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Moniri Javadhesari S, Koohi chapan M, Darvishi Harzevili F, Madzak C. Constructing a surface display plasmid encoding recombinant E7 protein of human papilloma virus type 18. Journal title 2023; 5 (1)
URL: http://pbp.medilam.ac.ir/article-1-171-en.html
URL: http://pbp.medilam.ac.ir/article-1-171-en.html
Solmaz Moniri Javadhesari * 
, Mohaddeseh Koohi chapan , Farshad Darvishi Harzevili , Catherine Madzak
, Mohaddeseh Koohi chapan , Farshad Darvishi Harzevili , Catherine Madzak
Department of Biology, Faculty of Basic Sciences, Azarbaijan Shahid Madani University, Tabriz, Iran , solmazmoniri@gmail.com
Abstract: (451 Views)
Background and objectives: High risk human papillomaviruses (HPV) are the main cause of urogenital cancers particularly cervix. The available prophylactic vaccines have no effect on the past HPV infections. E7 oncoprotein of HPV plays a critical role in carcinogenesis and recombinant E7 can stimulate T-cell based immune response against HPV that is a promising target as vaccine against previous HPV infections. This study aimed at the construction of a surface display vector encoding E7 protein of HPV18 to be expressed in yeast Yarrowia lipolytica in order to combine the advantages of both systems.
Materials and Methods: DNA from a HPV18-positive individual was applied for amplification of E7 encoding gene via nested-PCR. Both pINA1317-YLCWP110 plasmid and PCR products were double-digested using HindIII and SfiI. After purification, the double digested fragments were ligated together to create the recombinant pINA1317-YLCWP110-E7 plasmid. After transformation into E. coli, the positive transformants were subjected to molecular analysis. The accuracy of cloning was further assessed using Sanger sequencing.
Results: Plasmid DNA from positive transformants was analyzed by molecular methods. The E7 related band was detected in PCR, and restrictive cleavage confirmed the presence of E7 insert in the recombinant plasmid. In addition, Sanger sequencing of recombinant pINA1317-YLCWP110-E7 plasmid confirmed the accuracy of cloning; alignment of the sequencing data in gene bank verified the DNA sequence of E7 encoding gene, insert orientation, and accuracy of reading frame.
Conclusions: Prosperous cloning of E7 encoding fragment of HPV18 into surface display pINA1317-YLCWP110 plasmid was achieved. Molecular analysis and BLAST of Sanger sequencing data proved the accuracy of the sequence and cloning frame. The constructed recombinant pINA1317-YLCWP110-E7 plasmid can be used to express the E7 protein in the yeast Yarrowia lipolytica to be applicable as a vaccine, molecular marker or therapeutic aspects.
Materials and Methods: DNA from a HPV18-positive individual was applied for amplification of E7 encoding gene via nested-PCR. Both pINA1317-YLCWP110 plasmid and PCR products were double-digested using HindIII and SfiI. After purification, the double digested fragments were ligated together to create the recombinant pINA1317-YLCWP110-E7 plasmid. After transformation into E. coli, the positive transformants were subjected to molecular analysis. The accuracy of cloning was further assessed using Sanger sequencing.
Results: Plasmid DNA from positive transformants was analyzed by molecular methods. The E7 related band was detected in PCR, and restrictive cleavage confirmed the presence of E7 insert in the recombinant plasmid. In addition, Sanger sequencing of recombinant pINA1317-YLCWP110-E7 plasmid confirmed the accuracy of cloning; alignment of the sequencing data in gene bank verified the DNA sequence of E7 encoding gene, insert orientation, and accuracy of reading frame.
Conclusions: Prosperous cloning of E7 encoding fragment of HPV18 into surface display pINA1317-YLCWP110 plasmid was achieved. Molecular analysis and BLAST of Sanger sequencing data proved the accuracy of the sequence and cloning frame. The constructed recombinant pINA1317-YLCWP110-E7 plasmid can be used to express the E7 protein in the yeast Yarrowia lipolytica to be applicable as a vaccine, molecular marker or therapeutic aspects.
Type of Study: Research |
Subject:
Biotechnology
Received: 2022/10/14 | Accepted: 2022/11/22 | Published: 2022/11/22
Received: 2022/10/14 | Accepted: 2022/11/22 | Published: 2022/11/22
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