长白与青海八眉“二元杂交”猪种群PERV亚型分析

Analysis of subtypes of porcine endogenous retrovirus in hybrids of Landrace with Qinghai Bamei swine

目的研究长白与青海八眉"二元杂交"猪种群携带的内源性逆转录病毒(porcine endogenous retrovirus,PERV)亚型,为评价从猪到人异种移植的生物安全性提供依据。方法从长白与青海八眉"二元杂交"猪种群中随机采集33头个体外周血,应用PCR和RT-PCR技术分别检测PERV前病毒DNA和mRNA,研究该种群携带PERV的亚型,并对PCR扩增的灵敏性进行评估。克隆测序该猪种PERVenv基因,结果用NCBI中的BLAST软件进行分析。结果所检测的33头个体均带有PERV前病毒DNA,外周血中均有PERV mRNA表达,其中,69.7%(23/33)个体携带env-A,env-B和env-C三种囊膜蛋白基因,而其余的30.3%(10/33)个体只带有env-A和env-B两种囊膜蛋白基因,env-C基因缺失。灵敏性分析实验结果表明,PCR扩增PERV时,DNA模板不能少于15ng。克隆env-A,env-B和env-C基因,结果提交GenBank(GenBank登录号分别为DQ856326、DQ856327、DQ856328)。生物信息分析结果表明,该猪种env基因与GenBank登录其他猪种序列相比存在差异,其中env-A、env-B基因发生移码突变,蛋白合成提前终止。结论长白与青海八眉"二元杂交"猪种群携带PERV,69.7%个体为PERV-ABC亚型;缺失突变使PERVenv-A,env-B基因阅读框发生改变,有可能影响其感染特性,从生物安全性方面综合考虑,该猪种不宜作为异种移植供体。

To investigate the subtypes of porcine endogenous retrovirus （PERV） in hybrids of Landrace with Qinghai Bamei swine, so as to provide basic parameters to evaluate the biological safety of xenotransplantation of pig to human, the peripheral blood samples from 33 individuals were randomly collected from hybrids of Landrace with Qinghai Bamei swine, and PCR and RT-PCR were used to detect the provital DNA and mRNA of PERV respectively. The sensitivity of the PCR assay was evaluated using a positive control, and the PERV env gene was amplified, sequenced and analysed by using the BLAST software in National Center of Biotechnology Information （NCBI）. The results showed that the provital DNA and mRNA of PERV could be detected in all these 33 individuals by means of PCR and RT-PCR, in which 69.7% （23/33） of individuals tested carried the envelop protein genes envA, envB and envC , while the other 30.3% of individuals carried only envA and envB, but no envC. It was demonstrated that the quantity of DNA to be detected by PCR amplification of the PERV genes was not less than 15 ng. These PERV envA, envB and envC genes were sequenced and submitted to GenBank with GenBank accession numbers of DQ856326, DQ856327, DQ856328 respectively. The information analysis showed that there were significant differences in env genes comparing with other breeds in GenBank, in which frameshift mutation occurred in envA and envB genes, thus causing premature stop of protein synthesis. It is concluded that from the aspect of biological safety, this breed is not suitable as donor in xenotransplantation, because 69.7% of individuals are the subtypes of PERV-A, B,C, in which frameshift mutation may occur, thus changing the character of the cross-species transmmision of PERV.