Construction and identification of recombinant cytomegalovirus with human papillomavirus 58 E6E7 fusion gene
DU Yang-yang1, ZHANG Fang1, MIAO Zhi-ying1, ZHAO Hong1,MA Di1, WANG Jing1, LI Shu-ying1, ZHENG Chun-yang1, LI Jin-tao2
1. North China University of Science and Technology/Hebei Key Laboratory for Chronic Diseases, Tangshan Key Laboratory for Preclinical and Basic Research on Chronic Diseases, Tangshan 063210,China; 2.College of Life Science and Bio-engineering, Beijing University of Technology, Beijing 100124,China
Abstract：To investigate the conversion activity of Human papillomavirus58 mutant E6E7 fusion gene (HPV58 mE6E7) and establish Towne recombinant virus carrying HPV58 mE6E7 fusion gene, which used HPV58 mE6E7 as target and Towne cytomegalovirus bacterial artificial chromosome (SW102-T-BAC) as a vector. The GalK, mE6E7 and Wild type E6E7 (wE6E7) gene fragments with about 50 bp Towne ORF75 homologous arms were amplified and purified. GalK was transferred to the competent cells of SW102-T-BAC. The clones of SW102-T-ORF75-GalK-BAC were obtained by homologous recombination, the selection from containing GalK and chloramphenicol medium. The fragments of mE6E7 and wE6E7 were transferred to the competent cells of SW102-T-ORF75-GalK-BAC. The clones of SW102-T-ORF75-mE6E7-BAC and SW102-T-ORF75-wE6E7-BAC were obtained by homologous recombination, the selection from replaced GalK and chloramphenicol medium, respectively. The plasmids of SW102-T-ORF75-mE6E7-BAC and SW102-T-ORF75-wE6E7-BAC were extracted, and transfected into ARPE-19 cells (ARPE-19 cells transfected with SW102-T-BAC and untransfected used as control). The expression of mE6E7 and wE6E7 in transfected cells were verified by reverse transcription PCR and sequencing. The effect of T-ORF75-mE6E7 and T-ORF75-wE6E7 on ARPE-19 cells were observed. The transformation activity of mE6E7 and wE6E7 were analyzed by soft agar cloning. The clones of SW102-T-ORF75-mE6E7-BAC and SW102-T-ORF75-wE6E7-BAC were obtained. The expression of mE6E7 and wE6E7 in transfected cells were verified by reverse transcription PCR and sequencing. ARPE-19 cells of transfected SW102-T-ORF75-wE6E7-BAC grow overlapping and lost contact inhibition, and the morphology of the cells changed from the original long fusiform to round, swelling, and full of cytoplasm particles, the cells can form clones in soft agar. The cells of transfected SW102-T-ORF-mE6E7-BAC, SW102-T-BAC, and untransfected do not show those characteristics of transfected SW102-T-ORF75-wE6E7-BAC cells. The recombinant virus of T-ORF75-mE6E7 and T-ORF75-wE6E7 were obtained, and the transformation activity of recombinant virus of T-ORF75-mE6E7 has been eliminated, which provided a foundation for the research of HPV58 therapeutic vaccine.
杜阳阳, 张放, 苗智颖, 赵红, 马迪, 王静, 李淑英, 郑春阳, 李劲涛. 人乳头瘤病毒58型E6E7融合基因重组巨细胞病毒的构建及鉴定[J]. 中国人兽共患病学报, 2020, 36(4): 303-308,319.
DU Yang-yang, ZHANG Fang, MIAO Zhi-ying, ZHAO Hong,MA Di, WANG Jing, LI Shu-ying, ZHENG Chun-yang, LI Jin-tao. Construction and identification of recombinant cytomegalovirus with human papillomavirus 58 E6E7 fusion gene. Chinese Journal of Zoonoses, 2020, 36(4): 303-308,319.
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