{"title":"Expression of full and fragment-B of diphtheria toxin genes in Escherichia coli for generating of recombinant diphtheria vaccines","authors":"Shaimaa Abulmagd, A. E. Khattab, H. Zedan","doi":"10.7774/cevr.2022.11.1.12","DOIUrl":null,"url":null,"abstract":"Purpose In the present study, whole diphtheria toxin (dt) and fragment B (dtb) genes from Corynebacterium diphtheriae Park William were cloned into Escherichia coli, the purified expressed proteins were evaluated for ultimately using as a candidate vaccine. Materials and Methods The dt and dtb genes were isolated from bacterial strain ATCC (American Type Culture Collection) no. 13812. Plasmid pET29a+ was extracted by DNA-spin TM plasmid purification kit where genes were inserted using BamHI and HindIII-HF. Cloned pET29a+dt and pET29a+dtb plasmids were transformed into E. coli BL21(DE3)PlysS as expression host. The identity of the sequences was validated by blasting the sequence (BLASTn) against all the reported nucleotide sequences in the NCBI (National Center for Biotechnology Information) GenBank. Production of proteins in high yield by different types and parameters of fermentation to determine optimal conditions. Lastly, the purified concentrated rdtx and rdtb were injected to BALB/c mice and antibody titers were detected. Results The genetic transformation of E. coli DH5α and E. coli BL21 with the pET-29a(+) carrying the dt and dtb genes was confirmed by colony polymerase chain reaction assay and were positive to grow on Luria-Bertani/kanamycin medium. The open reading frame of dt and dtb sequences consisted of 1,600 bp and 1,000 bp, were found to be 100% identical to dt and dtb sequence of C. diphtheriae (accession number KX702999.1 and KX702993.1) respectively. The optimal condition for high cell density is fed-batch fermentation production to express the rdtx and rdtb at 280 and 240 Lf/mL, dissolved oxygen was about 24% and 22% and the dry cell weight of bacteria was 2.41 g/L and 2.18 g/L, respectively. Conclusion This study concluded with success in preparing genetically modified two strains for the production of a diphtheria vaccine, and to reach ideal production conditions to achieve the highest productivity.","PeriodicalId":51768,"journal":{"name":"Clinical and Experimental Vaccine Research","volume":null,"pages":null},"PeriodicalIF":2.1000,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Clinical and Experimental Vaccine Research","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.7774/cevr.2022.11.1.12","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"IMMUNOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Purpose In the present study, whole diphtheria toxin (dt) and fragment B (dtb) genes from Corynebacterium diphtheriae Park William were cloned into Escherichia coli, the purified expressed proteins were evaluated for ultimately using as a candidate vaccine. Materials and Methods The dt and dtb genes were isolated from bacterial strain ATCC (American Type Culture Collection) no. 13812. Plasmid pET29a+ was extracted by DNA-spin TM plasmid purification kit where genes were inserted using BamHI and HindIII-HF. Cloned pET29a+dt and pET29a+dtb plasmids were transformed into E. coli BL21(DE3)PlysS as expression host. The identity of the sequences was validated by blasting the sequence (BLASTn) against all the reported nucleotide sequences in the NCBI (National Center for Biotechnology Information) GenBank. Production of proteins in high yield by different types and parameters of fermentation to determine optimal conditions. Lastly, the purified concentrated rdtx and rdtb were injected to BALB/c mice and antibody titers were detected. Results The genetic transformation of E. coli DH5α and E. coli BL21 with the pET-29a(+) carrying the dt and dtb genes was confirmed by colony polymerase chain reaction assay and were positive to grow on Luria-Bertani/kanamycin medium. The open reading frame of dt and dtb sequences consisted of 1,600 bp and 1,000 bp, were found to be 100% identical to dt and dtb sequence of C. diphtheriae (accession number KX702999.1 and KX702993.1) respectively. The optimal condition for high cell density is fed-batch fermentation production to express the rdtx and rdtb at 280 and 240 Lf/mL, dissolved oxygen was about 24% and 22% and the dry cell weight of bacteria was 2.41 g/L and 2.18 g/L, respectively. Conclusion This study concluded with success in preparing genetically modified two strains for the production of a diphtheria vaccine, and to reach ideal production conditions to achieve the highest productivity.
期刊介绍:
Clin Exp Vaccine Res, the official English journal of the Korean Vaccine Society, is an international, peer reviewed, and open-access journal. It covers all areas related to vaccines and vaccination. Clin Exp Vaccine Res publishes editorials, review articles, special articles, original articles, case reports, brief communications, and correspondences covering a wide range of clinical and experimental subjects including vaccines and vaccination for human and animals against infectious diseases caused by viruses, bacteria, parasites and tumor. The scope of the journal is to disseminate information that may contribute to elaborate vaccine development and vaccination strategies targeting infectious diseases and tumors in human and animals. Relevant topics range from experimental approaches to (pre)clinical trials for the vaccine research based on, but not limited to, basic laboratory, translational, and (pre)clinical investigations, epidemiology of infectious diseases and progression of all aspects in the health related issues. It is published printed and open accessed online issues (https://ecevr.org) two times per year in 31 January and 31 July. Clin Exp Vaccine Res is linked to many international databases and is made freely available to institutions and individuals worldwide