The goal of the current study is to prepare polyclonal antibody against bovine intestinal peptide transporter I (bPepTI) in order to develop assay for immunological assessment of protein levels. Antigenicity of the en...The goal of the current study is to prepare polyclonal antibody against bovine intestinal peptide transporter I (bPepTI) in order to develop assay for immunological assessment of protein levels. Antigenicity of the entire bPepTI was analyzed with DNAStar, and a fragment with high antigenicity (bPepTI ORF 1369 - 1695) was selected, cloned in pGEX-6p-1 vector, resulting in a recombinant plasmid GST-BP, which verified by double enzyme digestion and sequenced, the recombinant plasmid was introduced to BL21. Exogenous expression was induced by IPTG and validated by Western blot analysis. The recombinant protein was isolated, purified and used for production of antiserum in mice. The specificity of antiserum was evaluated with immunobloting and titer was estimated with ELISA. Results indicate that the antibody against bPepTI was produced. The optimal GST-BP antigen embedding concentration was 0.5 μg/ml. The optimal dilution was 1:400. An indirect ELISA assay indicates the effective dilution was 1:102400.展开更多
文摘The goal of the current study is to prepare polyclonal antibody against bovine intestinal peptide transporter I (bPepTI) in order to develop assay for immunological assessment of protein levels. Antigenicity of the entire bPepTI was analyzed with DNAStar, and a fragment with high antigenicity (bPepTI ORF 1369 - 1695) was selected, cloned in pGEX-6p-1 vector, resulting in a recombinant plasmid GST-BP, which verified by double enzyme digestion and sequenced, the recombinant plasmid was introduced to BL21. Exogenous expression was induced by IPTG and validated by Western blot analysis. The recombinant protein was isolated, purified and used for production of antiserum in mice. The specificity of antiserum was evaluated with immunobloting and titer was estimated with ELISA. Results indicate that the antibody against bPepTI was produced. The optimal GST-BP antigen embedding concentration was 0.5 μg/ml. The optimal dilution was 1:400. An indirect ELISA assay indicates the effective dilution was 1:102400.
