EXPRESSION AND PURIFICATION OF TRIMERIC SPIKE-FOLDON RECOMBINANT PROTEIN IN EXPICHO-S AND CHO-DG44 CELLS AS SARS-COV-2 VACCINE CANDIDATE

• Main Author: Mahmuda
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/66808
• Institution: Institut Teknologi Bandung

The spike protein (S) is the main component of the virus that induces neutralizing antibodies against SARS-CoV-2 infection so that this protein can be the main antigen candidate in the development of the COVID-19 subunit vaccine. On the other hand, the large size of the S protein makes it difficult to produce the protein so that the small S protein fragments, namely the S1 and RBD domains, are widely used in the development of Coronavirus vaccines. Domain S1 has a higher immunogenicity than RBD so that S1 was chosen as a good antigen candidate. The S1 domain was fused with the foldon domain (Fd) of the C-terminus of the bacteriophage T4 fibritin for recombinant S1 trimerization according to its original structure and could increase the immunogenicity of the antigen. CHO cells are the most widely used mammalian expression system in the production of therapeutic recombinant proteins. However, to date the S1 expression of SARS-CoV 2 fused with Fd in CHO cells has not been reported. Therefore, this study focused on plasmid construction, expression, purification, and S1-Fd antigenicity assay on two CHO cell lines, namely ExpiCHO-S and CHO-DG44. Plasmid construction was carried out by ligating the S1-Fd gene which had been amplified by PCR method into the vector pcDNA3.1(+) (~ 5.3 kb). These plasmids were transformed into competent E. coli TOP10 cells by heat-shock method. Cell lines were transiently transfected and grown in an incubator shaker at 37 °C, with 8% CO2, and 130 rpm agitation. Next, the supernatant was analyzed and purified by the IMAC method. Gene expression analysis was performed using slot blotting, SDS-PAGE, and western blotting methods. The antigenicity test was carried out using indirect ELISA. In this study, the plasmid construction was successfully carried out by confirming the ~2.5 Kb band in the gel electrophoresis results and the presence of the insert gene in the DNA sequencing results. The results of slot blotting showed that the ExpiCHO-S and CHO-DG44 samples were suspected to contain recombinant protein, although the negative control CHO-DG44 showed inconsistent results. The SDS-PAGE results showed that the protein expressed in both cell lines was monomeric (~70 KDa). The results of western blotting showed that the recombinant protein was only detected in the ExpiCHO-S sample. The indirect ELISA results showed that samples of recombinant protein from ExpiCHO-S cells could only be recognized by antibodies derived from the serum of Covid-19 positive patients. Thus, it can be concluded that the S1-Fd protein in this study has been successfully produced, but the purity of the purification results is still low and only the recombinant antigen protein from ExpiCHO-S can be recognized by antibodies from the serum of Covid-19 positive patients. Therefore, further research should be carried out to optimize the production of S1-Fd from harvesting to purification and increase the antigenicity of the antigen candidate.