الفهرس 
Development and evaluation of immunogenicity of candidate recombinant diphtheria vaccine
CONTENTSOnly 14 pages are availabe for public view
 |  | from | 192 |  |  |
| | | from | 192 | | |
Abstract
Whole diphtheria toxin (dt) and fragment B (dtb) genes from
Corynebacterium diphtheriae Park William were cloned into Escherichia coli,
and the purified expressed proteins were evaluated for ultimately use as a
candidate vaccine.
The dt and dtb genes were isolated from Corynebacterium diphtheriae
strain ATCC (American Type Culture Collection) No. 13812. Plasmid pET29a+
was extracted by a 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 fermentation parameters to determine optimal conditions.
Lastly, BALB/c mice were injected with the purified concentrated rdtx and rdtb,
and antibody titers were detected.
The genetic transformation of E. coli DH5α and E. coli BL21 with the pET-
29a(+) carrying the dt and dtb genes was confirmed by polymerase chain reaction
assay. It was 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 and
were found to be 100% identical to the dt and dtb sequence of C. diphtheriae
(accession number KX702999.1 and KX702993.1) respectively.
In the pilot-scale fermenter, five L (New Brunswick) three cultures A, B,
and C represent batch fermentation, Agitation/Air/DO cascade, and Fed-batch
glucose/yeast extract with 2ml/min, respectively. rdtx and rdtb production were compared to traditional fermentation of C. diphtheriae to produce dtx. A
flocculation test determines the concentration of diphtheria protein in the culture
medium. The optimal condition of kinetic parameters for high cell density is fedbatch
fermentation production to express the rdtx and rdtb at 280 and 240 lf/mL;
dissolved oxygen was about 24% and 22%. The dry cell weight of bacteria was
2.41g/L and 2.18 g/L, respectively. Cultivation was carried out at 35°C till the
mid-logarithmic phase was reduced to 28°C for 24h. The pH of the media for
protein production was adjusted to 6.8 with 25%NH4OH; the agitation and
aeration rates in the fermenter were 700 rpm and 5 L/h, respectively. An
antifoaming agent was added to the fermenter to reduce foam. Diphtheria toxoid was purified by subjecting the diphtheria protein rdtx in the fermentation medium
to ammonium sulfate fractionation, followed by detoxification with
formaldehyde and then dialysis. Moreover, the characteristics of the purified
protein were analyzed by SDS-PAGE.
The immunogenicity of purified concentrated rdtx and rdtb were
determined by injecting to BALB/c mice and determining antibody titers. Our
results displayed that the IgG antibody titers produced in mice were equivalent to
those produced by DTP; the results above will provide technical support for the
future combined recombinant protein vaccine against diphtheria.
This study concluded the success in preparing two genetically engineered
strains for producing a diphtheria vaccine and optimizing production conditions to achieve the highest productivity.