猪髓样分化因子MyD88特异性shRNA干扰载体的构建筛选及干扰效果评价

doi:10.13523/j.cb.20150701

中国生物工程杂志

2015, Vol. 35

Issue (7): 1-7 DOI: 10.13523/j.cb.20150701

研究报告

猪髓样分化因子MyD88特异性shRNA干扰载体的构建筛选及干扰效果评价

宗鑫, 胡汪洋, 汪以真

浙江大学饲料科学研究所农业部动物营养与饲料重点开放实验室浙江省饲料及动物营养重点实验室杭州 310029

Construction and Evaluation of Porcine Myeloid Differentiation Factor 88(MyD88) shRNA Interference Vector

ZONG Xin, HU Wang-yang, WANG Yi-zhen

Institute of Food Science, Ministry of Agriculture Key Laboratory of Animal Nutrition and Feed Science, Zhejiang Provincial Laboratory of Feed and Animal Nutrition, Zhejiang University, Hangzhou 310029, China

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摘要：

髓样分化因子MyD88(myeloid differentiation factor 88)信号通路是一个具有多种调节功能的传导通路,在免疫反应、炎症反应及肿瘤的发生和发展过程中均发挥重要作用。构建猪(Sus scrofa)MyD88基因的shRNA干扰载体,并在转录水平和蛋白质表达水平对其干扰效果进行验证,以筛选出干扰效果最优的干扰载体。根据猪 MyD88 基因(GenBank登录号:KC766424.1)全长cDNA序列,利用Invitrogen公司在线设计软件设计出4对shRNA干扰序列,退火成双链后,分别将其插入到pYr-1.1载体中,构建 MyD88 基因的shRNA真核表达载体pYr-1.1-pigMyD88-sh1、pYr-1.1-pigMyD88-sh2、pYr-1.1-pig MyD88-sh3、pYr-1.1-pigMyD88-sh4,并通过双酶切和测序对其进行鉴定。构建成功后转染猪肺泡巨噬细胞3D4/2,通过Real-time PCR及Western blot验证 MyD88 基因的表达水平,以及对LPS刺激后炎症因子TNF-α基因表达水平的影响。结果表明,所构建的猪 MyD88 基因的特异性shRNA表达载体均可显著降低猪MyD88 mRNA和蛋白质的表达水平(P<0.05),干扰效率分别达到36%、67%、60%、69%;相比于未干扰组,LPS刺激MyD88沉默之后的巨噬细胞,炎症因子TNF-α基因表达水平显著下降(P<0.05),表明所构建猪MyD88干扰载体干扰效果较好。

关键词： 猪; 髓样分化因子MyD88; 载体构建; 干扰效果

Abstract:

The myeloid differentiation factor 88 (MyD88) signaling pathway with a variety of regulatory functions, plays a critical role in the occurrence and development of inflammation and tumor immune response. The purpose is to construct a shRNA interference vector of MyD88 gene for porcine, and verify the effect of interference at the level of transcription and protein expression, in order to choose a optimal interference vector. Four pairs of shRNA oligonucleotide sequences were designed and synthesized according to MyD88 gene sequence of pig in GenBank (Accession: KC766424.1) and then were annealed into double-strand and inserted into the plasmid of pYr-1.1 respectively to construct expression vectors, pYr-1.1-pigMyD88-sh1, pYr-1.1-pigMyD88-sh2, pYr-1.1-pigMyD88-sh3, pYr-1.1 -pigMyD88-sh4. After DNA sequencing, four shRNA expression vectors were transfected into pig alveolar macrophage cells. The interference effect of these vectors was confirmed by detecting the expression of MyD88 with Western blot, Real-time PCR and the TNF-α gene expression in MyD88-slienced macrophages after LPS stimulation. The results show that MyD88 shRNA vectors were successfully constructed, and the specific shRNA of porcine MyD88 gene could significantly depressed the expression of porcine MyD88 at the level of mRNA and protein (P<0.05), with interference effects respectively reaching 36%, 67%, 60%, 69%. In contrast to the control group, the gene expression of TNF-α was decreased significantly in MyD88-slienced macrophages after LPS stimulation. These results indicated that the interference effect of MyD88 shRNA vectors was great. These data will provide insight in the role of MyD88, and the basic information to explore the mechanism of immune response related diseases in pigs.

Key words: Porcine Myeloid differentiation factor 88 (MyD88) Construction of expression vector Interference effect

收稿日期: 2015-03-23 出版日期: 2015-07-25

ZTFLH:

Q782

基金资助:

国家自然科学基金面上资助项目(31172213)

通讯作者: 汪以真 E-mail: yzwang321@zju.edu.cn

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引用本文:

宗鑫, 胡汪洋, 汪以真. 猪髓样分化因子MyD88特异性shRNA干扰载体的构建筛选及干扰效果评价[J]. 中国生物工程杂志, 2015, 35(7): 1-7.

ZONG Xin, HU Wang-yang, WANG Yi-zhen. Construction and Evaluation of Porcine Myeloid Differentiation Factor 88(MyD88) shRNA Interference Vector. China Biotechnology, 2015, 35(7): 1-7.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150701 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I7/1

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