Establishment And Preliminary Identification Of HEK-293 Cell Lines Stably Expressing Porcine Aminopeptidase

Porcine epidemic diarrhea virus (PEDV) is the etiological agent of porcine epidemic diarrhea (PED), which is a highly contagious enteric disease in piglets and resulting especially in severe economic losses to the affected farms. Virus receptors existed in the cell membrane or intracellular bind to the virus that activate series of intracellular biochemical reactions and produce effectively relevant proteins from external stimulation. Cellular responses are caused after changes of the conformation when the virus bind to receptors. To study virus receptors can deeply reveal interaction mechanism of virus and host cells, thus to provide new ideas for effective virus prevention measures.Porcine Aminopeptidase N is the porcine target cell receptors of PEDV. PEDV can infect non-permissive ST cells when expression of porcine APN has been increased in the membrane, however, the virus cannot infect the ST cells themselves for the poor expression of porcine APN. In this study, based on these researches produce, porcine APN genes were cloned and sequenced to construct porcine APN expression plasmid pcDNA3.1-pAPN, and the stable HEK-293 cell lines expressing porcine APN were established and preliminary identificated.1. The cloning of Porcine Aminopeptidase N, construction of the pAPN expressing plasmid and HEK-293 cells stably expressing porcine APNAccording to a porcine APN mRNA sequence on GenBank (NM214277), a pair of primers Ps/Pa was designed, and the cDNA of porcine APN complete coding sequence was amplified by RT-PCR from total cellular RNA of piglets' intestinal tissue.The sequence was cloned into pMD19-T Simple vector and sequencing results showed that the cloned sequence of porcine APN was 2892bp in length and encoded 963 amino acids. The sequence had a homology as high as 99.79% with the reference sequence on GenBank database, while there is no sign of lack of base pairs, especially the critical segment shared a homology of 99.93% on the level of amino acid with only one amino acid mutation.The porcine APN fragment which has the same sequence as the sequenced segment before were directly subcloned into pcDNA3.1/myc-His(-)A using BamH I and Xho I restriction site to construct porcine APN eukaryotic expressing plasmid pcDNA3.1-pAPN. The constructed plasmid pcDNA3.1-pAPN was transfected into HEK-293 cells. After the selection of Geneticin. the HEK-293 cell lines stably expressing porcine APN was established.RT-PCR, IFA and Western blot assays were performed to verify the expression of pAPN even in the 20th generation in the constructed cell lines which named HEK293-pAPN.2. Identification of PEDV infection in HEK293-pAPN cell and the Growth kinetics of PEDV on HEK293-pAPN cell and Vero cellsThe HEK293-pAPN cells were inoculated with PEDV SC-L strain. Obvious cytopathic effects were observed after inoculation with PEDV. The cells were brighter, wrinkled and agminated, and black granular substance was increased, following by cell shedding off and cracking. PEDV specific gene was amplified by RT-PCR, and the PEDV could be detected by IFA and WB assays, while it could not be detected in HEK-293 cells untransfected with pcDNA3.1-pAPN. The results evidenced that the established HEK293-pAPN cell lines can be infected with PEDV, and the virus proliferated stably in these cells. HEK293-pAPN cell lines and Vero cells were infected with the same multiplicity of infection (MOI=0.1) of PEDV, and samples were collected at different post-infection time. The PEDV ORF3 genes of samples were amplified using fluorescence quantitative PCR method constructed in our laboratory and the copies were calculated by the equation of standard curve. The growth curve was then drawn by those results.The grewth kinetics of PEDV SC-L strain in the two kinds of cells was very similar. Rapid multiplication of the virus were in 80 to 100 hours post-inoculation, and stable virus proliferation began in 108 hours post-inoculation.The virus titer of Vero cells was slightly higher than HEK293-pAPN cells', which confirmed the reliability of the construction of cell lines for proliferation of PEDV. And the constructed cell lines could provide new tools for carrying out researches of the PEDV.