title: [Construction and transfection of eucaryotic expression recombinant vector containing truncated region of UL83 gene of human cytomegalovirus and it's sheltered effect as DNA vaccine].
abstract: To construct eucaryotic expression recombinant vector containing vivo truncated region of UL83 gene of human cytomegalovirus, realize its steady expression in Hep-2 cell, and study sheltered effect of the eucaryotic expression recombinant vector as DNA vaccine. A vivo truncated UL83 gene fragment encoding for truncated HCMV pp65 was obtained by PCR from human cytomegalovirus AD169 stock genome. By gene recombinant ways, the truncated UL83 gene fragment was cloned into eucaryotic expression vector pEGFP-C1 with reported gene coding GFP to construct recombinant vector pEGFP-C1-UL83. The recombinant vector pEGFP-C1-UL83 was tested by different methods including PCR, restriction digestion and gene sequencing. Test results showed the recombinant vector was constructed successfully. After pEGFP-C1-UL83 was transfected into Hep-2 cell by lipofectin mediation, expression of GFP and truncated pp65 fusion protein in Hep-2 cell was observed at different time points by fluorescence microscope. Results showed that quantity of fusion protein expression was the highest at 36h point. Then, Hep-2 cell was cultured selectively by RPMI-1640 containing G418 (200 microg/mL) to obtain a new cell stock of expressing truncated UL83 Gene fragment steadily. RT-PCR and Western blot results showed the truncated fragment of UL83 gene could be expressed steadily in Hep-2 cell. The result showed a new cell stock of expressing Tpp65 was established. This cell stock could be useful in some HCMV research fields, for example, it could be a tool in study of pp65 and HCMV infection, and it could provide a platform for the research into the therapy of HCMV infection. Immune sheltered effect of pEGFP-C1-UL83 as DNA vaccine was studied in vivo of HCMV congenital infection mouse model. The mouse model was immunized solely by pEGFP-C1-UL83, and was immunized jointly by pEGFP-C1-UL83 and its expression product. When the mouse was pregnant and brought to bed, differential antibody of anti-HCMV pp65 was tested by indirect ELISA in mother mouse, the infectious virus was separated with the method of virus separation, and pp65 antigen was checked up by indirect immunofluorescence staining in fetal mouse. Results showed differential antibody of anti-HCMV pp65 was produced in mouse model. Tilter of the antibody was from 1:2.51 to 1:50.79. Results of virus separation and pp65 checkup of fetal mouse brain tissue were negative. So the conclusion can be reached that pEGFP-C1-UL83 as DNA vaccine in vivo has sheltered effect which can prevent HCMV vertical transmission from mother mouse to her fetus.
authors.affiliation: Department of Microbiology, College of Basic Medicine, Anhui Medical Univercity, Hefei 230032, China. rongbaogao@163.com
authors.forename: Rong-Bao
authors.initials: RB
authors.lastname: Gao
authors.forename: Yan-Qiu
authors.initials: YQ
authors.lastname: Li
authors.forename: Ming-Li
authors.initials: ML
authors.lastname: Wang
mesh_terms: D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000276:immunology; D045744:Cell Line, Tumor; D003587:Cytomegalovirus / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D004274:DNA, Recombinant / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005057:Eukaryotic Cells / Q000378:metabolism*; D005260:Female; D017353:Gene Deletion; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051379:Mice; D010750:Phosphoproteins / Q000172:deficiency / Q000235:genetics* / Q000276:immunology; D011247:Pregnancy; D014162:Transfection; D014611:Vaccination; D019444:Vaccines, DNA / Q000235:genetics* / Q000276:immunology; D014763:Viral Matrix Proteins / Q000172:deficiency / Q000235:genetics* / Q000276:immunology
chemical_list: D000906:Antibodies; D004274:DNA, Recombinant; D010750:Phosphoproteins; D019444:Vaccines, DNA; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa