OPTIMIZATION OF SPIKE AND NSP3 PROTEIN FUSION EXPRESSION FROM SARS-COV-2 USING E.COLI BL21(DE3) AT 37°C AS INITIAL DEVELOPMENT OF MULTIEPITOPE COVID-19 VACCINE CANDIDATES

• Main Author: Gunawan, Cynthia
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/65550
• Institution: Institut Teknologi Bandung

Coronavirus disease-2019 (Covid-19) is an infectious respiratory disease caused by the SARS-CoV-2 virus which has a high transmission rate and death rate worldwide. One of the efforts to deal with the Covid-19 pandemic is vaccination. However, the currently available Covid-19 vaccine still has several shortcomings, so a new vaccine platform is needed that is able to handle various mutational variants of SARS-CoV-2 and can induce humoral and cellular immune systems. In previous studies, multiepitope vaccines have been designed in silico, but the results of these designs have not been expressed in vitro. Thus, in this study, we will optimize the expression of the S-NSP3 fusion protein from SARS-CoV-2 as the initial development of a multiepitope Covid-19 vaccine candidate. The purpose of this study was to determine the solubility of the S-NSP3 SARS-CoV-2 fusion protein and determine the concentration of IPTG with an incubation time that resulted in the most optimum number of the SARS-CoV-2 S-NSP3 protein fusion. The recombinant plasmid containing the gene encoding the S-NSP3 fusion protein from SARS-CoV-2 was transformed into E.coli BL21(DE3). The results of the transformation were confirmed by PCR and DNA sequencing methods. Optimization of fusion protein expression at 37oC was carried out by varying the incubation time of 1, 2, and 4 hours and varying the IPTG concentration of 0 mM, 0.05 mM, 0.1 mM, 0.25 mM, and 0.5 mM. Furthermore, the fusion protein was isolated using sonication, and cell lysates were analyzed by SDS-PAGE. The results of SDS-PAGE were analyzed using ImageJ and statistical significance was analyzed using One Way ANOVA and Post Hoc Test. The protein was purified with a Ni-NTA column in order to obtain a pure S-NSP3 protein fusion that would be used as a candidate for the multiepitope vaccine. From these results, it was found that the transformant E.coli BL21(DE3) colony was confirmed to carry the gene encoding the SARS-CoV-2 S-NSP3 protein fusion. Based on the results of expression optimization, a protein band measuring 34.5 kDa was obtained which was thought to be a SARS-CoV-2 S-NSP3 fusion protein and was in the insoluble phase (pellet). Incubation for 2 hours with 0.1 mM IPTG induction produced the most optimum and significant amount of protein (p<0.05) against other treatment variations. From the purification results, it was found that the protein was suspected to be a fusion protein of S-NSP3 SARS-CoV-2 from a pure insoluble phase based on the results of SDS-PAGE analysis. The conclusion of this study is that the multiepitope vaccine candidate protein was successfully expressed in the insoluble phase at 37oC. The results of this study are expected to be potential candidates for the development of a multiepitope vaccine for the Covid-19 pandemic in Indonesia.