| Foot-and-mouth disease(FMD)is an acute and highly infectious disease caused by foot-and-mouth disease virus(FMDV)that infects pigs,cattle,sheep and other cloven-hoofed animals,seriously affects the development of global breeding industry.Vaccine immunization is a common method to prevent FMD,but some antigen components of the traditional inactivated vaccine are easily destroyed during the preparation process,and the immunization maintenance period is short.Therefore,the development of efficient new FMD vaccine is a hot spot at present.VP1 is the main structural protein of FMDV,which has been confirmed plays a key role in inducing immune response,and is the candidate protein to develop FMD subunit vaccine.Based on the sequence of swine O-type FMDV VP1 gene,the fusion protein gene RP1 that repeatedly linked the dominant T and B cell epitope genes was obtained,in order to express the high immunogenicity antigen protein RP1 in CHO-K1 cells,and lay a foundation for the development of new FMD vaccine.In this study,the recombinant RP1 gene was cloned into the vector p CDH-CMV-MCS-EF1-Puro.After PCR,double enzyme digestion and gene sequence determination,the recombinant plasmid p CDH-CMV-MCS-EF1-Puro-RP1 was obtained.This recombinant plasmid was co-transfected into HEK-293 T cells with helper plasmids PLP1,PLP2 and PLP3 to obtain recombinant virus HIV-RP1.CHO-K1 cells were infected with HIV-RP1,and the recombinant cell CHO-K1-RP1 expressing the fusion protein RP1 was obtained by puromycin resistance screening.In order to identify the expression of RP1 gene in recombinant cells,RT-PCR and Indirect immunofluorescence assay(IFA)were performed in this study.RT-PCR results showed that a clear single band could be observed at about 1 500 bp,consistent with the expected fragment size.IFA results showed that green fluorescence could be observed in CHO-K1-RP1 cell under fluorescence microscopy.The results showed that the RP1 gene was successfully integrated into the genome of CHO-K1 cells,and the recombinant cell CHO-K1-RP1 expressing the fusion protein RP1 was obtained.In order to obtain the CHO-K1-RP1 monoclonal cell line that can stably and efficiently express the fusion protein RP1,multiple monoclonal cell lines were screened by limited dilution method and IFA,Western blot,q RT-PCR were detected to analyze the expression of RP1 gene in each cell line.IFA results showed that green fluorescence could be observed in some cells under fluorescence microscopy.Western blot results showed that a single band was observed at about 55 k Da in some samples,consistent with the expected protein molecular weight.q RT-PCR results showed that the RP1 gene was successfully amplified from the genome of the positive cell lines identified by IFA and Western blot.The results showed that the CHO-K1-RP1 monoclonal cell line 51 can efficiently express the fusion protein RP1.In this study,the monoclonal cell line 51 was continuously transferred to the 30 th generation,and cells were collected every 5 generations for denaturation.The cell samples were detected by Western blot with FMDV VP1 monoclonal antibody,and the stable expression of RP1 gene in CHO-K1-RP1 cell line was analyzed.The results showed that the specific bands were observed at about 55 k Da in the 5 th,10 th,15 th,20 th,25 th and30 th generation cell samples,which was consistent with the expected molecular weight.It showed that RP1 gene had been integrated into the genome of CHO-K1 stably,and the expressed products of each generation of cells had good antigenicity.In order to obtain a large amount of fusion protein RP1,the selected cell line 51 was cultured in suspension,and its concentration was detected by Western blot,and semi-quantitative analysis was performed by Image J.The results showed that the selected cell line 51 could adapt to the normal growth of low serum medium in shaking flask,and the concentration of RP1 in suspension culture reached 110 μg/m L.To study the immune effect of IL-4 on fusion protein RP1,the IL-4 gene of swine was amplified by RT-PCR and cloned into pc DNA3.1.After PCR,double enzyme digestion and gene sequence determination,the recombinant plasmid pc DNA3.1-IL-4 was obtained.In this study,the obtained fusion protein RP1 was mixed with a certain volume of ISA201 VG adjuvant to produce an oil emulsion vaccine.BALB/c mice were immunized to analyze the immunogenicity of RP1,and a combined immunization group of FMD subunit vaccine and different immunostimulants were set up.The experimental groups were as follows: Group I was FMD subunit vaccine,Group II was FMD subunit vaccine+CD154,Group III was FMD subunit vaccine+pc DNA3.1-IL-4,Group IV was FMD subunit vaccine+NAA,Group V was FMD inactivated vaccine,and Group VI was PBS control group.Each group was immunized three times by multiple subcutaneous injection.Blood samples were collected and isolated 7 days after each immunization,and the serum level of specific antibodies in mice was detected by indirect ELISA.The results showed that the FMD subunit vaccine prepared in this study could stimulate the mice to produce specific antibody of FMDV after the second immunization.Except for the PBS group,the FMDV antibody in each group had a certain increase after strengthening immunity,and the changes of FMDV antibody in FMD subunit vaccine+NAA group was significantly higher than that of FMD inactivated vaccine group(p<0.05).There was no significant difference in antibody levels between the FMD subunit vaccine group,FMD subunit vaccine+CD154group,FMD subunit vaccine+pc DNA3.1-IL-4 group and FMD inactivated vaccine group(p>0.05).It showed that the fusion protein RP1 expressed in this study had good immunogenicity,and the combination with NAA could produce high levels of FMDV-specific antibodies.In conclusion,the CHO-K1-RP1 suspension cell line that can stably and efficiently express FMDV epitope fusion VP1(fusion protein RP1)was screened and obtained in this study.The expression product of CHO-K1-RP1 was confirmed has good immunogenicity,which is expected to be a candidate protein for the development of FMD subunit vaccine. |
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