Detection Of Porcine Endogenous Retrovirus In Chinese Minipigs And Construction Of The Full-Length CDNA Of Wzs Minipig Porcine Endogenous Retrovirus Genome

Porcine endogenous retrovirus (PERV) is a member of Mammalian Type C Retroviridae, integrating into the genome of porcines by the form of previrus DNA and replicating with the replication of cell chromosome. In 1997, Patience's research confirmed PERV could infect human-derived cells in vitro. As a result, the safety of heterotransplantation was on the agenda.So we detected PERV in Chinese minipigs by the method established by our laboratory, aimed at understanding the situation of PERV existing in Chinese minipig, sifting and breeding minipig groups without PERV, providing safe and reliable organs for transplantation. 259 blood samples were collected including ten unit in six provinces and detected by PCR.The results indicated that PERV was universal in all minipigs with positive rate of 100%, including 95.37% subtype B, 67.18% subtype A and 40.93% subtype C. We didn't find PERV negative individuals up to now. The main structure proteins of PERV including Gag, Pol and Env, could not only exist in the genome of minipig, but also be transcriped into RNA. The expression rate of subtype A was higher than subtypeB, while the expression of subtype C was not detected. What'special was that multiple infection was universal with a rate of 78.38%.Being the universality of PERV existing in Chinese minipig, It is difficult to sift and breed minipig groups without PERV. However, reverse genetics brought a new era for RNA virus research. It was a technique to rescue RNA virus from cDNA clone, through which the genetic manipulation on the genome of RNA virus was realized. Its core techniques included cloning the full-length cDNA of virus genome and preparing infectious transcripts. While the full-length cDNA clone of virus genome was key step.Therefore, we might find out method eliminating PERV infection by researching PERV molecular biology character using reverse genetics.To this day, 10 full-length cDNA clones of PERV had been constructed, most through establishing cDNA library, which was time-consuming and laborious. This research tried to establish RT-PCR to amplify PERV-WZS genome, construct the full-length cDNA of PERV-WZS genome, thus to make some basis for constructing the infectious cDNA clone of PERV-WZS, and for studying of mechanism on PERV infection.With the optimization of the components of reaction buffer and amplification parameter, RT-PCR for PERV genome amplification was finally set up. Using the techniques, cDNA fragments ranging from 1 kb to 2.5 kb could be amplified. Then the full-length cDNA clone of PERV-WZS was constructed by gene recombination in vitro.To prove the integration of the full-length cDNA clone, primers were devised to amplify 5 fragments that covered the whole genome of PERV-WZS. The fragments with the length ranging from 500 bp to 3500 bp could be amplified, with the length as expected. To identify whether there are absence, insert or not in the constructed full-length cDNA clone of PERV-WZS,we sent fragments containing coding regions for sequencing.The result showed that there were no absence and insert in the constructed full-length cDNA clone. To identify whether the devised external sequences were introduced into the constructed full-length cDNA clone or not, 5'end and 3'end of the constructed full-length cDNA clone were sequenced. The sequencing showed that the devised external sequences were introduced into he constructed full-length cDNA clone.In a word, the full-length cDNA clone of PERV-WZS was constructed correctly. This research will make some basis for constructing the infectious cDNA clone of PERV-WZS and studying the mechanism on PERV infection.