【摘要】：通過熒光素酶實(shí)驗(yàn)驗(yàn)證宿主CD276為日本血吸蟲mir-125b的靶基因,并分析感染血吸蟲后宿主CD276基因在不同組織中的表達(dá)情況。利用PCR擴(kuò)增mir-125b與CD276互作的核酸序列,并將目的片段克隆至表達(dá)熒光素酶報(bào)告基因質(zhì)粒的下游。將重組質(zhì)粒轉(zhuǎn)染到HEK293T細(xì)胞內(nèi),檢測熒光素酶報(bào)告基因表達(dá)。進(jìn)一步利用熒光定量PCR技術(shù)檢測小鼠巨噬細(xì)胞在轉(zhuǎn)染血吸蟲mir-125b mimic后,靶基因CD276的表達(dá)情況,同時(shí)檢測感染血吸蟲不同時(shí)間段、小鼠不同組織中CD276的表達(dá)情況。熒光素酶實(shí)驗(yàn)表明轉(zhuǎn)染mir-125b mimic和重組質(zhì)粒后細(xì)胞的熒光素酶報(bào)告基因的表達(dá)極顯著降低(P0.01),提示mir-125b可與CD276相關(guān)區(qū)域互作,抑制報(bào)告基因的表達(dá);血吸蟲mir-125b mimic轉(zhuǎn)染小鼠巨噬細(xì)胞后導(dǎo)致其宿主CD276基因的表達(dá)降低,進(jìn)一步提示宿主CD276可能為血吸蟲mir-125b靶基因;熒光定量PCR分析表明感染日本血吸蟲后的不同感染時(shí)期,小鼠淋巴細(xì)胞、肝臟及脾臟中的CD276的表達(dá)水平顯著低于對(duì)照組(P0.01),進(jìn)一步表明蟲源性mir-125b在體內(nèi)影響宿主CD276基因的表達(dá)。日本血吸蟲mir-125b可能靶向調(diào)控宿主CD276基因的表達(dá),在蟲體與宿主互作中發(fā)揮重要調(diào)控功能。
[Abstract]:The host CD276 is the target gene of the mir-125b of Schistosoma japonicum by the luciferase experiment, and the expression of the host CD276 gene in different tissues after the infection of the schistosome is analyzed. The nucleic acid sequence interacting with the CD276 was amplified by PCR and the target fragment was cloned downstream of the expression luciferase reporter plasmid. The recombinant plasmid was transfected into the HEK293T cell to detect the luciferase reporter gene expression. The expression of the target gene CD276 was detected by the fluorescence quantitative PCR technique, and the expression of CD276 in different tissues of the mice was detected. Luciferase experiments show that the expression of the luciferase reporter gene of the transfected mir-125b mimomic and the recombinant plasmid is significantly reduced (P0.01), suggesting that the mir-125b can interact with the relevant region of the CD276 to inhibit the expression of the reporter gene; The expression of the host CD276 gene was reduced after the mouse macrophages were transfected with the schistosome mir-125b momic, and the host CD276 could be the target gene of the mir-125b of the SCHISTOSOMA. The fluorescence quantitative PCR analysis showed that the mouse lymphocytes, The expression of CD276 in the liver and spleen was significantly lower than that in the control group (P0.01), and the expression of the host CD276 gene in the host was further indicated. The Schistosoma japonicum mir-125b may target the expression of the host CD276 gene, and play an important role in the interaction of the worm and the host.
【作者單位】： 天津農(nóng)學(xué)院;中國農(nóng)業(yè)科學(xué)院上海獸醫(yī)研究所農(nóng)業(yè)部動(dòng)物寄生蟲學(xué)重點(diǎn)實(shí)驗(yàn)室;
【基金】：國家自然科學(xué)基金(31502056和31672550) 中國農(nóng)業(yè)科學(xué)院科技創(chuàng)新工程
【分類號(hào)】：S852.7

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