Liposome-mediated IL-28 and IL-29 expression in A549 cells and antiviral effect of IL-28 and IL-29 on WISH cells
Liposome-mediated IL-28 and IL-29 expression in A549 cells and antiviral effect of IL-28 and IL-29 on WISH cells
复制标题
DOI：
10.1111/j.1745-7254.2006.00292.x
复制
发表时间：
2006-04-01
影响因子：
8.2
通讯作者：
He, SH
中科院分区：
文献类型：
--
作者：
Li, MC;Wang, HY;He, SH
目的：目的构建白细胞介素（IL）-28 A、IL-28 B和IL-29的重组表达载体，并在人A549细胞中表达，分析其对水泡性口炎病毒（VSV）感染的人永生化羊膜上皮细胞系（WISH细胞）的抗病毒活性。方法：从经poly I：C活化的人外周血单个核细胞（PBMC）中提取细胞总RNA。通过逆转录聚合酶链反应（RT-PCR）扩增编码人IL-28 A、IL-28 B和IL-29的cDNA，并插入pcDNA3.1/ V5-His-TOPO载体中。将这些重组载体通过脂质体介导的基因转移方法转染入人A549细胞。采用半定量RT-PCR和Western blotting检测IL-28 A、IL-28 B和IL-29的mRNA和蛋白表达。使用VSV作为攻击病毒，通过对WISH细胞的细胞病变效应减少测定来确定IL-28 A、IL-28 B和IL-29的抗病毒活性。结果：克隆的IL-28 A、IL-28 B和IL-29基因序列与GenBank中已发表的IL-28 A、IL-28 B和IL-29基因序列完全一致。结果表明，IL-28 A、IL-28 B和IL-29基因在转染细胞中显著转录。通过Western印迹分析证实所有3种白细胞介素在A549细胞中的表达。由A549细胞表达的IL-28和IL-29，如干扰素（IFN）α-2b，能够保护WISH细胞免受VSV感染。结论：成功克隆了IL-28和IL-29的cDNA，并将其转染真核细胞进行表达。用该载体转染产生了与IFN-α相似的特异性抗病毒活性，这将为这些细胞因子在人体中的功能研究提供新的工具。
Aim: To construct the recombinant vectors carrying interleukin (IL)-28A, IL-28B and IL-29 cDNAs and express them in human A549 cells, and analyze their antiviral activity in vesicular stomatitis virus (VSV)-infected human immortalized amnion epithelial cell line (WISH cells). Methods: Total cell RNA was extracted from human peripheral blood mononuclear cells (PBMC) activated with poly I:C. The cDNAs encoding human IL-28A, IL-28B, and IL-29 were amplified by reverse-transcription polymerase chain reaction (RT-PCR) and inserted into pcDNA3.1/ V5-His-TOPO vectors. These recombinant vectors were transfected into human A549 cells by a liposome-mediated gene transfer method. Semiquantitative RT-PCR and Western blotting were used to detect the mRNA and protein expression of IL-28A, IL-28B, and IL-29. The antiviral activity of IL-28A, IL-28B, and IL-29 was determined by a cytopathic effect reduction assay on WISH cells using VSV as a challenge virus. Results: The DNA sequences of the inserts were identical to the published sequences encoding IL-28A, IL-28B, and IL-29 in GenBank. It was demonstrated that IL-28 A, IL-28B, and IL-29 genes were markedly transcribed in transfected cells. Expression of all 3 interleukins in A549 cells was confirmed by Western blot analysis. IL-28 and IL-29 expressed by A549 cells, like interferon (IFN) alpha-2b, were able to protect WISH cells against VSV infection. Conclusion: IL-28 and IL-29 cDNAs were successfully cloned and expressed in eukaryotic cells via transfection with pcDNA3.1/V5-His-TOPO-IL-28/IL-29. Transfection with this vector produced a specific antiviral activity similar to that of IFN-alpha, which will provide a new tool for the functional study of these cytokines in humans.