Expression And Characterization Of The Recombinant Epitopes Of HSV-gB&gD Protein

Herpes simplex virus (HSV) keratitis is a severe ocular disease which cause blindness over worldwide. After primary infection, HSV can establish a lifelong latent infection in trigeminal ganglion, which causes recurrent disease. Therefor, it is with significant importance to develop a vaccine to prevent HSV infection, latency and recurrence. In this study, we analyzed the epitopes of HSV-gB&gD protein with epitope prediction software. A novel gene, named X, encoding 9 predicted epitopes of gB&gD protein was designed and synthesized using chemical method. X gene was cloned into vector PET-28a(+), expressed in E.coli BL21(DE3) and analyzed by Western Blotting.Material and Method1,Material(1) Vector and Bacteria:vector PET-28a and E.coli BL21(DE3) were donated by Mr. Fang, from Biochip Lab, CMU. The E.coli BL21(DE3) competent cell was purchased from TaKaLa Biotechnology Co., Ltd.(2) Main Reagent:Pyrobest DNA Polymerase, IPTG, Kanamycin and DL2000 DNA Marker were purchased from TaKaLa Biotechnology Co., Ltd. UNIQ-10 Colume Vector Extraction Kit and UNIQ-10 Colume DNA Gel Extraction Kit were purchased from Shanghai Sangon Biological Engineering Technology& Services Co., Ltd. Xhol & EcoRI endonuclease, T4 DNA Ligase were purchased from New England Biolabs Co,. Ltd.2,Method(1) Design and Synthesize the recombined epitope gene:We analysied the epitopes in HSV gB, gD protein with software laser gene DNASTAR, designe the sequence of recombined epitopes, and a piece of His sequence was also added in the recombined gene, which was named X. And X gene was synthesized by Sangon Bio Co., Ltd.(2) Construction of X-gene expression vector:X-gene was amplified by PCR, of which the produce was extracted. The amplified X-gene and vector PET-28a(+) separately reacted with XhoI & EcoRI endonuclease, linked the two pieces up with T4 DNA Ligase then acquired the expression vector PET-X. After transforming PET-X vector into BL21(DE3), we filtrated positive clone by PCR and restriction endonuclease reaction.(3) Expression of X-gene:Cultured positive cloned BL21(DE3), preserve sample lml as negative control. Then added IPTG and continue to culture the bacteria, preserve the sample after adding IPTG 1h,2h,3h. Check samples with SDS-PAGE electrophoresis.(4) Identification of recombined protein:Identified recombined protein by Western Blotting, using Penta-His Antibody and HRP-Rabbit Anti-Mouse IgG.Result1. Design and Synthesize the recombined epitope geneWe selected 9 epitopes from gB, gD protein to design the recombined gene, X-gene, which contains 1299bp, coding 433 amino acids. The recombined protein was calculated about 47.5kD.2. The PCR of X-geneThe PCR produce was tested with agarose gel electrophoresis, which showed a bright band, located near about 1300bp. DNA sequencing result showed that the PCR produce was the X gene.3. Construction and Identification of X-gene expression vectorAfter X-gene expression vector reacting with XhoI & EcoRI endonuclease, we got two bright bands in agarose gel electrophoresis, one was with vector's size, the other was about 1300bp size. DNA sequencing result showed that the X-gene was correctly inserted. (by TaKaLa Biotech. Co., Ltd.)4. The expression of recombined proteinSDS-PAGE electrophoresis showed a new bright band in group 1h,2h,3h, located near the 50kD of the marker (about 47kD).5. The Western Blotting test of recombined protein XIt showed anti-His antibody cognition bands in group 1h,2h,3h, while the negative control with no band. It was proved that the recombined protein X was successfully expressed.Conclusion1. Created a new recombined HSV epitopes gene.2. Expressed and identified recombinant epitopes protein, which provides a new potential protein for developing vaccine against HSV infection.