Construction of the Porcine Endogenous Retrovirus Envelope Glycoprotein A and B Specific Antibody

Xenotransplantation using porcine organs could potentially associate with the risk of pathogenic infections, because human tropic porcine endogenous retrovirus (PERV) particles could be released from pig cells or organs. While there is no evidence of PERV transmission to human, safety issues become a paramount concern. For the prevention of this transmission, specific immunological tools must be provided for PERV transmission detection. In this study we described the expression of PERV envelope proteins and the production of a specific antibody against PERV envelope (Env) glycoprotein. The nucleotide sequence harboring the partial region of glycoprotein 70 was cloned into the pET vector and envelope protein was expressed in E. coli. Approximately 42 kDa recombinant Env protein (PERV Env-aa357) was purified by the Ni-affinity column. For antibody production, mice were immunized with the recombinant PERV Env-aa357. The generated anti-serum was tested using Western blot and immunocytochemical assay. We found that anti-PERV Env serum displayed the specificity against the PERV Envs (PERV-A and PERV-B) expressed not only in E. coli but also in mammalian cells, and PERV particles within the porcine cell lines (PK 15 and PK-1). Taken together, PERV antibody could be useful for detecting PERV infection or xenotransplantation transmission.

Molecular Characterization of Porcine Endogenous Retrovirus gag Genes from Pigs in Korea

Xenotransplantation, as a potential solution to the shortage of human organs, is associated with a number of concerns including immunologic rejection and xenogenic infection. While the pigs are considered the most suitable organ source for xenotransplantation, there is a potential public health risk due to zoonosis. Among the known porcine zoonotic microbes, Porcine Endogenous Retrovirus (PERV) is the most considerable virus. PERV belongs to the Gammaretrovirus and has been divided into three groups (A, B, and C). To characterize the gag of PERVs, we isolated the genomic DNAs from three pig breeds (Birkshire, Duroc, and Yorkshire) and two types of SPF miniature pigs. About 1.5 kb fragments covering full length of gag were amplified and cloned into T-vector. A total of 38 clones were obtained and sequenced. Nucleotide sequences were analyzed and phylogenetic trees were constructed from the nucleotide and deduced amino acids. PERV-A, -B and -C were present in the proportion of 47, 19 and 34%, respectively. Regardless of origin or subgroups, gag clones showed highly homology in nucleotide and deduced amino acid sequences. Deduced amino acids sequence alignments showed typical conserve sequences, Cys-His box and processing sites. Among analyzed clones, about 28% of isolates had the correct open reading frame. To test the functional expression of Gag protein, gag was subcloned into expression vector and confirmed its expression in HeLa cell. This research provides the fundamental information about molecular characteristics of gag gene and functional Gag protein related xenotropic PERVs.