Related Research Of Porcine Endogenous Retrovirus

As a kind of laboratory animal, the pig is being used more and more in the medical experiments. However, the porcine endogenous retroviruses' (PERV) can infect human cells in vitro, so it raised wide concerns regarding the pathogen safety of pig to human xenotransplantation and biologies from pig's cell or tissue.It's essential to establish a method for detection of PERV. So that, three pair primers were synthesized according to the sequence of gag, pol and env gene of PERV. Polymerase chain reaction (PCR) assays were performed for detection of PERV in genomic DNA using above primers. We also performed reverse transcription PCR (RT-PCR) for detection of PERV specific mRNA. The results showed that PERV DNA and PERV mRNA were existed in all of the four tested cell lines. The sizes of amplified fragments are identical with predicted. These methods may be suitable for monitoring-the PERV existence in DNA and expression of mRNA in culture cells.In order to investigate the existence and expression of PERV in miniature swine of China, we synthesized three pairs of specific primer (gag, pol, env) and three pairs of type-specific primer (A, B, C), then using the PCR and RT-PCR methods, we detected the PERV of DNA and RNA samples derived from the peripheral blood lymphocytes. The results showed that the PERV existed and expressed in all samples, and the expression of PERVbelonged to PERV-A and PERV-B, but the expression of PERV-C wasn't detected in allsamples. This research may be helpful to develop the miniature swine of China and to assessthe pathogen safety of PERV.The previous reports showed the envelope gene contains three subtype. For the sake of this, we cloned and analysised the envelope gene of porcine endogenous retrovirus from theminiature swine of China. Total RNA was extracted from the peripheral blood lymphocyte of miniature swine by TRIzol Reagent , the envelope gene was amplified using the RT-PCR method, then cloned into pGEM-T vectors. Sequence analysis shows that we have obtained the 2004bp proper fragment containing a integrity open reading frame(ORF) that is made up of 1983 base pair and coding 660 amino acids. The envelope gene of PERV was cloned successfully, which is facility to produce the recombination protein for further research.In order to obtain a model of the HEK293 cells infected by PERV and further research the biological characters of the PERV, we firstly have established the G418-resislant HEK293 cell line. The plasmid pIRESneo was transfected into the HEK293 cells with the lipofectin, then eliminated the untransfected cells using the G418 selective culture medium. Since the cells transfected by the neo gene express G418-resistant products, they can grow in the G418 selective culture medium. The identification of the G4I8 resistant HEK293 cell was conducted by polymerase chain reaction with the specific primers of neo gene. The result showed that the G418-resistant HEK293 cell line was established successfully. Then the G418-resistant HEK293 cell and PK-15 cell were co-cultured in a system, after 6 weeks, the PK-15 cell in co-culture system was eliminate.d using the G418 selective culture medium. The infected HEK293 cells were identified using above methods. The results showed that the PERV can integrated in HEK293 cell genome and can transferred into mRNA of PERV. We also performed another PCR with the species-specific primers for pgt34 intron of porcine a-l,3-galactoyltransferase gene. No specific fragment was amplified in infected HEK293 cell, but the control cell of PK-15 can amplified specific fragment. These demonstrate that the PK-15 cell was eliminated completely in co-culture system. These results showed that we have established a model of HEK293 cell infected by porcine endogenous retrovirus.