锚定表达猪IFN-λ3重组植物乳杆菌的构建及其抗PEDV效果的检测

Construction of recombinant Lactobacillus plantarum with surface displayed porcine IFN-λ3 and its functional resistence to PEDV

为构建锚定表达猪IFN-λ3的重组植物乳杆菌(L.plantarum),并验证其抗病毒活性,本研究将猪IFN-λ3基因插入大肠杆菌-乳酸菌穿梭锚定表达载体pSIP-409-pgsA'中构建重组质粒pSIP-409-pgsA'-IFN-λ3,将其电转化至植物乳杆菌NC8中获得重组pSIP-409-pgsA'-IFN-λ3/L.plantarum。经清酒乳杆菌素(SPPIP)诱导后用His标签单克隆抗体经westernblot、间接免疫荧光检测目的蛋白的锚定表达情况。结果显示,猪IFN-λ3在重组植物乳杆菌表面锚定表达。利用重组菌刺激细胞后通过荧光定量PCR测定IPEC-J2/PEDV系统中猪流行性腹泻病毒(PEDV)载量,并检测该重组菌刺激细胞后干扰素刺激基因(ISGs)的相对转录水平。结果显示,该重组菌能够显著抑制PEDV在IPEC-J2中的复制,并能诱导细胞中ISGs(OASL、IFITMs)的高转录水平,具有抗病毒作用。本研究为PEDV的预防和治疗提供可行方案。

To construct a recombinant lactic acid bacterium that anchoring expression porcine IFN-λ3 and verify its antiviral activity,the porcine IFN-λ3 gene was inserted into the E.coli-lactic acid shuttle anchoring expression vector pSIP-409-pgsA',and electrotransformed into Lactobacillus plantarum NC8 to construct recombinant pSIP-409-pgsA'-IFN-λ3/L.plantarum.The anchored expression of the target protein was detected by western blot and indirect immunofluorescence assay with His tag monoclonal antibody.The results indicated that porcine IFN-λ3 was successfully anchored on suface of the recombinant L.plantarum.The PEDV viral load was determinated by qPCR in IPEC-J2/PEDV system stimulatied by the recombinant lactic acid bacteria.The relative transcription levels of the interferon-stimulated genes(ISGs)with antiviral activity were detected after stimulation of the cells with the recombinant bacteria.The recombinant L.plantarum strain with antiviral effect was able to significantly inhibit the replication of PEDV in IPEC-J2,and triggered high transcription of ISG genes(especially OASL and IFITM3 gene),which provided a viable solution for the prevention and treatment of PEDV infection.