR and western blot were used to detect the mRNA and protein expression of the CD46 in transfected cells.MDDCs were transfected with the pVAX1-CD46 and the shRNA recombinant plasmid pLL3.7-CD46-shRNA that was the best silencing of the CD46 gene respectively,and followed infected BVDV.Real-time PCR was used to detect mRNA transcriptional levels of cellular phenotype and cytokines in the CD46 gene-modified DCs infected with BVDV.The results showed that,compared with untransfected cells,the mRNA transcriptional levels of CD40,CD80,CD86 and MHCII in CD46 gene overexpression MDDCs were significantly increased (p＜0.05);CD40 mRNA transcriptional levels in CD46 gene silencing MDDCs was also increased significantly (p＜0.05);CD86 mRNA transcriptional level was not changed (p＞0.05);CD80 and MHC-Ⅱ mRNA transcriptional levels were significantly decreased (p＜0.05).Compared with untransfected cells,the levels of IL-10 and IL-12 mRNA in CD46 gene overexpressed MDDCS were not significantly changed(p＞0.05),while their mRNA transcriptional levels were significantly decreased in CD46 gene silencing MDDCs (p＜0.05).This study revealed a new perspective for the effect of CD46 molecule on maturation of dendritic cells infected with BVDV and proliferation and differentiation of naive T cells,providing new ideas for addressing the immunosuppressive effects of DCs to BVDV,which is of great significance to control the occurrence of BVD.