S蛋白来源的B细胞表位抗体对PEDV感染的影响研究

Porcine epidemic diarrhea (PED) is an economically important, acute enteric disease of swine with high mortality in neonatal piglets. Porcine epidemic diarrhea virus (PEDV), the causative agent of PED, is a member of the Coronaviridae family. PEDV is currently reemerging in immunized swine herds.The reason why PEDV vaccine can not effectively protect suckling pigs from PEDV infection is still unknown. S protein was the main protein that is capable of inducing PEDV-neutralizing antibodies. We have previously obtained three PEDV that can be steadily passed down on Vero cell. In this study, we will analyze in vivo and in vitro the function of antibodies in PEDV infection. Porcine aminopeptidase (pAPN) gene was cloned into eukaryotic expression vector pIRES2-EGFP. The positive plasmid contained pAPN gene was transferred into Vero cell to construct a Vero cell line expressing pAPN gene. Monoclonal antibodies (McAbs) of B-cell epitope peptides originated from spike protein was prepared by inosculate SP2/0 and spleen cell of mice immuned with epitope peptides. Piglets will be inoculated with epitope peptides or with McAbs to analyze in vivo the function of antibodies in PEDV infection. Neutral red neutralization test will be used to test in vitro the function of antibodies. The results of this study will provide a useful basis for PEDV vaccine research.

猪流行性腹泻（PED）是引起世界各养猪国家特别是我国仔猪早期死亡的重要肠道传染病。其病原为猪流行性腹泻病毒（PEDV）。当前，在PED疫苗免疫的猪场，该病仍经常发生，疫苗未能有效保护机体免于PEDV感染的原因，目前尚未明确。本研究拟在已经成功获得3株在Vero细胞上能稳定传代的PEDV基础上，将PEDV的细胞感染受体-猪氨基肽酶（pAPN）基因克隆至真核表达载体pIRES2-EGFP，转染Vero细胞，建立能高效增殖PEDV的Vero细胞系；并在此基础上，用PEDV刺激机体产生中和抗体的主要蛋白-S蛋白的各优势抗原表位多肽分别免疫BALB/c小鼠，制备单克隆抗体；通过细胞中性红染色法中和试验、用多肽免疫仔猪、对仔猪静脉注射单克隆抗体等体内外试验，研究这些抗体在PEDV感染过程中的作用，进而阐明S蛋白来源的抗体对PEDV感染的影响机制。为PED的防治工作提供有力的科学依据。

为了构建能够表达猪pAPN-SPC段的真核细胞表达载体pEGFP-N1-pAPN-SPC, 并在Vero细胞中进行表达。本试验从猪肠道黏膜基因组中扩增出pAPN-SPC段基因,插到带有EGFP的真核细胞表达载体pEGFP-N1中,经BamHI及EcoRI酶切鉴定正确后转染Vero细胞, 用荧光显微镜检测细胞中增强型绿色荧光蛋白(EGFP)的表达,用RT-PCR和Western-blot检测PAPN-SPC段基因的表达情况，用IPMA和荧光定量PCR检测对病毒增殖量的影响。成功构建真核表达载体pEGFP-N1-pAPN-SPC,且在Vero细胞中得到了有效表达，为研究pAPN-SPC段蛋白对病毒增殖效果的影响奠定了基础。