A newly cloned pig dolichyl‐phosphate mannosyl‐transferase for preventing the transmission of porcine endogenous retrovirus to human cells
A newly cloned pig dolichyl‐phosphate mannosyl‐transferase for preventing the transmission of porcine endogenous retrovirus to human cells
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DOI：
10.1111/j.1432-2277.2009.00999.x
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发表时间：
2010-04
影响因子：
3.1
通讯作者：
A. Yamamoto;S. Nakatsu;A. Kondo;Tetsuya Asato;M. Okabe;M. Fukuzawa;S. Miyagawa
中科院分区：
文献类型：
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作者：
A. Yamamoto;S. Nakatsu;A. Kondo;Tetsuya Asato;M. Okabe;M. Fukuzawa;S. Miyagawa
猪内源性逆转录病毒(PERV)是临床异种移植成功的主要问题。在我们之前的研究中，减少高甘露糖型N-葡聚糖的含量被证明在降低PERV感染性方面非常有效。本研究对与N-连锁糖合成早期相关的甘露糖酯转移酶D-P-M进行了研究。新分离到猪D-P-M基因的编码区。应用针对D-P-M的RNA干扰(SiRNA)技术，将其导入含有Lac Z基因和PERV-B的猪内皮细胞系PEC(Z)/PB中，以降低高甘露糖型N-糖链的水平。与模型株相比，临时PEC(Z)/PB细胞株对猪D-P-M的表达降低，在Lac Z伪型分析中，每株细胞对人PERV的感染性明显破坏。将h1-RNA基因启动子pSXGH-siRNA导入PEC(Z)/PB。已建立的带有pSXGH-siRNA的PEC(Z)/PB克隆明显地下调了PERV的感染性，这从猪D-P-M的mRNA水平的降低可见一斑。降低D-P-M酶的活性是解决临床异种移植中PERV感染问题的一种潜在的有用方法。
Porcine endogenous retrovirus (PERV) is a major problem associated with successful clinical xenotransplantation. In our previous study, reducing the high mannose type of N‐glycan content proved to be very effective in downregulating PERV infectivity. In this study, dolichyl‐phosphate mannosyltransferase (D‐P‐M), an enzyme related to the early stages of N‐linked sugar synthesis was studied. The pig cDNA of the encoding D‐P‐M was newly isolated. The RNA interference (siRNA) for the D‐P‐M was applied and transfected to PEC(Z)/PB cells, a pig endothelial cell line with the Lac Z gene and PERV‐B, to reduce the levels of high mannose type N‐glycans. Compared with the mock line, the temporary PEC(Z)/PB lines showed a decreased mRNA expression for pig D‐P‐M, and each line then showed a clear destruction of PERV infectivity to human cells in the Lac Z pseudotype assay. The PEC(Z)/PB was next transfected with pSXGH‐siRNA, H1‐RNA gene promoter. The established PEC(Z)/PB clones with pSXGH‐siRNA clearly led to the downregulation of PERV infectivity, as evidenced by the decreased levels of the mRNA for pig D‐P‐M. Reducing D‐P‐M enzyme activity represents a potentially useful approach to address the problem of PERV infections in clinical xenotransplantations.