| Classical swine fever(CSF)is an acute,heat,and highly contagious disease caused by a classical swine fever virus(CSFV).In 1992,it was classified as a Class A infectious disease by the Office international des epizooties(OIE),causing great losses to the pig industry.At present,the main measure of my defense control CSF is to inoculate the attenuated vaccine of pigs,and the attenuated vaccine of pigs and rabbits is at risk of virulence,and it is impossible to distinguish between infected and vaccinted animals(DIVA)is very detrimental to the purification of CSF.Therefore,the development of a new type of swine fever labeled vaccine is particularly important.In this study,multiple specific epitopes were sequenced in tandem with the CSFV E2 gene in the transmembrane region to form the recombinant gene CSFV E2 B,and the recombinant gene CSFV E2 B was cloned into the green fluorescent protein(GFP)-labeled and Phytophthora.The recombinant plasmid p EB-CSFV E2B-6His-cop GFP(T2A)puro was constructed by using the lentiviral expression vector p EB-cop GFP(T2A)puro of the resistance gene,and was identified by PCR,double digestion and sequencing.The successfully constructed recombinant plasmid p EB-CSFV E2B-6His-cop GFP(T2A)puro was co-transfected into HEK-293 T cells with helper plasmids PLP1,PLP2 and PLPV for lentiviral packaging,and subjected to fluorescence microscopy 48 h after transfection.Observe the green fluorescence excitation in the cells.The results showed that green fluorescence was observed in more than 90% of the cells,indicating that the recombinant lentivirus HIV-1-p EB-E2 B was successfully packaged.CHO-K1 cells were infected with recombinant lentivirus HIV-1-p EB-E2 B,and the infected cells were subcultured.The expression of fusion protein CSFV E2 B in green fluorescent CHO-K1 cells was detected by laser confocal technique.The results showed that the expression of GFP was detected in thenucleus,and the expression of the fusion protein CSFV E2 B was detected in both the nucleus and the cytoplasm,indicating that the recombinant gene CSFV E2 B was successfully integrated into the CHO-K1 cell genome.The reactance of the expressed fusion protein was detected by indirect immunofluorescence assay(IFA)using the CSFV E2 monoclonal antibody as the primary antibody.The results showed that red fluorescence was observed in all cells,indicating that the fusion protein has good reactogenicity.The above results indicate that the recombinant cell CHO-K1-CSFV E2 B expressing the fusion protein CSFV E2 B was successfully obtained.In order to further obtain a recombinant monoclonal cell strain stably and efficiently expressing the fusion protein CSFV E2 B,we used a limiting dilution method combined with puromycin pressure screening to observe the fluorescence intensity of the recombinant cell CHO-K1-CSFV E2 B in real time,and obtained 4 cell states.Good,high fluorescence intensity CHO-K1-CSFV E2 B recombinant monoclonal cells.Furthermore,the expression level of the fusion protein of the above four recombinant monoclonal cells was detected by Western blot(WB)semi-quantitative method,and the cell line with the highest expression level of fusion protein was screened,and the concentration of fusion protein in the culture was up to 68 μg/m L.The transcription level of the fusion protein gene m RNA in the above four recombinant monoclonal cells was detected by q RT-PCR.The results are consistent with the results of the semi-quantitative test.The results showed that a recombinant monoclonal cell CHO-K1-CSFV E2B(+)expressing the fusion protein CSFV E2 B was successfully constructed.The recombinant monoclonal cell strain was subcultured,and the transcription level of the human pac and protein gene m RNA in the 5th,10 th,15th and 20 th generation cells was detected by q RT-PCR.The results showed that the transcription levels of the fusion protein gene m RNA in each generation of cells were basically the same.The recombinant gene CSFV E2 B was stably expressed in the monoclonal cell line CHO-K1-CSFV E2B(+).In summary,this study screened a recombinant monoclonal cell CHO-K1-CSFV E2B(+)stably and efficiently expressing the fusion protein CSFV E2 B,and the fusion protein expressed by the cell has good reactogenicity.This study is intended to provide a reference for the study of new subunit vaccines for swine fever. |
