Expression of full and fragment-B of diphtheria toxin genes inEscherichia colifor generating of recombinant diphtheria vaccines

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.

Recent development of oral vaccines (Review)

“…Therefore, these antigens are often combined with various adjuvants such as chitosan/aluminum, glucan and squalene-based adjuvants or carriers ( 91 ). Recombinant vaccines against HBsAg ( 92 ), tetanus toxin ( 93 ), diphtheria toxin ( 94 ) and pertussis toxin ( 95 ) have been developed and can be produced on a large scale. Furthermore, research into the development of oral forms of these recombinant vaccines is being conducted.…”

Hosts and Heterologous Expression Strategies of Recombinant Toxins for Therapeutic Purposes

“…The authors demonstrated that the proteins were expressed in discrete quantities (0.9–1.12 mg/mL) under batch culture conditions implemented by fed-batch, improving biomasses with the addition of glucose and yeast extract as carbon sources [ 21 ]. A mutant, full-length form of diphtheria toxin (Glu148Ser), 800-fold less cytotoxic than a wild-type toxin, was cloned and expressed into E. coli K-12 under BL-1EK-1 conditions, and the protein has been recovered from the periplasmic extracts [ 22 ].…”