Construction And Screening Of Stable Expression BHK-21 Cell Line With Classical Swine Fever Virus E2 And Multiple Epitope Genes

Classical swine fever(CSF)is an acute,febrile,highly contagious disease caused by classical swine fever virus(CSFV),and is an OIE(World Organisation for Animal Health)-listed disease.CSFV is widely prevalent in the country and even in the world and it is seriously endangering the pig industry.And the Ministry of Agriculture of China proposed in 2017,that it is necessary to complete the purification of classical swine fever in China by 2020.At present,the vaccine used to control the CSF epidemic in China is mainly attenuated vaccines,which is not conducive to the purification and prevention and control of CSF.Therefore,it is particularly important to develop new vaccines that are beneficial to the purification of CSF.In this study,genetic engineering technology was used to clone the CSFV E2 gene with good immunogenicity and four specific linear neutralizing epitopes TAVSPTTLR in tandem and cloned into the lentiviral vector pCDH-CMV-MCS-EF1-copGFP-T2A-Puro;the constructed recombinant plasmid pCDH-CMV-MCS-EF1-copGFP-T2A-Puro-E2(+)was subjected to PCR,enzyme digestion,and gene sequencing.The results showed that the CSFV E2 gene and four antigenic epitopes TAVSPTTLR were successfully cloned into the pCDH-CMV-MCS-EF1-copGFP-T2A-Puro vector,A lentiviral recombinant vector pCDH-CMV-MCS-EF1-copGFP-T2A-Puro-E2(+)containing the CSFV E2 gene and the four antigenic epitopes TAVSPTTLR was obtained.Then,the lentiviral recombinant expression vector pCDH-CMV-MCS-EF1-copGFP-T2A-Puro-E2(+)was co-transfected with the helper plasmids PMD2.G and psPAX2 into the packaging cell HEK-293 T,after 48 hours of transfection,the transfected HEK-293 T cells were observed under fluorescence microscope and typical green fluorescent cells were seen,suggesting that the recombinant lentivirus HIV-1-CSFV E2(+)containing the target gene CSFV E2(+)was packaged successfully.The BHK-21 cells were infected with the culture supernatant of the recombinant lentivirus HIV-1-CSFV E2(+)cells.The infected BHK-21 cells were serially passaged for 5 passages,and green fluorescence was observed in each passage of the cells.The green-fluorescent BHK-21 cells were detected by RT-PCR,and CSFV E2(+)was positive.This indicates that the CSFV E2(+)gene carried by the lentiviral vector has been successfully transduced into the BHK-21 cell genome.The recombinant cell line BHK-21-CSFV E2(+)expressing the CSFV E2(+)gene was successfully obtained.To screen recombinant cell lines that highly express CSFV E2(+)protein.Through the real-time observation of the fluorescence intensity of the recombinant cell line BHK-21-CSFV E2(+)combined with puromycin resistance,the recombinant fluorescence cell line BHK-21-CSFV E2(+)was screened,the recombinant fluorescence cell line BHK-21-CSFV E2(+)was re-screened by the limiting dilution method,Eleven BHK-21-CSFV E2(+)recombinant single cells with good morphology and growth status and high fluorescence intensity were obtained.Subsequently,we detected the CSFV E2(+)mRNA transcription and protein expression levels in 11 recombinant BHK-21-CSFV E2(+)single cells by qRT-PCR,SDS-PAGE and Western-blot.The results showed that the transcription level of the CSFV E2(+)gene in these 11 strains of BHK-21-CSFV E2(+)single cells differed by 5 times,and the expression level of CSFV E2(+)protein was several-fold different.To further investigate the stability of the integration of the CSFV E2(+)gene in the BHK-21-CSFV E2(+)recombinant single-cell genome,we selected the BHK-21-CSFV E2(+)recombinant single cell with the highest expression of CSFV E2(+)protein.,the mRNA levels of BHK-21-CSFV E2(+)recombinant single-cell genomics were detected by RT-PCR on the 5th,10 th,20th,and 30 th generations,respectively.The results showed that the CSFV E2(+)mRNA was detected in the 5th to 30 th generation BHK-21-CSFV E2(+)recombinant single cells,it was shown that the CSFV E2(+)gene has been successfully integrated into the genome of the BHK-21-CSFV E2(+)recombinant cell and has been successfully passaged.In summary,this study established a lentiviral vector containing the CSFV E2(+)gene,Co-transfected HEK-293 T cells with helper plasmids packaged the HIV-1 recombinant lentivirus HIV-1-CSFV E2(+)containing the CSFV E2(+)gene.Then,BHK-21 cells were infected with the recombinant lentivirus,and finally a recombinant BHK-21-CSFV E2(+)recombinant single cell that expresses the CSFV E2(+)protein was selected.It laid the foundation for the further development of the new CSF vaccine.