Volume 5, Issue 1 (6-2023)
pbp 2023, 5(1): 58-66 |
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Moniri Javadhesari S, Koohi chapan M, Darvishi Harzevili F, Madzak C. Constructing a Surface Display Vector Encoding E7 Protein of HPV18. pbp 2023; 5 (1) :58-66
URL: http://pbp.medilam.ac.ir/article-1-195-en.html
URL: http://pbp.medilam.ac.ir/article-1-195-en.html
Solmaz Moniri Javadhesari * 
1, Mohaddeseh Koohi chapan2 , Farshad Darvishi Harzevili3 , Catherine Madzak4
1, Mohaddeseh Koohi chapan2 , Farshad Darvishi Harzevili3 , Catherine Madzak4
1- Department of Biology, Faculty of Basic Sciences, Azarbaijan Shahid Madani University, Tabriz, Iran , solmazmoniri@gmail.com
2- Department of Biology, Faculty of Basic Sciences, Azarbaijan Shahid Madani University, Tabriz, Iran
3- Department of Microbiology, Faculty of Biological sciences, Alzahra University, Tehran, Iran
4- INRAE France’s New National Research Institute for Agriculture, Food and Environment. Paris-Saclay University, Paris, France.
2- Department of Biology, Faculty of Basic Sciences, Azarbaijan Shahid Madani University, Tabriz, Iran
3- Department of Microbiology, Faculty of Biological sciences, Alzahra University, Tehran, Iran
4- INRAE France’s New National Research Institute for Agriculture, Food and Environment. Paris-Saclay University, Paris, France.
Abstract: (676 Views)
Objectives: This study aimed 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.
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: 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.
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: 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.
Received: 2023/07/18 | Accepted: 2023/03/1 | Published: 2023/05/31
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