Kentucky State University
Expression and Purification of Peptide Antigens Containing Genetic Adjuvants for the Generation of Antibodies Used in Protein Analysis
Institution
Kentucky State University
Faculty Advisor/ Mentor
Greg Graf; Charles Bennett
Abstract
The novel strategy to be employed was the use of a genetic adjuvant to increase the antigenicity of peptides. We designed an antigen with three major components: a 6 histamine tag; P2/P30 T epitopes of tetanus toxin; and amino acids 1-79 of ABCG1 or 1- 86 of ABCG5. A pET28a bacterial protein expression vector was used which contained a 6-His tag and T7 promoter. Cloning oligos were designed for insertion of the plasmid. We selected the restriction sites and added them to the 5’ and 3’ oligonucleotides, then cloned the G1 and G5 cDNAs encoding the peptide fragment into the EcoRI and HindIII sites of pET28a. The cloned plasmids were transformed into BL21 (DE3) of E.Coli cells, cultured the bacteria, and induced with IPTG to increase protein expression. A western blot was performed and lyced the cells to see whether the protein was in the cells or secreted into the medium. The results indicated that the expressed protein was present in the cells. The purified peptides were shipped to ProSci for rabbit immunization. After eight weeks the immunized rabbits gave raise to antibodies which were sent back for testing. We used immunofluorescence and immunoblot analyses to test the antibodies. The native antigen was recognized in the immune system of the rabbits and useful antibodies were produced.
Expression and Purification of Peptide Antigens Containing Genetic Adjuvants for the Generation of Antibodies Used in Protein Analysis
The novel strategy to be employed was the use of a genetic adjuvant to increase the antigenicity of peptides. We designed an antigen with three major components: a 6 histamine tag; P2/P30 T epitopes of tetanus toxin; and amino acids 1-79 of ABCG1 or 1- 86 of ABCG5. A pET28a bacterial protein expression vector was used which contained a 6-His tag and T7 promoter. Cloning oligos were designed for insertion of the plasmid. We selected the restriction sites and added them to the 5’ and 3’ oligonucleotides, then cloned the G1 and G5 cDNAs encoding the peptide fragment into the EcoRI and HindIII sites of pET28a. The cloned plasmids were transformed into BL21 (DE3) of E.Coli cells, cultured the bacteria, and induced with IPTG to increase protein expression. A western blot was performed and lyced the cells to see whether the protein was in the cells or secreted into the medium. The results indicated that the expressed protein was present in the cells. The purified peptides were shipped to ProSci for rabbit immunization. After eight weeks the immunized rabbits gave raise to antibodies which were sent back for testing. We used immunofluorescence and immunoblot analyses to test the antibodies. The native antigen was recognized in the immune system of the rabbits and useful antibodies were produced.