Prokaryotic expressions and polyclonal antibody preparations of duck transporter associated with antigen processing (TAP) 1 and TAP 2
Author(s): ZHANG Xiao-na, WU Shao-lian, WEN Hui-qiang, LIAN Chuan-jiang, CHEN Hong-yan, HAN Ling-xia, Research Team of Laboratory Animal and Comparative Medicine, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences
Pages: 40-43
Year: 2015 Issue: 11
Journal: Heilongjiang Animal Science and Veterinary Medicine
Keyword: duck; transporter associated with antigen processing; peptide binding domain; prokaryotic expression; polyclonal antibody;
Abstract: HBK duck is China’s first specific pathogen free( SPF) duck bred by the national seed center for poultry laboratory animals,which has been closely reared for nine generations.The duck transporter associated with antigen processing( TAP) genes locate in the major histocompatibility complex( MHC) region,and the dimer composed of TAP1 and TAP2,plays an important role on transport of antigenic peptides from the cytosol to the lumen of the endoplasmic reticulum( ER).To express the duck TAP proteins and prepare their specific polyclonal antibodies,the PCR assay was used to amplify the whole gene of TAP1 and the predicted peptide binding domain( PBD) of the TAP2 gene from the B line of HBK-SPF ducks.The PCR products were named as TAP1 and TAP2 PBD,and then were cloned into the prokaryotic expression vector p ET-30a( +) to obtain the recombinant plasmids p ET-30-TAP1 and p ET-30-TAP2 PBD.The recombinant plasmids were transformed into E.coli BL-21( DE3),and then the expressed recombinant proteins were identified by SDS-PAGE and Western blot after IPTG induced expression.The Balb/c mice were immunized with the recombinant proteins from the SDS-PAGE gel slice to prepare polyclonal antibodies.The results indicate that the two kinds of target proteins were correctly expressed.The result indicates that the duck TAP1 and TAP2 polyclonal antibodies with high specificity are prepared,which can be used to study the structure and function of waterfowl TAP.
Pages: 40-43
Year: 2015 Issue: 11
Journal: Heilongjiang Animal Science and Veterinary Medicine
Keyword: duck; transporter associated with antigen processing; peptide binding domain; prokaryotic expression; polyclonal antibody;
Abstract: HBK duck is China’s first specific pathogen free( SPF) duck bred by the national seed center for poultry laboratory animals,which has been closely reared for nine generations.The duck transporter associated with antigen processing( TAP) genes locate in the major histocompatibility complex( MHC) region,and the dimer composed of TAP1 and TAP2,plays an important role on transport of antigenic peptides from the cytosol to the lumen of the endoplasmic reticulum( ER).To express the duck TAP proteins and prepare their specific polyclonal antibodies,the PCR assay was used to amplify the whole gene of TAP1 and the predicted peptide binding domain( PBD) of the TAP2 gene from the B line of HBK-SPF ducks.The PCR products were named as TAP1 and TAP2 PBD,and then were cloned into the prokaryotic expression vector p ET-30a( +) to obtain the recombinant plasmids p ET-30-TAP1 and p ET-30-TAP2 PBD.The recombinant plasmids were transformed into E.coli BL-21( DE3),and then the expressed recombinant proteins were identified by SDS-PAGE and Western blot after IPTG induced expression.The Balb/c mice were immunized with the recombinant proteins from the SDS-PAGE gel slice to prepare polyclonal antibodies.The results indicate that the two kinds of target proteins were correctly expressed.The result indicates that the duck TAP1 and TAP2 polyclonal antibodies with high specificity are prepared,which can be used to study the structure and function of waterfowl TAP.
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