Expressing The Fusion Protein Of PRRS Virus GP5and GFP In Mammalian Cells And Its Application

Porcine reproductive and respiratory syndrome (PRRS) is a contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV). PRRS mainly caused reproductive disorder, including of premature, abortion, fetal death, mummy and porcine respiratory syndrome at variety of ages and high mortality. PRRSV has a restricted cell tropism in its host (pig). PRRSV enter into cells were dependent on cell receptors.Several candidate molecules have been identified to be the receptors/co-receptors for PRRSV entry including heparin sulfate, CD163, sialoadhesin and vimtin. The relationship of these receptors were general as follows:In order to make future exploration of the mechanism of PRRSV infection, this research take C-terminal of NMHC Ⅱ B as target to develop3parts experiment: HS adsorpted PRRSV to PAM cells; Sn mediated swallow of PRRSV; CD163and vimentin maybe assist Sn swallow, PRRSV strip in shell and viral genome RNA released into the role of cell plasma.GP5is one of the main structure protein of PRRSV, and it plays an important role in the process of infection and immune of PRRSV. Therefore, there is an important significance to study receptors of GP5on the surface of various cells to control the spread and morbidity of PRRS. This mammalian cells expression system was used in the experiment, and the results was detected by flow cytometry (FCM). The detect gene and the report gene become an organic whole by expressing the fusion protein of GP5and GFP, which can reduce the adsorption of nonspecific and to improve the specificity of detection methods. In addition, using mammals eukaryotic expression can keep the natural structure and biological activity of the protein.The GP5gene of PRRSV was cloned into the eukaryotic expression vector of pEGFP-Nl by reverse transcription-the polymerase chain reaction (RT-PCR) from the Marc-145cells infected by PRRSV, to study and detect the distribution of PRRSV receptor on the cell surface further more. The recombinant plasmid of pEGFP-N1-GP5was used as template to proliferate the fusion gene of GP5and GFP, and then the fusion gene was cloned into the eukaryotic expression vector of pcDNA3.1/V5-His A. The expression plasmid of peDNA3.1-N,-GP5+GFP was transfected into HEK-293T cells, and the fusion protein of GP5and GFP can be detected by an inverted fluorescence microscope after48h. A nickel column was used for purifying the fusion protein, and then the detection of the fusion protein of purification was by the immune imprinting (western blot).The single-celled levitation liquid of pig pulmonary macrophage, Marc-145cells, sheep pulmonary macrophage, PK-15, Vero, MDBK, MCF-7and HEK-293T cells was prepared for the experiment. They were incubated with the purification protein in4℃for an hour, then were analyzed by flow cytometry to detect the positive cells. The results indicate that there are receptors of GP5on the surface of the pig pulmonary macrophage(PAM) and marc-145cells, while, there are no receptors of GP5on the surface of sheep pulmonary macrophage, PK-15, Vero, MDBK, MCF-7and HEK-293T cells. A conclusion can be obtained that high valence of the pig serum can prevent the GP5protein from combining with receptors on the surface of the host cells through the serum block experiment. A new way that PRRSV can be prevented from combining with receptors by using the immune serum can be used to control the infection of PRRSV, and then gain a purpose of controlling the occurrence, spread and transmission of PRRS.