Amplification And Analysis Of The Whole Genome Of Perv Strain From Guangxi Bama Minipig And Construction Of Its Full-Length Complementary DNA Clone

Using pigs as the transplant source in xenotransplantation has the potential to tackle the severe shortage of human organ donors, and is now considered to be one of the valid measures for treating end-stage organ diseases.Mini pigs in Bama, Guangxi, a locally specific variety which breed by way of inbreeding within enclosed environment, are excellent animals for lab experimentation with genetic stability and can be used as a safe and ideal organ donors in xenotransplantation. However, these pigs usually have porcine endogenous retrovirus (PERV) in vivo and can integrate into the host genome in the form of provirus, and can duplicate with the duplication of the cell chromosome. The discovery that PERV can infect human cells of various kinds has cause wide concern about biosafety of xenotransplantation, so it is necessary to carry out more fundamental research on the PERV in the mini pigs in Bama, Guangxi (PERV-BM), find out its infection mechanism so as to provide information for the evaluation of pathogen safety.In this study, by using the technology of polymerase chain reaction (PCR) and next-generation sequencing (NGS), four of full length cDNA overlapping fragments that cover the whole viral genome were obtained, after which, a full-length cDNA sequence of PERV-BM genome,8774bps in length, was obtained by assembling the3’-end cDNA with the5’-end from genomic DNA. Phylogenetic analysis indicated that PERV-BM may be belong to subtype A viruses. Sequence analysis showed that the promoter and enhancer positions of PERV-BM5’UTR region were located in400-467bps and313-432bps, respectively. Amino acid sequence analysis discovered that15possible antigenic epitopes were occurred in the predicted amino acid sequence of the env protein from the virus, which contained20phosphorylation sites of Ser,7phosphorylation sites of Thr,9phosphorylation sites of Tyr, and10possible glycosylation sites. And the transmembrane domain of this env protein was located in between position600and622amino acid residues. According to the hypothesis of molecular clock, we compared the3’-LTR (long terminal repeats) sequence of the PERV-BM obtained from this study to the other8PERV-BM strains previously amplified by our group, and speculated that the PERV-BM would be originated from7.1×106years ago.Based on the genome sequencing result of this PERV-BM, the full-length cDNA cloning was tructed. All the4cDNA overlapping fragments were amplified by RT-PCR and cloned into the binary vector pBluescript II SK (-) in a certain order with the help of the intermidiate vector plasmid pMD18-T to construct the full-length cDNA clone. Inserts in the positive clones were submitted to DNA sequencing, then submitted to the sequence alignment with all the screened sequences of PERV in GenBank. The results showed that15of conserved base mutations in the target cDNA clone were found. To eliminate the potential impact of the mutations for the next virus rescue, gene site-directed mutagenesis and chemosynthesis had been successfully performed, and those15mutations were restored. The modified PERV-BM was then registered at GenBank, getting the accession number HM159246. For easier identification, we induced site mutation during the cDNA clone construction by replacing the base adenine (A) with cytosine (C) at the8500bps site of PERV-BM using site-specific mutagenesis technology. The single site of restricting enzymes Aat II was also induced into the mutant virus. The sequence of the recombinant plasmid was confirmed to have been constructed as expected by double restricting enzyme digestion Not I/Nhe I, Nhe VCal I, and Not1/Cal I, following the DNA sequencing, and it was named pBluescript II SK (-)-PERV-BM. After that, the study constructed the PERV infectious cDNA clone by using the reverse genetics approaches, and finished the preliminary identification. In sum, the fruitful results of this study will provid a solid foundation for downstream studies, the DNA level study of the transmission mechanism of PERV as well as the viral replication and transcription during post infection in human cells in vitro.