| Avian influenza virus subtype H9N2(AIV)is one of the most important pathogens that seriously affects the chicken industry in China,causing reduced performance and immunosuppression in chickens.Mixed infections can also cause high mortality.Currently,the disease is mainly prevented and controlled by inactivated vaccines.In order to improve the mucosal protection against H9N2 subtype AIV infection,we developed a recombinant live attenuated H9N2 subtype AIV vaccine rTX-NS1128(mut)with exchanging the packaging signals between HA gene and NS1 gene and truncated NS1 gene,which can effectively prevent recombination between the vaccine strain and wild virus.The vaccine can also induce mucosal immunity and humoral immunity,which have good immune protection efficacy.However,the replication ability of the vaccine on MDCK cells was significantly reduced compared with that of rTX.In this study,we constructed an MDCK cell line stably expressing the full-length NS1 gene of H9N2 AIV,determined the replication ability of rTX-NS1-128(mut)on this cell line,and evaluated the immune efficacy of the vaccine produced by this cell line.Our work lays the foundation for the development of a safe and high-yielding H9N2 AIV attenuated vaccine.1 Construction of MDCK cell line stably expressing H9N2 AIV full-length NS1 geneThe full-length H9N2 AIV NS1 gene was amplified by PCR and cloned into the phage-bsd vector to construct the phage-bsd-NS1-Flag plasmid by homologous recombination.MDCK polyclonal cell lines expressing NS 1-Flag protein were obtained by lentiviral infection and screened with blasticidin.The results showed that MDCK monoclonal cell line 2G8D5 expressing NS1-Flag protein was obtained.The expression of NS1-Flag protein was stable in different generations of cell lines after 30 generations.Compared with MDCK cells,cell line 2G8D5 could effectively inhibit the level of interferon induced by SeV(Sendai virus,SeV).Stable expression of NS1-Flag protein did not affect the cellular activity of cell line 2G8D5.Therefore,we obtained an MDCK monoclonal cell line that can stably express NS1-Flag protein,which can effectively inhibit interferon levels and has good growth status.2 Evaluation of the immune potency of rTX-NS1-128(mut)cultured on MDCK cell line 2G8D5The proliferation ability and biological properties of the vaccine candidate rTXNS1-128(mut)on MDCK cell line 2G8D5 after passaging were determined by indirect immunofluorescence,and the full length of the vaccine candidate rTX-NS1-128(mut)HA after passaging was PCR amplified and sequenced for sequence analysis.The proliferation ability results showed that the TCID50 of rTX-NS1-128(mut)on MDCK cell line 2G8D5 was 7±0.17 Log10TCID50/0.1 mL,while the TCID50 of the virus on wild-type MDCK cells was 5.75±0.21.The growth curve showed that within 12-72 h past infection,the titers of rTX-NS1-128(mut)replicated on the MDCK cell line 2G8D5100-fold higher than that on wild-type MDCK.Passage stability results showed no significant differences in TCID50,EID50 and HA among the viruses of different generations on MDCK cell line 2G8D5.There was also no significant difference in TCID50 after repeated freeze-thawing and after different times of treatment at different temperatures.HA gene sequencing analysis showed that there was no mutations in HA of viruses passed on MDCK cell line 2G8D5.Therefore,MDCK cell line 2G8D5 was effective in increasing the yield of vaccine candidate rTX-NS1-128(mut)and the vaccine candidate was stable in the passages on the modified cell line.SPF chickens was immunized intranasally with the vaccine candidate strain rTXNS1-128(mut)cultured on MDCK cell line 2G8D5 to determine the situation of virus shedding,induced cellular,mucosal and humoral immune response and the immune protection rate of the vaccine against A/Chicken/Taixing/10/2010.The virus shedding results showed that the 3rd day after immunization,a small number of chickens showed virus shedding in the larynx and cloaca in cell-origin and chicken embryo-origin rTXNS1-128(mut),and only a small amount of virus shedding in the larynx on the 5th day after immunization.The most important is that no virus shedding was detected on the 7th day,which indicates the pathogenicity of the vaccine rTX-NS1-128(mut)did not change after cell passages.The immune response assay showed that both cell-origin and chicken embryo-origin rTX-NS1-128(mut)could induce HI and IgY antibodies,and there was no significant difference between cell-origin and chicken embryo-origin rTX-NS1-128(mut)groups,both of which were lower than that of the rTX inactivated vaccine.The IgA levels induced in cell-origin and chicken embryo-origin rTX-NS1128(mut)were significantly higher than that in the rTX inactivated vaccine,and the levels of IL-4,IL-5 and IFN-y were also significantly higher than that in the rTX inactivated vaccine.The challenge protection test showed that the challenge control group had virus shedding on the 3rd,the 5th,and the 7th day after infection with A/Chicken/Taixing/10/2010(H9N2).However,the groups vaccinated with cell-origin and chicken embryo-origin rTX-NS1-128(mut)had 2/10 and 2/10 virus shedding respectively on the 3rd day,and the rest of the time had no virus shedding.Therefore,the vaccine candidate rTX-NS1-128(mut)cultured on cell line 2G8D5 still had good immune protection efficacy.In summary,the stable expression of H9N2 AIV full-length NS1 gene in MDCK cell line 2G8D5 and its application for vaccine candidate rTX-NS1-128(mut)production can not only effectively solve the problem of low yield,but also does not change the safety,stability and immune protection efficacy of the vaccine candidate,which has the potential to be put into production application. |
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