人白介素10重组腺相关病毒血清型2/1杂合载体的构建及转导供肝的效果

Construction of an adeno-associated viral vector serotype 2/1 containing human interleukin-10 and its expression in donor liver

目的:构建含人白介素10(hIL-10)的腺相关病毒血清型2/1杂合载体(AAV2/1),并观察其在供肝移植物中的表达.方法:采用RT-PCR方法从人外周血淋巴细胞中扩增hIL-10cDNA,将其克隆对pMD18-T载体中,测序后,再经酶切定向插入真核表达载体pSNAV,用脂质体介导转染BHK21细胞,经G418筛选得到含ITR—hIL-10-ITR的BHK21细胞株,用携带rep2-cap1基因的辅助病毒(rHSV/r2c1)感染该细胞株.通过细胞孵育、裂解和病毒纯化,得到含hIL-10的AAV2/1载体.在供肝灌洗、保存中应用该重组病毒载体转染供肝移植物,移植后检测其在移植物中的表达.结果:RT-PCR扩增的hIL-10cDNA序列与GenBank中hIL-10cDNA序列一致,构建了rAAV2/1-hIL-10表达载体.转染供肝移植物后可检测到hIL-10,持续24wk表达.移植后24wk,实验组供肝中hIL-10的表达比空白对照组和rAAV2/1-GFP对照组显著增高(219.15±45.83ng/Lvs40.02,38.64ng/L,P<0.05).结论:成功构建了rAAV2/1-hIL-10表达载体系统,并在大鼠供肝中持续表达hIL-10,为IL-10在器官移植中的生物学作用机制及临床应用的深入研究奠定基础.

AIM: To construct an adeno-associated viral vector sero-type 2/1 (AAV2/1) containing human interteukin-10 (hIL-10) gene and to observe its expression in donor liver. METHODS: hIL-10cONA amplified by reverse transcription polymerase chain reaction (RT-PCR) from human peripheral blood mononuclear cells was cloned into vector pMD18-T. After confirming the sequence, hIL-10cDNA was isolated and inserted into eukaryotic expression vector pSNAV. The recombinant plasmid pSNAV-hIL-10 was transfected into BHK21 cells. BHK21 cells which contained ITR-hIL-10-ITR were obtained by G418 screening. Then the cells were transfected with rHSV/r2c1 containing rep2-cap1 gene. The cells were cultured and purified to obtain rAAV2/1 -hIL-10. The expression of hIL-10 gene was detected after this vector was transfer into donor liver in vivo. RESULTS: The sequence of cloned hIL-1 cDNA was identical with that published on GenBank. A new adeno-asso-ciated virus vector containing hIL-10cDNA was constructed. And the transcription and expression of hIL-10 were detected in donor liver for 24 weeks. hIL-10 was significantly expressed in test group than that in empty and rAAV2/1-GFP controls 24 wk after transferred (219.15±45.83 ng/L vs 40.02, 38.64 ng/L, P<0.05). CONCLUSION: The adeno-associated viral vector serotype 2/1 of hIL-10 is successfully established, which provides the basis for applying IL-10 in clinical organ transplantation.