مقالات پذیرفته شده در ششمین کنگره بین المللی زیست پزشکی
Expression of SARS-CoV-2 receptor binding domain (RBD) in prokaryotic system
Expression of SARS-CoV-2 receptor binding domain (RBD) in prokaryotic system
Samaneh Jahandar-Lashaki,1,*Safar Farajnia,2Morteza Milani,3Rana Yousefzadeh,4
1. 1-Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran , 2-Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran 2. Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran 3. Medical Biotechnology Department, Faculty of Advanced Medical Science, Tabriz University of Medical Sciences, Tabriz, Iran 4. Central Medical Laboratory, Tabriz University of Medical Sciences, Tabriz, Iran
Introduction: The receptor binding domain (RBD) is a part of the SARS-CoV-2 S protein, which binds to the host receptor and facilitates viral entry. RBD expressed in eukaryotic cells cannot meet the high expression yield and cost requirements for therapeutics and vaccines. There have been numerous studies suggesting that the RBD expressed by E. coli may induce protective immunity. Furthermore, RBD expressed in E. coli has been used for worldwide research purposes as a cost-effective antigen. In this study, we expressed RBD in the E. coli BL21 strain and evaluated the purified protein by SDS page and ELISA.
Methods: The plasmid and the sequence coding for RBD were treated with restriction enzymes. The RBD fragment was ligated into pET28a (+) using T4 DNA ligase. Then the expression construct was transformed into E. coli BL21, cultured in LB media, and induced with isopropyl b-D-1-thiogalactoside. A 15% SDS-PAGE was used to analyze the expression. For purification, the cells were lysed by sonication and recombinant RBD was purified by the NiNTA column. Finally, the recombinant RBD was refolded by the gradual elimination of urea by dialysis. The quality of purified RBD was assessed by SDS-PAGE analysis and ELISA.
Results: To produce a large amount of antigen for the biopanning process, the DNA sequence of the RBD protein of SARS-CoV-2 spike protein was synthesized and cloned into the PET-28a vector. Results of PCR analysis and DNA sequencing confirmed the recombinant PET-28a sequence at the appropriate size of ~ 4000 bp as shown in supplementary data 1. The recombinant protein was expressed in E. coli strain BL21, purified by NINTA column, and refolded by dialysis. The SDS-PAGE results showed 95% purification of the RBD protein. To ensure the accurate antigenicity and function of the RBD protein an ELISA test on pooled COVID-19 serum samples was performed and the results revealed a high binding affinity of the serum antibodies to the RBD protein.
Conclusion: In conclusion, we expressed high yields of SARS-CoV-2 receptor binding domain protein in E. coli strain BL21. Our results indicate that expressed RBD is functional and can induce immune responses.
Keywords: Receptor binding domain (RBD), protein expression, E. coli BL21, COVID-19, SARS-CoV-2.