Designation and silencing function of the small interfering RNA of HLA-A2

doi:10.3969/j.issn.2095-4344.2014.29.003

Abstract

Abstract:

BACKGROUND: Human leukocyte antigen (HLA), the major histocompatibility complex of human, plays an important function in the transplant rejection. Decreasing the expression of HLA will prolong the survival time of transplants.

OBJECTIVE: To design small interfering RNA (siRNA) of HLA-A2 and to detect the effect of siRNA-HLA-A2 on the expression of HLA-A2.

METHODS: Four kinds of siRNA-HLA-A2 domains were designed, and recombinant lentivirus expression vector were formed. The 293T cells, highly expressing HLA-A2, were infected in vitro. Then the knockout efficacy of four domains was detected to select the highly efficient siRNA-HLA-A2 target sequences. The human embryo lung fibroblasts were cultured in vitro and infected with the lentivirus carrying the target sequence. The infecting efficiency of LV-siRNA-HLA-A2 was observed under the fluorescence microscope and the silence function of this siRNA in human embryo lung fibroblasts was detected by western blot analysis.

RESULTS AND CONCLUSION: According to the mRNA sequence of HLA-A2 in Genbank, three siRNAs were designed and synthesized. In vitro, the over expression of HLA-A2 in 293K cells was successfully silenced. The HLA-A2 expression in human embryo lung fibroblasts was also efficiently silenced after the human embryo lung fibroblasts were infected by the highly efficient siRNA of HLA-A2. The efficacy was up to 80%.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: HLA antigens, RNA interference, gene silencing, fibroblasts

CLC Number:

R318

Liu Jian-sheng, Zhao Gang, Pei Dan, Zheng De-yu. Designation and silencing function of the small interfering RNA of HLA-A2[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(29): 4605-4610.

Figures/Tables 1

2.1 HLA-A2基因克隆和表达载体的构建和表达在5个PCR扩增的温度中，均得到HLA-A2 cDNA的目的片段，长度约1 100 bp，其中64 ℃扩增效率最好(图1)。切取64 ℃所代表的目的条带进行玻璃奶回收，回收的PCR产物与T载体经EcoRⅠ酶切后，经T4 DNA连接酶连接获得pGEM-T easy-HLA-A2，序列测定显示与Gene bank中的序列完全一致。将pGEM-T easy-HLA-A2与pEGFP-C1载体应用BamHⅠ和EcoRⅠ双酶切后，玻璃奶回收目的片段，经T4 DNA连接酶连接获得重组载体。pEGFP-HLA-A2转化感受态细胞，提取质粒酶切鉴定表明融合蛋白表达载体pEGFP-HLA-A2构建成功，测序结果表明HLA序列完全正确。 2.2 pEGFP-HLA-A2在体外的表达在Lipofectamine 2000介导下，pEGFP-HLA-A2和pEGFP-C1均能高效的转染293T细胞。收集细胞总蛋白，用HLA-A2抗体进行Western Blot检测，结果表明pEGFP-HLA-A2转染293T细胞能表达HLA-A2蛋白，而转染pEGFP-C1空载体则无HLA-A2的表达(图2)。 2.3 表达siRNA慢病毒载体的构建和病毒包装退火的siRNA双链序列全长64 bp，接入pGCL-EGFP载体用PCR鉴定可扩增出预期的360 bp片段，pGCL空载体扩增产物为300 bp(图3)。测序结果表明插入的siRNA序列完全正确。表达siRNA分子的慢病毒在包装细胞能表达绿色荧光蛋白。48 h后在倒置荧光显微镜下检测绿色荧光，通过对比不同稀释度的假病毒颗粒溶液对细胞的感染效率，得知经离心浓缩的病毒滴度>108/mL。 2.4 不同靶点siRNA干扰效率检测在HLA-A cDNA质粒转染293T细胞的基础上，加入适量表达不同靶点siRNA干扰病毒溶液，用Western blot检测HLA的表达水平，结果表明，1号和2号靶点病毒的敲减效率较好(图4)。本实验对含2号靶点的病毒载体进行大包装和纯化浓缩。对比观察不同MOI值的病毒溶液对人胚肺成纤维细胞的感染情况，发现MOI值为20时细胞状态良好，病毒感染效率在80%以上。 2.5 慢病毒感染人胚肺成纤维细胞人成纤维细胞感染72-96 h后荧光显微镜下观察，可见感染率达到80%以上(图5)。在感染1周后，收集细胞，提取细胞总蛋白，经Western blot检测显示，感染siRNA-HLA-A2后，人成纤维细胞中HLA-A2的表达在有的组几乎检测不到，有的仅能看到淡淡的条带(图6)。将Western blot结果进行灰度扫描，应用HLA-A2的条带灰度除以内参照β-actin条带的灰度，可以得出HLA-A2的相对表达量，可见HLA-A2在siRNA-HLA- A2感染人胚肺成纤维细胞中的表达量仅为0.041±0.005，不足正常培养细胞(0.139±0.021)或感染siRNA阳性对照组(0.143±0.018)的30%，差异有显著性意义(P < 0.01)，而正常培养的细胞和阳性对照组之间则差异无显著性意义。说明siRNA-HLA-A2感染后能有效的抑制HLA-A2的表达。"

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