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The Construction of BAC-HSV-1 Strain HF with GFP Reporter Gene and the Research of Its Infectious Progeny Virus

Author: LiuXinJing
Tutor: XuYuMing；HanZhiQiang
School: Zhengzhou University
Course: Neurology
Keywords: Herpes simplex virus type 1 HF strains Bacterial artificial chromosome Homologous recombination
CLC: R373
Type: Master's thesis
Year: 2011
Downloads: 61
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Abstract

Herpes simplex virus type Ⅰ (Herpes simplex virus type Ⅰ, HSV-1) is an important human pathogen that can cause skin infections from mild to fatal encephalitis different levels of a variety of diseases. HSV-1 genome is a base containing 152 kbp linear double-stranded DNA, whose genome comprises at least 75 genes encoding proteins. Although the majority of HSV-1 structural gene has been studied well, but there are still many viral gene function is not clarified, while its cis-acting elements to regulate gene expression and the interaction between genes and not very clear , the difficulty of the problem is reflected in the final large and complex HSV-1 genome. Its large and complex genomes in eukaryotic cells to the genetic manipulation, such as point mutations in certain genes targeting and become very difficult to knock, to the detriment of the HSV-1 gene function. In recent years, the development of bacterial artificial chromosome based Pets large fragments of foreign DNA technology allows the new HSV-1 genome to BAC (Bacterial artificial chromosome) in the form of the plasmid in E. coli stored, proliferation, and genetic modification . And in eukaryotic cells, compared to the operation performed, HSV-1 genome in the form of the plasmid in E. coli in a simple and fast operation, good security. BAC-HSV-1 plasmid was transfected into eukaryotic cells, after reconstruction of infectious progeny virus HSV-1, HSV-1 and thus can promote the entire genome of the function of different genes. In this study, the E. coli F factor that BAC, in eukaryotic cells by homologous recombination successfully constructed HSV-1 genome BAC-HSV-1, as part of its restructuring replication of HSV-1 non-essential gene , and the ends of BAC containing the loxP sites in the same direction, the Cre enzyme, it can produce recombinant virus progeny of BAC free, such that each of herpes simplex virus and Genetic Analysis of gene function analysis elevated to a new stage for further study of the nature of the disease strain has laid a solid foundation. Purpose 1. Construct carried by the HSV-1 BAC plasmid and carrying green fluorescent protein (Green fluorescent protein, GFP) of recombinant BAC-HSV-1 HF strain of infectious progeny virus; 2. Discussion BAC-HSV-1HF recombinant virus strains replication proliferation; 3. explore BAC-HSV-1HF recombinant virus strains BAC component operability. Method 1. Constructed BAC-HSV-1 recombinant plasmid (1) Construction of HSV-1 carrying plasmid homology arms around C223-plasmid homology arms HSV-1 UL43-47 template design flanking sequence primers homologous with HSV-1 recombinant homologous arms around the left and right homology arms cloned into plasmid C223 SacI and NotI sites of plasmid homology arms around C223-DNA. (2) C223-left homology arms by homologous recombination with the HSV-1 C223-MluI digested about homology arms, to make it linear, liposome encapsulation method C223-linearized DNA fragment homologous arms turn left transfected into Vero cells after 24 hours, with a MOI = 0.01 The HSV-1 infected Vero cells at 37. C, 5% CO2 incubator, by homologous recombination in eukaryotic cells produce GFP reporter gene containing BAC-HSV-1 recombinant virus. (3) plaque purification filter green fluorescent reporter gene BAC-HSV-1 recombinant virus, Hirt extraction of BAC-HSV-1 circular DNA, recombinant virus circular DNA was transformed to DH10B competent bacteria were picked BAC-HSV -1 recombinant plasmid clones were identified by PCR and restriction enzyme BAC-HSV-1 recombinant plasmid. 2 plasmid type BAC-HSV-1 progeny virus reconstruction and characterization of (1) recombinant virus BAC-HSV-1 reconstruction and characterization of the experimental group BAC-HSV-1 and HSV-1 control group of genomic DNA to lipid Body embedding each transfection 2ugDNA to Vero cells after 48 hours cells were observed under an inverted fluorescence microscope lesions and green fluorescent protein expression. Supernatant collected after 72 hours, half of the tissue culture infectious dose (50% tissue culture infective dose, TCID50) virus titer determination groups, will reap experimental and control groups of progeny virus re-infection with MOI = 0.01 Vero cells, TCID50 titer determination groups. The virus titer groups as mean ± standard deviation (x ± s) said, using SPSS16.0 software analysis between the two groups were compared using t test, with P lt; 0.05 was considered statistically significant. (2) recombinant virus BAC-HSV-1 BAC operability studies in experimental group A group given MOI = 2 Vero cell replication-defective recombinant adenovirus Adv-Cre, 18h, with its MOI = 0.01 of BAC-HSV- a recombinant virus infected Vero cells; experimental group B given MOI = 2 Vero cell replication-defective recombinant adenovirus Adv, 18h, with its MOI = 0.01 of BAC-HSV-1 infected Vero cells. Cell growth was observed, the cells appeared to be complete CPE supernatant collected after lesions, lesions of the collected cell supernatant at -80. C and 37. Freezing and thawing three times between C, 3000rpm/min centrifuged for 10 minutes, the supernatant from each 10ul Vero cells were infected, 24h, the fluorescence 3000rpm/min centrifuged for 10 minutes, the supernatant from each 10ul Vero cells were infected, 24h after , GFP expression was observed under a fluorescence microscope case. Results 1. Successfully constructed HSV-1 HF strains BAC-HSV-1 plasmid - virus shuttle system; 2. BAC-HSV-1 recombinant virus replication in Vero cell proliferation ability of HSV-1 virus and the original no difference compared statistically significant (P = 0.079); 3. successful reconstruction without BAC components BAC-HSV-1HFΔBAC progeny virus. Conclusion The recombinant BAC-HSV-1 can plasmid / virus in the form of eukaryotic / prokaryotic cells between the shuttle; BAC-HSV-1 recombinant virus replication in Vero cell proliferation and the original compared to HSV-1 virus was no significant difference; The Cre / loxP system to remove BAC-HSV-1 in the BAC component, BAC-HSV-1 of BAC element operable.

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