Expression Of Bovine Viral Diarrhea Virus E0 Protein In MDBK Cell Mediated By Lentivirus

Bovine viral diarrhea virus?BVDV?belongs to the genus Pestivirus in the Flaviviridae family and is a virus that has a widely infectious host spectrum,such as bovine,sheep,deer and swine and so on.There is a high genomic homologous between the members of Flaviviridae,and have serologic cross reaction.They can break the host specificity and occurrence of cross infection,thus increasing the difficulty of diagnosis and disease prevention of the pathogens.Sequence analysis indicted that BVDV E0 gene has a higher conservative,and E0 protein has good immunogenicity.In addition,E0 protein has RNase activity.Therefore,it becomes the main target protein in the study of virus infection mechanism,and it also opens up a new way to prevent and cure bovine viral diarrhea diseases.At the same time E0 protein is a good candidate for the production of genetic engineering diagnostic antigen and gene engineering subunit vaccine.In this study,22 strains of BVDV E0 gene sequences were obtained and analyzed from GenBank,including 12 strains of bovine origined BVDV: Oregon C24V?AF091605.1?,NADL?AJ133738.1?,Singer?DQ088995.2?,SD1?M96751.1?,Osloss?M96687.1?,Changchun 184?JN704144.1?,JZ05-1?GQ888686.2?,SH2210-17?HG426493.1?,GS5?KJ541471.1?,Powder?JN380089.1?,Riems?AY078406.1?,10JJ-SKR?KC757383.1?.6 strains of sheep origined BVDV: VM?U75982.1?,21372?U75976.1?,R2727?U00891.1?,Ind51966?EU371402.1?,47?U75978.1?,21800?U75977.1?.3 strains of swine origined BVDV: SD0803?JN400273.1?,ZM-95?AF526381.3?,SH-28?HQ258810.1?.And 1 strain of deer origined BVDV: CCSYD?FJ555203.1?.After sequence alignment,the E0 gene sequence of Changchun 184 strain,which is the earliest isolated and the main prevalent strain in Chain,was selected and synthesized.In order to construct lentiviral vector of BVDV E0 gene and detect its expressing in MDBK cell.The template was amplified by the polymerase chain reaction with P1/P2,P3/P2 and P4/P2 primers,which introduce of IgK signal peptide,his tag and Nhe?restriction enzyme cutting site,and ligated into the plasmid of pZJ-CMV-eGFP.Nucleotide sequence analysis of the pZJ-CMV-eGFP-IgK-E0,pZJ-CMV-eGFP-IgK-E0 and pZJ-CMV-eGFP-E0 indicated they were 765 bp,765 bp and 705 bp long,respectively.Recombinant plasmids were transfected into HEK-293 T cell with two other helper plasmids?pVsvg and pAX₂?to pack lentivirus,and then concentrated and titrated.Western blot analysis of MDBK cell culture supernatant suggested that the IgK signal peptide group and predicted signal peptide group expression and secretion of E0 protein,the recombinant E0 protein has a molecular weight of about 40 ku.In order to detect the copy numbers of E0 gene which integrated in MDBK cell genome,Oligo6.0 software was used to design a pair of primer for SYBR Green? quantitative PCR,and pGSI-E0 recombinant plasmid was used as standard plasmid.After reaction condition optimization,there was no nonspecific amplification for CSFV,EHDV-5,BTV-1,5,20.And the detection limit of this quantitative PCR method was 20 copies/ ?L.The standard deviation of Ct value was between 0.018 to 0.055,coefficient of variation was between 0.11 to 0.16% in intra-assay.The standard deviation of Ct value was between 0.037 to 0.096,coefficient of variation was between 0.17 to 0.28% in inter-assay.It was indicated that the quantitative PCR method established in this study was specificity,sensitivity and repeatability.MDBK cell of pZJ-CMV-eGFP-Pre-E0 and pZJ-CMV-eGFP-IgK-E0 group were passaged continuous for 21 generations.Cell numbers was counted for each generation after digestion,and was adjusted to 1×10⁶?/L.Every 2 generation cells was collected as samples,two copies each generation,1 of them was analysised by flow cytometric,the other one was used to extract genome,and detect E0 gene copy number.Passage stability analysis showed that fluorescence rate and E0 gene copies numbers were decreased significantly after twelfth and fifteenth generation,respectively.And there was no significant change of fluorescence rate before and after freezing.Results showed that the two groups of MDBK cell lines have good subculture,freezing stability.In conclusion,two kinds of MDBK cell lines that secretory expression of BVDV E0 protein were successfully constructed and quantitative PCR method for detection of BVDV E0 gene was established in this study,which not only laid the foundation for the study of biological function of E0 protein,provided a new way to prevention and control this disease,but also provided a chance to investigate the possibility of been used as genetic engineering diagnostic antigen and gene engineering subunit vaccine of BVDV E0 protein.