| OBJECTIVE:Newcastle disease virus(NDV)has shown promising application as a natural oncolytic drug in the treatment of cancer.IP10 is a chemokine that specifically chemotizes immune cells such as CD4~+,CD8~+and NK cells,and they have specific killing effect on tumor cells.To further improve the oncolytic effect of Newcastle disease virus,we used reverse genetics technology to construct and rescue the recombinant Newcastle disease virus(rNDV-IP10)that expresses the IP10 protein.We then tested whether rNDV-IP10 had better ability to kill tumor cells.METHODS:(1)We used homologous recombinase to insert the genes of interferon gamma-inducible protein 10(IP10)and enhanced green fluorescent protein(EGFP)into Newcastle disease virus transcription vector.The recombinant and helper plasmids pBS-NP,pBS-P and pBS-L were then co-transfected into BSR-T7/5 cells.We successfully rescued the recombinant Newcastle disease virus named rNDV-IP10.(2)The titer of virus at different time points was measured by primary chicken embryo fibroblast(PCEF),and then the growth kinetics of the virus was measured.We detected the expression of foreign gene IP10 in hamster kidney cells infected with recombinant Newcastle disease virus by indirect immunofluorescence.(3)Hepatoma cells were infected with NDV and rNDV-IP10,and mRNA expression of IP10 gene was detected by fluorescence quantitative PCR.Western blot and ELISA were used to detect the IP10 expression of recombinant Newcastle disease virus in hepatoma cells and cell culture supernatants.We tested the effect of foreign genes on the virulence of recombinant Newcastle disease virus by CCK8 test.(4)We used H22 liver cancer cells to establish experimental animal tumor models.After the tumor model was established,it was randomly divided into three groups and injected with PBS,NDV and rNDV-IP10 respectively.The tumor volume was then measured.(5)On the 12th day after the injection of the drug,the mice were sacrificed to take tumor tissues.Flow cytometry was used to detect the levels of CD4~+and CD8~+lymphocytes in tumor tissue.The lymphocyte infiltration in tumor tissues was measured by HE staining and immunohistochemistry.The expression of CD31 in tumor tissues was detected by immunohistochemistry.Light brown or dark brown were positive reaction particles.RESULTS:(1)We successfully constructed a recombinant Newcastle disease virus vector pMD18-IP10 carrying the IP10 and EGFP genes.We used reverse genetics to rescue the recombinant Newcastle disease virus rNDV-IP10.(2)From the virus growth curve,it can be observed that the insertion of the foreign gene has no effect on the growth replication of the recombinant Newcastle disease virus rNDV-IP10.We used indirect immunofluorescence to visualize specific red fluorescence in hamster kidney cells infected with rNDV-IP10.(3)Hepatoma cells infected with rNDV-IP10 can transcribe the mRNA of the IP10 gene and can translate the IP10 protein.The translated IP10protein can be successfully secreted into the extracellular fluid.The insertion of exogenous genes had no effect on the virulence of Newcastle disease virus.(4)We successfully established a mouse liver cancer tumor model.The tumor growth of rNDV-IP10 group was slower than that of NDV group and PBS group,and the tumor size was smaller(P<0.05).(5)HE and immunohistochemistry results showed that lymphocyte infiltration in the rNDV-IP10 group was greater than that in the NDV and PBS groups(P<0.05).The proportion of CD4+and CD8+cells in the rNDV-IP10 group was greater than that in the NDV and PBS groups(P<0.05).Density of neovascularization was down-regulated in the rNDV-IP10 group compared with the NDV and PBS groups(P<0.01).CONCLUSIONS:(1)We successfully rescued rNDV-IP10,a recombinant Newcastle disease virus carrying the IP10 gene.(2)The biological characteristics of rNDV-IP10 have not been affected by foreign genes and can successfully translate the foreign gene IP10.(3)rNDV-IP10 has a better anti-tumor effect in a liver cancer animal model. |
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