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The Studies on the Oncolytic Herpes Simplex Virus Type 1

Author: LiYongZuo
Tutor: MaZhengHai
School: Xinjiang University
Course: Biochemistry and Molecular Biology
Keywords: Herpes simplex virus type 1 Bacterial artificial chromosome Oncolytic virus Latency-associated transcript promoter Neural tissue-specific
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 28
Quote: 0
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Abstract


Herpes simplex virus type 1 (Herpes simplex virus type 1, HSV-1) infection with natural neurotropic and efficient, has been widely used in cancer treatment research and clinical trials. The current development of oncolytic HSV-1 amplicon (Amplicon), replication-defective virus (Replication-defective virus) and conditions selective replication of the virus (Conditional-replicative virus) into three categories. The first two types of HSV-1 in the cell can not be copied, oncolytic effect is limited to local tissue, and replicating oncolytic HSV-1 replication in tumor cells, tumor cells after lysis and release of progeny virus replication cycle is completed, followed by infection Close to the tumor cells. At present, some replicating oncolytic HSV-1 has entered clinical trials, and achieved some encouraging results in cancer therapy. Preliminary studies have been deleted HSV-1 inverted repeats within the recombinant virus HSV-1/BN. The This study reporter gene luciferase (Luciferase) and green fluorescent protein (EGFP, enhanced green fluorescent protein) build into the shuttle plasmid pKO5/BN carrying the luciferase gene the shuttle plasmid pKO5/LUC and carrying green fluorescent protein gene the shuttle plasmid pKO5/EGFP, and by a variety of restriction enzyme digestion shuttle plasmid, are expected to match the bands, the shuttle plasmid was constructed correctly. Containing HSV-1 bacterial artificial chromosome (Bacterial artificial chromosome, BAC-HSV) E. coli shuttle plasmid electroporation, antibiotic resistance and biochemical screening recombinant strain homologous recombination. Colonies identified by PCR and Southern blotting detection are obtained recombinant DNA and is expected to match the strip. Recombinant DNA transfection of Vero cells distinct cell pathological changes (cell pathological changes, CPE) plaques were picked, ultrasound again after the release of the virus infected Vero cells for purification and enrichment of the recombinant virus. PCR and Southern blot analysis of recombinant virus DNA are consistent with the estimated size of the strip, the proof of the recombinant virus is correct, named HSV / BN / Luc and HSV / BN / GFP. The titer of recombinant virus detection up to the 1.53 × 108pfu/mL. Wild-type HSV-1 the HSV / BN / Luc and HSV / BN / GFP were infected Vero cells, the results showed that the wild-type HSV-1 and similar to the replication-competent recombinant virus in Vero cells. HSV / BN / LUC detecting the level of expression of the luciferase gene after infection of Vero cells, the results show that the luciferase associated virus replication and expression. The above results indicate that the deleted HSV-1 internal inverted repeat sequence does not affect viral replication in the region as an exogenous gene is inserted into the bit point. Type 1 herpes simplex virus latency-associated transcript promoter (Latency associated transcriptspromoter, LATP) remain active during latent infection of the virus in the nerve tissue. In this study, PCR amplification LATP of HSV-1, and the LATP were inserted before the green fluorescent protein gene and the luciferase gene, eukaryotic expression plasmid pcDNA / of LATP / EGFP and pGL3/LATP/LUC. pcDNA / LATP / EGFP transfected 293T cells, detected the expression of green fluorescent protein gene, LATP promoter activity activity activity lower than pCMV; then to the pcDNA / LATP / EGFP and pGL3/LATP/LUC were transfected to dye HeLa cells and glioma cells SK-N-sh, results show that the expression of green fluorescent protein gene and the luciferase gene in SK-N-SH cells was significantly higher than the level of expression in HeLa cells, indicating LATP has nerve tissue specificity, and can be used as a nerve tissue specific expression elements.

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