| As we all know, donor organs are most deficient in clinical therapies. People are fixing their eyes on heterotransplantation, in which porcines are the most suitable donor up to now. However, PERV, integrating into the genome of porcines by the form of pre-virus DNA, could infect human-derived cells in vitro. As a result, the safety of heterotransplantation was on the agenda. China is rich in minipig resources and a lot of trials on heterotransplation are carried out from porcines to human beings although the infectivity, expression and biological characteristic of PERV in porcins are rarely reported. As a result, its most necessary in order to understand the PERV existence situation, privide safe and reliable organs for transplantation, construct cell-infected models and genome clone for further PERV biological characteristics and infectivity mechanism. Our research work includes the following:(1) Existance status of PERV in minipigs specific in China259 blood saples were collected including ten units from six provinces and detected by PCR. The results indicated that PERV were generic in all minipigs with possitive rate of 100%, among those were subtype B(95.37%), then subtype A(67.18%)and then subtype C(42.08%). No PERV negative individuals were found up to now. The main structure proteins including gag, pol, and env, could be transcriped into RNA. The expression rate of subtype A was higher than B, while no expression was detected of subtype C. What's special in Chinese minipigs was that combination of PERV infection was generic with a rate of 78.38%, which may suggested a possibility of rearrangement of various viruses.(2) HEK293 cell models infected with PERVBased on HEK293 cell strains with G418 resistance, we co-cultured the cells and PBL of WZS, BMXZ, BM and GZ minipigs and screened by G418. Systematic evaluation was executed on the cell models by PCR and RT-PCR after removel of PBL. We finally constructed 3 HEK293 cell models infected by PERV, and verified the infectivity of PERV from porcine PBL to human-derived cells in vitro, which helped in PERV biological characteristics and pathogen safety research..(3) Whole genome sequencing of PERV-WZSWe'd sequenced the genome of PERV derived from WZS miniature pig, Bama miniature pig and HEK293 cell strains using PCR and RACE technologies. The length of the genome was 8639bp, 8595bp and 8689bp respectively. They belong to subtype A viruses unanimously. Genetic evolution analysis showed that PERV-WZS was the closer to PERV-A subtype than to C and B subtype. The promoter and enhancer position of PERV-WZS 5'UTR region, analyzed by sequence comparison and bioinformatics, was located in -67—+1 and -97—-59. PERV-Env protein was supposed to have 18 possibleantigen epitopes and 7 possible glycosylated sites. Homology between HEK293 cell-derived 293-PERV-WZS and PERV-WZS was 94.6%, and the major differences were fixed in 5' UTR and gag gene. The 5'UTR of PERV-BM was obviously different from that of PERV-WZS, which may be resulted from a relative short period of evolution. (4) Constrution the Full-length cDNA cloning of PERV-WZS genomeFive pairs of overlapping primers were designed to amplify the cDNA fragments of the full length of PERV-WZS genome by RT-PCR based on the known genome sequence. The subsequent fragments were cloned into pGEM-T and pMD18-T for connection in order, and then cloned into low copy number rector plasmid vector pWSK29 to form pWSKWZS. We thus established the basis for cDNA cloning of PERV infectivity, and for the mechanism research of PERV infectivity and integration into the genomes.
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