Efficient expression system of sars-cov-2 receptor binding domain (rbd), methods for purification and use thereof

Abstract

The present invention relates to the production of the receptor binding domain (RBD) of the Spike glycoprotein 1 of the SARS-CoV-2 in mammalian cell expression systems, and the successive method of purification thereof. A recombinant plasmid containing the coding sequence of said RBD is produced and transfected in said mammalian cells, for example, Expi293. A high level of the protein is secreted in the medium and subsequently purified using the N-terminal tag, that can be removed by a specific protease. The present invention also includes a recombinant expression vector carrying the RBD gene, the successive methods for protein purification, the strategy for establishing a stable cell line producing the RBD, methods of use of the recombinant protein in formulating a pharmaceutical composition, including but not limited to, vaccines for preventing SARS-CoV-2 induced diseases.

1 . A vaccine composition for SARS-CoV-2 comprising a recombinant protein formed by transfecting an expression vector for producing at least a portion of a receptor-binding domain (RBD) sequence of SARS-CoV-2 spike glycoprotein into a host cell, harvesting an expressed, recombinant RBD protein, and removing an affinity tag of the expressed, recombinant RBD protein to obtain a tag-free RBD protein;
 wherein the expression vector comprises a polynucleotide sequence encoding a receptor-binding domain (RBD) sequence of SARS-CoV-2 spike glycoprotein that is at least 80 percent identical to SEQ ID NO. 2.   
     
     
         2 . The vaccine composition for SARS-CoV-2 according to  claim 1 , further comprising a pharmaceutically acceptable excipient. 
     
     
         3 . The vaccine composition for SARS-CoV-2 according to  claim 1 , wherein the expression vector further comprises:
 at least one polynucleotide sequence encoding at least one signal peptide sequence;   one or more polynucleotide sequences encoding one or more affinity tags;   at least one polynucleotide sequence encoding a protease recognition site sequence; and   one or more polynucleotide sequences encoding one or more reporter sequences.   
     
     
         4 . The vaccine composition for SARS-CoV-2 according to  claim 1 , wherein the expression vector has a bicistronic structure. 
     
     
         5 . The vaccine composition for SARS-CoV-2 according to  claim 3 ,
 wherein the polynucleotide sequence encoding the protease recognition site sequence is positioned between the polynucleotide sequence encoding the affinity tag and the polynucleotide sequence encoding the receptor-binding domain sequence such that a protease is capable of cleaving the affinity tag from the expressed, recombinant RBD sequence to create the tag-free RBD sequence; and   wherein the polynucleotide sequence further includes a polynucleotide sequence for an internal ribosome entry site operatively linked between the receptor-binding domain (RBD) polynucleotide sequence and the reporter polynucleotide sequence.   
     
     
         6 . The vaccine composition for SARS-CoV-2 according to  claim 3 , wherein the signal peptide sequence is selected from human serum albumin, human granulocyte colony-stimulating factor or combination thereof. 
     
     
         7 . The vaccine composition for SARS-CoV-2 according to  claim 3 , wherein the affinity tag is selected from chitin binding protein, maltose binding protein, streptavidin, poly histidine, or combination thereof. 
     
     
         8 . The vaccine composition for SARS-CoV-2 according to  claim 3 , wherein the protease recognition site sequence can be recognized by a tobacco etch virus protease. 
     
     
         9 . The vaccine composition for SARS-CoV-2 according to  claim 3 , wherein the reporter sequence is selected from enhanced yellow fluorescent protein, enhanced red fluorescent protein, enhanced green fluorescent protein, beta-galactosidase, or combination thereof. 
     
     
         10 . The vaccine composition for SARS-CoV-2 according to  claim 3 , wherein the expression vector further comprises one or more nucleotide signal sequence selected from SV40 poly(A) nucleotide sequences, HSV TK poly(A) nucleotide sequences, or a combination thereof. 
     
     
         11 . The vaccine composition for SARS-CoV-2 according to  claim 3 , wherein the expression vector further comprises one or more polynucleotide sequences encoding a promoter sequence selected from an SV40 promoter, an ampR promoter, a CMV promoter or a combination thereof. 
     
     
         12 . The vaccine composition of  claim 1 , wherein the vaccine composition further comprises a polypeptide selected from spike protein (S), nucleocapsid (N) protein of SARS-CoV-2, matrix protein (M) of SARS-CoV-2, envelope (E) protein of SARS-CoV-2, or a combination thereof; and an immunological adjuvant. 
     
     
         13 . A method comprising administering to a subject an effective amount of the vaccine composition of  claim 1 , so as to induce a virus-specific antibody. 
     
     
         14 . The method of  claim 13 , wherein the antibody is effective against a wild type SARS-CoV-2 2019-nCov strain, a beta strain (B.1.351), a delta strain (B.1.617.2), an omicron strain (B.1.1.529) or a combination thereof.