Stable Co-expression Of A2 Haplotype Duck TAP1 And TAP2 Proteins In A Eukaryotic System

The transporter associated with antigen processing(TAP) belongs to the ABC(ATP-binding cassette) family of transporters, TAP is a heterodimeric protein consisting of TAP1 and TAP2,which are close linkage on major histocompatibility complex(MHC) molecules, TAP is located on MHC I class molecules as to different animals, but exact location of TAP on MHC molecules is different. TAP translocates peptides from the cytosol into the lumen of the endoplasmic reticulum(ER), during which four chaperones are necessary, they are calreticulin, calnexin, tapasin and the oxidoreductase ERp57, then these peptides will bind to MHC molecules, formed MHC-peptide complexes are displayed on the cell through the Golgi apparatus, induced cytotoxic T cell immune response. The absence and mutation of TAP protein will affect the presentation of antigenic peptides, ultimately will impair the immune response.In this study, TAP1 and TAP2 were amplified from duck spleen by RT-PCR, sequence analysis showed that TAP2 is polymorphic higher than TAP1. TAP2 genotype of breeding the third generation haplotypes duck was monitored about genetic stability, the results showed that MHC genotype of haplotype duck remained stable. After sequence alignment, the number of SNP of A2 strain is at most, then TAP1 and TAP2 proteins of A2 haplotype duck were analyzed with bioinformatics.The coding region of full-length TAP1 and TAP2 peptide binding regions of A2 haplotype duck were amplified by RT-PCR from duck spleen and cloned into p ET-30 a vector, prokaryotic expression vector p ET-A2TAP1 and p ET-A2TAP2 PBD were successfully constructed and transformed into E. coli BL21(DE3), the two recombinant proteins were expressed by IPTG and two specific bands(51 k Da and 22 k Da) were obtained in the form of inclusion body. In order to prepare and purify mouse polyclonal antiserum, six-week-old female BALB/c mice were immunized by recombinant protein.The coding region of full-length TAP1 and TAP2 of A2 strain were cloned into the eukaryotic expression vector p IRES, the eukaryotic expression plasmid p IRES-A2TAP1-A2TAP2 was successfully constructed and transfected into RMA-S cell using Amaxa Cell Line Nucleofector Kit L by electric transfection. The cells were selected under G418, ultimately stable co-expression A2TAP1 protein and A2TAP2 protein RMA-S cell line were obtained and named RMA-S-A2TAP1-A2TAP2. The expression of A2TAP1 and A2TAP2 gene in the purified 15 th generation cell line were identified by RT-PCR and indirect immunofluorescence assay(IFA) and the results indicated that the recombinant proteins A2TAP1 and A2TAP2 were co-expressed in RMA-S cells, and the two proteins had immunological activities.The study will provide material conditions for the study of TAP biological functions at the cellular level.