Expression And Indection Of Human HLA-DRB1*0901 On Porcine Kindey Cell

The increasing success in allotransplantation has generated a severe shortage of organs available for transplantation. The alternative to using animals as organ donors has focused on the pig as a source for xenegrafts. Although interspecies incompatibilities exist, pig organs are considered to be most suited for xenotransplantation because of similarities in size and physiology of pigs and humans.Because of interspecies incompatibilities, there will happen pig-to-human xenograft rejection, including hyperacute rejection, acute vascular rejection, and chronic rejection. Hyperacute rejection is mediated by nature antibody existed in recipient, which react with the galactosylα1,3-galactose (αGal) epitopes on the endothelium of the graft. With the exception of humans, apes, and Old World monkeys, almost all mammalian cells express galactosylα1,3-galactose antigen. Acute vascular rejection is medicated by anti-Gal and anti-non Gal antibodies. There is little knowledge about chronic rejection, but it has been demonstrated that the porcine MHC molecules can effectively induce a strong human T-cell response, through direct or indirect antigen recognition. Many researches were done on overcoming xenotransplantation immuno-rejection, such as, knocking outαGal antigen of procine cells, genetic modifying the pig organs for human complement regulator, and using immunosuppress drugs. Researchers of Taiwan University Hospital have produced the human HLA DPw0401 transgenic pig and found HLA DPw0401 molecule can attenuate human-to-pig xenogenic cellular response. The genetic loci involved in the rejection of foreign organs are known as the major histocompatibility complex (MHC).The human MHC is called human leukocyte antigen(HLA) which is divided into HLA classⅠ,classⅡand classⅢ. The HLA classⅠmolecules include HLA-A,-B,-C,-E,-F,-G,etc. The HLA classⅡmolecules include DR,DP,DQ. The HLA classⅢmolecules include C2,C4A,C4B which is irrelevant with HLA. The HLA molecules play an important role at regulating immunoreaction.The aim of this study is to investigate the expression of human HLA-DRB1 molecule on pig kidney cell. This research will lay foundation for further investigation the role of HLA-DRB1 on xenograft immunological rejection.First part: identified HLA-DRB1*09Objective: To identify sample whose HLA-DRB1 genotype is 09.Methods: The samples'genomic DNA was extracted by salting-out method and typed by PCR-SSO and PCR-SBT.Result: Individual whose HLA-DRB1 genotype is 09 was found.Conclusion: HLA typing methods can quick and accurately identify HLA genotype. The individual's HLA-DRB1 locus is 090102/010101.Second part: Construction and identification of the eukaryotic co-expression vector of human HLA-DRB1*0901 and HLA-DRA with pVITRO2 Objective: To construct the eukaryotic co-expression vector of human HLA-DRB1*0901 and HLA-DRA with Pvitro2 and identify its expression in kidney cell of porcine(LLC-PK1).Methods: RT-PCR was used to amplify the HLA-DRB1*0901 and HLA-DRA cDNA from total RNA of leukocyte in peripheral blood. The HLA-DRB1*0901 DNA fragment(digested with BglⅡ) was inserted into the multiple cloning site 1 of vector pVITRO2. after identified its direction, the HLA-DRB1*0901 DNA fragment(digested with BamHⅠ) was inserted into the multiple cloning site2 of vector pVITRO2-dra. Then, the co-expression vector pVITRO2-dra-drb was transfected into the LLC-PK1, and western-blotting was used to identified its expression.Results: The restriction endonucleases digestion and PCR suggested the co- expression vector pVITRO2-dra-drb was constructed successfully. Western-blotting identified the protein HLA-DRB1. Conclusion: The co-expression vector pVITRO2-dra-drb can express human HLA-DRB1*0901 in LLC-PK1.