Design, expression, and purification of a multi-epitope vaccine against Helicobacter Pylori based on Melittin as an adjuvant
2021
Jafari, Elham | Mahmoodi, Shirin
Helicobacter Pylori, a Gram-negative bacterium in the human stomach, causes adenocarcinoma and MALT (mucosa-associated lymphoid tissue) lymphoma in addition to infection and gastric ulcer. With regard to Helicobacter Pylori prevalence rate and widespread, producing an effective vaccine against this bacterium appears reasonable and necessary. Today, vaccine design by immunoinformatics is a promising solution in vaccine field. In the present study, potential immunodominant CD4⁺ T cell epitopes of UreB, HpaA, and NapA antigens were selected with a focus on IFN-γ secretion inducing ability. After joining the selected epitopes with KK and GPGPG linkers, sequence of Melittin, the major active protein of honey bee venom, was put in C-terminal by DPRVPSS linker as adjuvant. After reverse translation and codon optimization, the designed vaccine was cloned into pET-23a vector. The final construct was estimated as antigenic (71 & 74%) and non-allergenic with molecular weight of 36.785KD. The instability index (II) and codon frequency distribution were predicted to be 26.5 and 92%, respectively. The pET-23a vector transformed to the E.coli BL21 (DE3) strain. The evaluation of expression by SDS-PAGE analysis showed that the optimized expression is in SOB medium 8 h after induction by 0.5 mM IPTG. Finally, purification was performed by Ni-NTA affinity chromatography and Western blot analysis validated the purified protein. Future research is needed to investigate the designed vaccine efficiency against H. pylori, and also it's potential as a gastric cancer-preventive candidate.
اظهر المزيد [+] اقل [-]الكلمات المفتاحية الخاصة بالمكنز الزراعي (أجروفوك)
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