The Construction Of BAC-HSV-1 Strain HF With GFP Reporter Gene And The Research Of Its Infectious Progeny Virus

Herpes simplex virus typeⅠ(HSV-1), an important human pathogen, could cause many infections disease with different severity from mild skin infections to deadly encephalitis. The genome of HSV-1 is a double-stranded linear DNA molecule, which is 152-kb in length,including at least 75 kinds encoding gene. Although most genes function have been declarated, there are still a lot of genes function have not been clarified, while the role of the cis-regulatory elements that how to control gene expression and the interactions between gene and gene also not been clearly illuminated. The difficulty of those problems finally focus on the large and complex genomes of HSV-1, Its vast and complex genomes making the genetic operations in eukaryotic cells such as point-mutation and targeting gene knock-out very difficult, largly hindering the research of HSV-1. Bacterial artificial chromosome (BAC) is a bacterial cloning system based on bacterial F factor. BAC offers a number of advantages over other cloning systems, including:(i) the potential to incorporate 100-300kb foreign inserted fragments; (ii) could be inherited stably:genomes cloned into BAC vectors seldom have the loss or recombination or chimeric phenomenon occoured during the passage process; (iiii) It could be manipulated in the E.coli. Construction of the BAC -HSV-1 shuttle vector system may facilitate the operation on any gene in HSV-1 genome, and it provides an important platform for studying gene functions and construction of vectors.In this study, the BAC plasmid was inserted in the non-essential genes of HSV-1 by homologous recombination, the plasmid of BAC -HSV-1 with GFP reporter gene was successfully constructed, meanwhile, the BAC flanked by loxP sites,which could delete the BAC after the function of Cre recombinase and then the BAC-HSV-1HFΔBAC could be produced, which will be benificial in further research on HSV-1 virus.Objectives 1. To construct BAC-HSV-1 strain HF plasmid-virus shuttle system;2. To Explore the characteristics of BAC-HSV-1 recombinant virus;3. To Explore the operability of BAC component of BAC-HSV-1.Methods1. Construct the recombinant plasmid BAC-HSV-1(1) Construct the C223-LRarm carrying the homologous sequences of HSV-1PCR amplify the UL43 and UL47 homologous sequences of HSV-1, and then insert the fragments to the SacI and NotI sites of C223 to construct the C223-LRarm.(2) Homologous recombination between C223-LRarm and HSV-1Liposome2000 embedding method was used to transfect HSV-1 genome and the plasmid C223- LRarm after being linear digestion with Mlul to Vero cells.24 hours later, HSV-1 infected the Vero cells at MOI=1, keeping the 37℃,5% CO2 conditions of incubation. the recombinant BAC-HSV-1with GFP reporter gene was generated by homologous recombination in the eucaryote cells.(3) Plaque purificationThe positive CPE were taken by plaque purification, and The recombined circular DNA was extracted with the hirt method. Electricity transformation was used to transfect the BAC -HSV-1 to DH10B, then the monoclone was confirmed both by MluI enzyme digestion and PCR.2. The research of BAC-HSV-1 progeny virions(1) The reconstruct and the research of BAC-HSV-1 progeny virionsGroup and the control group cells were given BAC-HSV-1 plasmid and HSV-1 genomic DNA respectively to produce the BAC-HSV-1 and HSV-1 progeny virions.48 hours later, observe cells cytopathic effect and the GFP expression by fluorescence microscopy. The progeny virions were given at MOI=0.1 to Vero cells and then the method of TCID50 was used to titer the drops of virons between two groups 48 hours later. The results of the statistical analyses are shown as mean±standard deviation. The analyses were conducted with SPSS 16.0 software. The means of the different groups were compared using a t-test, and a value of p<0.05 was considered statistically significant. (2) The operability research of BAC-HSV-1Group A Vero cells was infected Ade-Cre virus at MOI=2,18 hours later, infected the cells by BAC-HSV-1 virus at MOI=0.01, Group B Vero cells was infected Ade virus at MOI=2,18 hours later, infected the cells by BAC-HSV-1 virus at MOI=0.01, harvest virus after cells appearing completely cytopathic effect.Infect the Vero cells by the supernatant of group A and group B respectively,24 hours later, observe the GFP expression by fluorescence microscopy.Results1. BAC-HSV-1 plasmid-virus shuttle system was established.2. The BAC-HSV-1 recombinant virus has strong ability of reproduction. The drops of progeny virions were calculated by the method of TCID50, No significant difference of virions between two groups was observed (P>0.05)3. The progeny BAC-HSV-IHFΔBAC virus without BAC was established.ConclusionBAC-HSV-1 plasmid-virus shuttle system was established using BAC-HSV-1 recombinant viruses based on homologous recombination in eukaryotic cells. The BAC-HSV-1 recombinant virus has strong ability of reproduction and there has no statistically difference in charateristics between recombined virus and provirus. The progeny BAC-HSV-1HFΔBAC virus without BAC was established mediated by Cre/loxP site recombination, illuminating the BAC component of BAC-HSV-1 is operable.