【作者】 阳玉彪；

【导师】 罗廷荣； 章金刚；

【作者基本信息】 广西大学 ， 预防兽医学， 2005， 硕士

【摘要】 猪内源性反转录病毒(PERV)为反转录病毒科(Retroviridae)哺乳动物C型反转录病毒属(Mammalian Type C Retroviridae)成员,它是以前病毒DNA的形式整合进猪细胞基因组中,并随细胞染色体复制而复制的反转录病毒。1997年,Patience等发现PERV在体外可以感染人的多种细胞,引起了人们对PERV与猪-人异种移植病原安全性的广泛关注。 因此,本研究利用本实验室已建立的PERV检测方法对我国小型猪中PERV的存在情况进行调查,旨在筛选培育无PERV猪群,为异种移植提供安全可靠的供体源。我们采集了6个省市10个单位、7个品种、17个猪群259份血样,通过PCR检测发现:我国小型猪基因组中普遍存在PERV,其阳性率达100%。其中以B亚型最高(95.37%),其次为A亚型(67.18%)、C亚型(40.93%),目前为止没有筛选到无PERV的阴性猪个体。PERV主要结构蛋白基因gag、pol、env不仅在小型猪基因组中存在,而且都能够转录为RNA。除了检测不到PERV-C亚型病毒的表达外,大部分A、B亚型病毒都能够检测到表达,且A亚型的表达率高于B亚型。值得注意的是我国小型猪中各种PERV亚型病毒混合存在的现象十分普遍,有78.38%存在2种以上PERV亚型病毒混合感染。 鉴于PERV存在的普遍性与广泛性,筛选培育天然无PERV猪群存在很更多还原

【Abstract】 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.更多还原