本文選題：豬小腸上皮細胞 + 白細胞分化抗原基因(CD)��； 參考：《農(nóng)業(yè)生物技術(shù)學(xué)報》2016年03期

【摘要】：白細胞分化抗原14(cluster of differentiation antigen 14,CD14)在先天性免疫和適應(yīng)性免疫反應(yīng)中都發(fā)揮重要調(diào)控作用,本研究旨在構(gòu)建豬(Sus scrofa)CD14基因的短發(fā)夾RNA(short hairpin RNA,sh RNA)干擾載體,包裝成慢病毒,轉(zhuǎn)染豬小腸上皮細胞,在細胞水平上進行相關(guān)功能探討。本研究參照CD14基因(Gen Bank登錄號:EF626695.1)全長編碼區(qū)序列,設(shè)計4個靶向豬CD14基因的sh RNA干擾序列,將其克隆至慢病毒表達載體質(zhì)粒中,分別命名為p GLV3-CD14-1、p GLV3-CD14-2、p GLV3-CD14-3和p GLV3-CD14-4,以及陰性對照p GLV3-CD14-NC。包裝成功后轉(zhuǎn)染豬小腸上皮細胞系IPEC-J2,利用q RTPCR檢測其干擾效率。選擇干擾效率最高的慢病毒進行藥篩,獲得CD14基因穩(wěn)定沉默的豬小腸上皮細胞系。大腸桿菌(Escherichia coli)黏附實驗檢測大腸桿菌F18ab和F18ac對CD14基因沉默小腸上皮細胞黏附能力的變化。結(jié)果表明,本研究成功構(gòu)建4個sh RNA表達載體,包裝成的慢病毒均能降低豬CD14m RNA表達水平,其中p GLV3-CD14-3慢病毒的干擾效果最好,其干擾效率達到94.6%;CD14基因沉默后F18ab對小腸上皮細胞的黏附能力顯著增強,而F18ac雖然也有上調(diào),但并未產(chǎn)生顯著性差異。研究結(jié)果表明,CD14基因沉默后的小腸上皮細胞對大腸桿菌F18ab的黏附能力顯著增強,CD14基因可能在小腸上皮細胞抵御大腸桿菌F18ab感染過程中發(fā)揮了重要的調(diào)控作用。慢病毒介導(dǎo)的CD14沉默的豬小腸上皮細胞系的建立,不僅為在細胞水平研究CD14基因功能提供了有效模型,也為進一步探究其所在的Toll樣受體/白介素-1受體(toll-like receptors/interleukin-1 receptor,TLRs/IL-1R)信號通路在豬腸道病原微生物引起的免疫反應(yīng)和抵御病原入侵的調(diào)控機制奠定了基礎(chǔ)。
[Abstract]:Leukocyte differentiation antigen 14 (cluster of differentiation antigen 14, CD14) plays an important role in both congenital and adaptive immune responses. The aim of this study is to construct the short hairpin RNA (short hairpin RNA) of the swine (Sus scrofa) CD14 gene (short hairpin RNA), which is packaged into lentivirus, transfected to pig small intestinal epithelial cells, and in cell water. On the basis of the CD14 gene (Gen Bank login number: EF626695.1) full length coding region, 4 sh RNA interference sequences targeting the CD14 gene of swine were designed and cloned into the lentivirus vector plasmid, named P GLV3-CD14-1, P GLV3-CD14-2, P, and negative controls, respectively. After the LV3-CD14-NC. package was successfully transfected to the pig small intestinal epithelial cell line IPEC-J2, the interference efficiency was detected by Q RTPCR. The sieves with the highest interference efficiency were selected to obtain the stable and silent pig small intestinal epithelial cell line of the CD14 gene. The adhesion test of Escherichia coli (Escherichia coli) was used to detect the silencing of the CD14 gene of the Escherichia coli F18ab and F18ac. The changes in the adhesion ability of small intestinal epithelial cells showed that 4 sh RNA expression vectors were successfully constructed in this study. The packaged lentivirus could reduce the expression level of CD14m RNA in pigs. The interference effect of P GLV3-CD14-3 lentivirus was the best and the interference efficiency reached 94.6%, and the adhesion ability of F18ab to small intestinal epithelial cells after the CD14 gene was silent was significant. The results showed that the adhesion ability of small intestinal epithelial cells to Escherichia coli F18ab was significantly enhanced after the CD14 gene silencing, and the CD14 gene may play an important role in preventing the F18ab infection of Escherichia coli in the intestinal epithelial cells. The CD14 gene may play an important role in the F18ab infection of the Escherichia coli. The slow virus mediated CD1. The establishment of the 4 silent pig intestinal epithelial cell line not only provides an effective model for the study of CD14 gene function at the cellular level, but also further explores the immune response and counterpart of the Toll like receptor / interleukin -1 receptor (Toll-like receptors/interleukin-1 receptor, TLRs/IL-1R) signaling pathway in the pig intestinal pathogenic microorganism. The regulatory mechanism of the invasion of the pathogen has laid the foundation.
【作者單位】： 揚州大學(xué)動物科學(xué)與技術(shù)學(xué)院;揚州大學(xué)獸醫(yī)學(xué)院;江蘇省種豬繁育和健康養(yǎng)殖工程技術(shù)研究中心;
【基金】：國家自然科學(xué)基金(No.31572360和No.31372285) 轉(zhuǎn)基因生物新品種培育科技重大專項(No.2014ZX08006-001B) 江蘇省科技支撐計劃(No.BE2015329和No.BE2014357) 江蘇高校優(yōu)勢學(xué)科建設(shè)工程資助項目(PAPD)
【分類號】：S852.6;Q78

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