| Herpes simplex virus type 1(HSV-1) has been widely applied on the tumor therapeutic and preclinic trials due to the nature of neurotropism and the ability to infect dividing cells as well as nondeviding cells. At present,Three type oncolytic HSV-1 are in use: amplicons, replication-defective and conditional-replicative virus. The previous two types can't replicate and spread in cultured cells and in vivo , so their ability to lyse tumor cells is confined to local tissue, while conditional-replicative HSV-1 can replicate in tumour cells, then lyse the cells and release the virus, the released virus keep on infecting the nearby tumor cells. At present, several conditional-replicative HSV-1 has come into preclinic trials and gotten some promising results of tumor therapy.In previous studies, we have obtained the recombinant HSV-1/BN which has deleted the inverted repeat sequences within the HSV-1 genome. In this study, luciferase gene and EGFP (enhanced green fluorescence protein) gene were cloned into the shuttle vector pKO5-B/N respectively, and then we got the shuttle vector pKO5/LUC and pKO5/EGFP. The restriction endonucleases analysis of the shuttle vectors showed the expected band, so the shuttle vectors are correct. Then the shuttle vectors were electroporated into the E.coli cells containing the HSV-1 BAC (Bacterial artificial chromosome) respectively, the recombinant bacterium strains were screened through the properties of antibiotic resistance and biochemistry. The analysis of colony PCR and Southern blotting of the recombinant DNAs showed the expected band, so the recombinant DNAs are correct. Then the recombinant DNAs were transfected into Vero cells respectively, the plaques which have the obvious cell pathological changes (CPE) were picked and sonicated, then the viruses infected Vero cells to proliferate and purify the recombinant virus HSV/BN/LUC and HSV/BN/GFP. The analysis of PCR and Southern blotting of the recombinant virus DNAs showed the expected band, so the recombinant viruses are correct. And the titers of the recombinant virus come to 1.53×108pfu/mL. the wild-type HSV-1, HSV/BN/LUC and HSV/BN/GFP infected Vero cells respectively, the analysis of the proliferation ability of the viruses indicated that the proliferative ability of the recombinant viruses are similar to wild-type virus'. And the Vero cells infected HSV/BN/LUC were expressing luciferase gene along with the virus proliferation. The results suggested the deletion of invert repeat sequence in the HSV-1 does not affect the proliferation of the virus replication, and the recombinant virus can be treat as vector to accommodate foreign gene.The herpes simplex virus 1 latency associated transcripts promoter (LATP) is still keeping its activity during HSV-1 latency in the neurons. PCR was used to clone the LATP fragment, then the LATP fragments were sub-cloned into the front of the enhanced green fluorescent protein gene and luciferase gene respectively, and we got the recombinant plasmid pcDNA/LATP/EGFP and pGL3/LATP/LUC. Then pcDNA/LATP/EGFP were transfected into the 293T cells, the expression of EGFP was observed through fluorescence microscope, the result indicated that LATP take on the promoter activity and its activity were lower than PCMV. Then pcDNA/LATP/EGFP and pGL3/LATP/LUC were transfected into HeLa cell line and glioma cell line SK-N-SH respectively, the result showed that the expression level of luciferase gene in the SK-N-SH cells was significant higher than in the HeLa cells (p<0.01), the results suggest that LATP possess the specificity of nervous tissue, and LATP can be used as a specifical promoter of nervous tissue.
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