Compositions and methods for identifying and sorting antigen-specific B cells
AssigneesEmory University, Rutgers, The State University of New Jersey
InventorsArash Grakoui, Joseph Marcotrigiano
Life of this patent
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Abstract
The present disclosure relates to constructs useful in expressing biotinylated monomers and tetramers produced from these monomers. Specifically, the disclosure provides a construct useful in expressing biotinylated antigen monomers, said construct comprising a viral protein of an ectodomain of Hepatitis C Virus (HCV) envelope glycoprotein E2 or Human Immunodeficiency Virus (HIV) gp140. Further disclosed are methods for production and use of these tetramers in identifying and isolating antigen specific B cells and cloning antibodies thereto.
Claims10
A vector construct useful in expressing biotinylated antigen monomers, said construct comprising:
- a segment encoding a viral protein or functional fragment thereof;
- a segment encoding a biotinylation site inserted downstream of the segment encoding the viral protein or functional fragment thereof,
- wherein the viral protein has the HCV E2 sequence of
- THTVGGSAAQTTGRLTSLFDMGPRQKIQLVNTNGSWHINRTALNCNDSLHTGFI ASLFYTHSFNSSGCPERMSACRSIEAFRVGWGALQYEDNVTNPEDMRPYCWHYPPRQC GVVSAKTVCGPVYCFTPSPVVVGTTDRLGAPTYTWGENETDVFLLNSTRPPLGSWFGCT WMNSSGYTKTCGAPPCRTRADFNASTDLLCPTDCFRKHPDTTYLKCGSGPWLTPRCLID YPYRLWHYPCTVNYTIFKIRMYVGGVEHRLTAACNFTRGDRCNLEDRDRS (SEQ ID NO:
- 4), and
- wherein the biotinylation site has the amino acid sequence of GLNDIFEAQKIEWHE (SEQ ID NO:
- 2).
A vector construct useful in expressing biotinylated antigen monomers, said construct comprising:
- a segment encoding a viral protein or functional fragment thereof;
- a segment encoding a biotinylation site inserted downstream of the segment encoding the viral protein or functional fragment thereof;
- a segment encoding a cleavage site, segment encoding a tag for affinity purification and reporter gene inserted downstream of the segment encoding the viral protein or functional fragment thereof;
- a segment encoding signal sequence upstream from the viral protein or functional fragment thereof;
- wherein the viral protein has the HCV E2 sequence of
- THTVGGSAAQTTGRLTSLFDMGPRQKIQLVNTNGSWHINRTALNCNDSLHTGFI ASLFYTHSFNSSGCPERMSACRSIEAFRVGWGALQYEDNVTNPEDMRPYCWHYPPRQC GVVSAKTVCGPVYCFTPSPVVVGTTDRLGAPTYTWGENETDVFLLNSTRPPLGSWFGCT WMNSSGYTKTCGAPPCRTRADFNASTDLLCPTDCFRKHPDTTYLKCGSGPWLTPRCLID YPYRLWHYPCTVNYTIFKIRMYVGGVEHRLTAACNFTRGDRCNLEDRDRS (SEQ ID NO:
- 4);
- and
- wherein the biotinylation site has the amino acid sequence of GLNDIFEAQKIEWHE (SEQ ID NO:
- 2).
An HCV envelope glycoprotein E2 monomer vector construct, said construct comprising:
- a segment encoding an ectodomain of HCV envelope glycoprotein E2;
- a segment encoding a biotinylation site inserted downstream of the segment encoding the glycoprotein,
- a segment encoding a cleavage site, a segment encoding a tag for affinity purification and a reporter gene inserted downstream of the segment encoding the glycoprotein;
- a segment encoding a signal sequence upstream from the glycoprotein;
- and
- wherein the viral protein has the HCV E2 sequence of
- THTVGGSAAQTTGRLTSLFDMGPRQKIQLVNTNGSWHINRTALNCNDSLHTGFIASLFY THSFNSSGCPERMSACRSIEAFRVGWGALQYEDNVTNPEDMRPYCWHYPPRQCGVVSA KTVCGPVYCFTPSPVVVGTTDRLGAPTYTWGENETDVFLLNSTRPPLGSWFGCTWMNS SGYTKTCGAPPCRTRADFNASTDLLCPTDCFRKHPDTTYLKCGSGPWLTPRCLIDYPYR LWHYPCTVNYTIFKIRMYVGGVEHRLTAACNFTRGDRCNLEDRDRS (SEQ ID NO:
- 4).
A tetramer comprising monomer viral proteins having the HCV E2 sequence of
- THTVGGSAAQTTGRLTSLFDMGPRQKIQLVNTNGSWHINRTALNCNDSLHTGFIASLFY THSFNSSGCPERMSACRSIEAFRVGWGALQYEDNVTNPEDMRPYCWHYPPRQCGVVSA KTVCGPVYCFTPSPVVVGTTDRLGAPTYTWGENETDVFLLNSTRPPLGSWFGCTWMNS SGYTKTCGAPPCRTRADFNASTDLLCPTDCFRKHPDTTYLKCGSGPWLTPRCLIDYPYR LWHYPCTVNYTIFKIRMYVGGVEHRLTAACNFTRGDRCNLEDRDRS (SEQ ID NO:
- 4).
Dependencies are inferred from the claim text.