Designing of a multi-epitope protein composed of essentialvirulence factors of SARS-Cov-۲ virus and evaluation of itsimmunogenicity in animal model

Background and Aim : The current COVID-۱۹ pandemic resulted in high mortality andmorbidity in the world. In the pandemia of SARS-Cov-۲ virus, there was no vaccine for treatmentor prevention of the virus, and this showed the need for studies to develop an effective vaccineagainst the virus. Therefore, we designed a multi-epitope of essential virulence factors of the virusincluding the proteins S, N, nsp۳, and nsp۸ to inhibit the virus from several pathways andexpressed it in a prokaryotic system to evaluate its immunogenicity in the animal modelMethods : In this study, by bioinformatic studies, a multi-epitope composed of proteins S, nsp۳,nsp۸, and N in SARS-Cov-۲ was designed to stimulate both humoral and cellular responses. Thefinal synthesized gene was ligated into pET۲۸a expression vector and transformed into the BL۲۱(DE۳) host by heat shock protocol. After culture of the transformed bacteria on the Luria bertani(LB) medium, the recombinant plasmids were screened using PCR, enzymatic digestion, andsequencing. The expression of the multi-peptide was performed by adding IPTG inducer and wasevaluated by SDS-PAGE and Western blot. The recombinant protein was purified by Nickel resin.After inoculation of mice with the recombinant protein, evaluation of humoral response was doneby ELISA.Results : Two B-cell epitopes, ۳ HTL epitopes, and ۳ CTL epitopes were selected from S, N, andNsp۳ proteins to design the vaccine construct, and about Nsp۸ protein, one was selected from eachB-cell, HTL, and CTL epitope. After synthesis of the multi-epitope gene, its expression wassuccessfully performed. Evaluation of the expression of the protein by SDS-PAGE and westernblot showed its expression with a size of about ۴۵ KD, which was purified by nickel column withhigh purity. The results of the evaluation of humoral responses showed that the group of micereceiving the purified protein was able to significantly induce IgG and IgA responses compared tothe control group (PBS group).Conclusion : Considering the significant immune responses induced by the multi-epitope protein,it is recommended that a challenge mice model design to evaluate the protective efficacy ofinduced immune responses against the experimental infection.

کلیدواژه ها:

COVID-۱۹ ، SARS-Cov-۲ ، bioinformatic studies ، multi-epitope ، vaccine candidate ، humoral responses