梅花鹿Fibulin 5编码区cDNA的克隆及功能研究

cDNA coding region cloning and functional analysis of Fibulin 5 in sika deer

为研究梅花鹿扣针蛋白5(Fibulin 5, FBLN5)在鹿茸再生过程中的作用,本研究通过RT-PCR获得了梅花鹿FBLN 5的cDNA编码区,并对其进行生物信息学分析。同时利用RNA干扰技术(RNA interference, RNAi)下调了梅花鹿角柄骨膜致敏区(Potentiated pedicle periosteum, PPP)细胞中FBLN5基因表达,并研究了基因表达下调后PPP细胞条件培养液对HUVEC细胞增殖的影响。结果显示:(1)梅花鹿FBLN5 cDNA编码区长1 347 bp,编码448个氨基酸;(2)生物信息学软件分析显示,梅花鹿FBLN5蛋白含有一个跨膜区,一段23个氨基酸的信号肽序列,vWA_Matrilin、cEGF和EGF_CA等重要的功能结构域;(3)构建了梅花鹿FBLN5基因的RNAi重组质粒并获得了一条能有效下调目的基因效率达88.72%的靶序列pLVTHM-FBLN2,MTT结果显示PPP细胞FBLN5基因表达量下调后,所获细胞条件培养液可显著促HUVEC细胞增殖,推测FBLN5可能参与了鹿茸再生过程中的血管生成及稳态维持等过程。

The cDNA coding region of Fibulin 5(FBLN 5) of sika deer was cloned to investigate the function of FBLN5 in antler regeneration RT-qPCR was used to analyze the sequence and using bioinformatic tools RNAi vectors of FBLN5 were constructed to down regulate the FBLN5 gene expression in PPP cells. Conditioned medium both from down regulated and negative control groups were made and added to HUVEC cells to test the biofuntion of FBLN5 in PPP cells by MTT assay. The results revealed that:(1) The length of cDNA of FBLN5 is 1 347 bp, which encodes a protein of 272 amino acids.(2)Bioinformatics predicts that FBLN5 is a hydrophilic protein with one transmembrane region and a signal peptide including 23 amino acids. FBLN5 has three kinds of domains: vWA_Matrilin, cEGF and EGF_CA. We predicted the tertiary structure of sika deer FBLN5.(3)The RNAi sequence pLVTHM-FBLN2 was most effective.It down regulated the expression of FBLN5 in PPP cells up to 88.72%. MTT assay revealed that the proliferation of HUVEC cells was stimulated by up to 35.7% as cultured with whole conditioned medium of pLVTHM-FBLN2 group in construct with negative control group. We concluded that FBLN5 may be involved in angiogenesis and homeostasis in the process of antler regeneration.