| Oncolytic virus is a kind of tumor-killing virus with replication ability,it can specifically recognize and infect tumor cells,and eventually cause swelling of cells and destroy tumor cells,but it cannot replicate in normal cells without killing effect.Theoretically,it has a higher anti-tumor effect and lower side effects.Newcastle disease virus(NDV),as a new type of viral vector,can play an anti-tumor role by expressing foreign genes,such as increasing the apoptosis level of tumor cells by expressing pro-apoptotic proteins.By inhibiting innate immune response and increasing fusion capacity to increase viral replication and intercellular transmission,tumor-specific lymphocytes are activated by the expression of tumor-associated antigens.Anti-tumor antibodies were expressed to activate cytotoxic responses regulated by antibody-dependent cells,and cytokines were expressed to activate and recruit lymphocytes and dendritic cells.It was found that the co-expression of two foreign genes by recombinant Newcastle disease virus vector(r NDV)could co-regulate the antitumor activity of the virus,and the antitumor activity was better than that of expressing a single foreign gene alone.In addition,the insertion site of foreign gene affects the expression level of foreign protein,and then affects the anti-tumor activity of recombinant virus.However,when r NDV expresses two foreign genes,the optimal combination of foreign gene insertion sites is not clear.In this study,a recombinant viral plasmid expressing single fluorescent protein gene and a recombinant viral plasmid expressing double fluorescent protein gene were constructed by using the attenuated strain of r Clone30 as the skeleton of the virus,and the platform of reverse genetic manipulation was used to construct the recombinant viral plasmid expressing the double fluorescent protein gene.The recombinant virus with infectious expression of single fluorescent protein and the recombinant virus expressing double fluorescent protein were saved.After identification and characterization,the recombinant virus was transfected into four kinds of tumor cells.Immunofluorescence test and flow cytometry were used to detect the expression level of fluorescent protein,and the difference of fluorescence intensity was used to determine the expression level of foreign gene.At the same time,the optimal combination of foreign gene insertion sites in the expression of two foreign genes by r NDV was discussed.The experimental results are as follows:According to the difference of site selection in the genome of Newcastle disease virus(NDV),the weak strain of r Clone30 was used as the skeleton of the virus,and the suitable restriction site was selected through the virus map.The recombinant viral plasmids were constructed to insert foreign fluorescent protein gene EGFP or RFP at the NP/P site and / or P/M site.By using liposome 3000 transfection reagent to explore the ratio of transcription plasmid and auxiliary plasmid,the recombinant virus with infectious expression of EGFP and / or RFP was saved by reverse genetic technique platform.The RNA,of the recombinant virus was extracted by RT-PCR test,and the stable existence of the foreign gene in the recombinant virus was confirmed.The recombinant Newcastle disease virus(NDV)expressing a single foreign gene included r Clone30-EGFP(NP/P),r Clone30-EGFP(P/M)and r Clone30-RFP(P/M).Recombinant Newcastle disease virus(NDV)expressing double foreign genes included r Clone30-EGFP(NP/P)-RFP(P/M)and r Clone30-RFP-EGFP(P/M).The recombinant virus agglutinated chicken red blood cells,infected chicken fibroblast cells,infected SPF chicken embryos and infected chicks.The results of HA,TCID50,EID50,MDT and ICPI tests confirmed that the rescued recombinant virus was in viral titer.The virulence and virulence of the virus were still weak.By drawing and comparing the growth curves of the recombinant virus and the parent virus,it was proved that the recombinant virus and the parent virus had similar growth kinetics.The results showed that there was no significant difference in growth dynamics and molecular biological characteristics between the five recombinant Newcastle disease viruses and their parent viruses.Four kinds of tumor cells(Hep G2,A549,Hela and U251)were cultured in vitro,and five recombinant Newcastle disease virues(NDV)were transfected into these four tumor cells respectively.The expression level of EGFP protein and / or RFP protein in recombinant virus was detected by immunofluorescence assay and flow cytometry.The difference of fluorescent intensity of fluorescent protein confirmed that the expression level of fluorescent protein in recombinant virus r Clone30-EGFP(NP/P)-RFP(P/M)and r Clone30-RFP-EGFP(P/M)was significantly higher than that in r Clone30-EGFP(NP/P),r Clone30-EGFP(P/M)and r Clone30-RFP(P/M).The expression level of exogenous fluorescent protein in recombinant virus r Clone30-EGFP(NP/P)-RFP(P/M)was higher than that in r Clone30-RFP-EGFP(P/M).At the same time,it was found that the expression level of exogenous genes of the recombinant viruses in Hep G2 cells was higher than that in other three similar cells(A549,Hela and U251).The results showed that the recombinant virus expressing double gene at the level of foreign gene expression was better than the recombinant virus expressing single gene.When two foreign genes were inserted into NP/P site and P/M site respectively,the expression level of exogenous protein was the highest.As mentioned above,the recombinant Newcastle disease virus expressing single foreign gene and expressing double foreign gene has been successfully constructed and saved,and the recombinant Newcastle disease virus has similar growth kinetics and biological characteristics compared with the parent virus.The optimal insertion sites of exogenous genes were determined when r NDV expressed two foreign genes.This study lays a foundation for further research on anti-tumor activity of recombinant Newcastle disease virus by effectively expressing a variety of foreign genes. |
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