Screening of siRNA Targeted for TLR9 Gene and Its Effect on the Proliferation Profile of BHV-1

The aim of this study was to investigate the role of Toll-like receptors 9 (TLR9) of the bovine embryonic tracheal (EBTr) cells in the innate immune response mediated by bovine herpesvirus type 1(BHV-1). Using small interfering RNA (siRNA) technology, three siRNA interference sequences target for TLR9 were designed and synthesized in this study. After siRNA-TLR9 interference, the expression levels of TLR9 gene were detected by Real-time PCR. After 48 h, the expression levels of TLR9 mRNA induced by siRNA-TLR9A, siRNA-TLR9B and siRNA-TLR9C were reduced to 60.90%, 24.05% and 40.75%, respectively. Comparing with the control group, the expression levels of TLR9 mRNA were significantly inhibited by siRNA-TLR9A fragments at 12 to 72 h (P < 0.05), and after transfecting the best fragments and infecting with BHV-1, the BHV-1 DNA copy numbers of siRNA-TLR9A group were lower than BHV-1 DNA of the control group at 6 to 72 h. The specific siRNA fragments target for TLR9 were successfully screened out in this test, and demonstrated that inhibition of TLR9 expressions could reduce the BHV-1 proliferation in EBTr cells.     

BHV-1在牛胚气管细胞中的增殖规律研究

为研究牛疱疹病毒Ⅰ型(BHV-1)在牛胚气管细胞(EBTr)中的生长特性和增殖规律,参考文献设计合成特异性引物和探针,建立了TaqMan实时荧光定量PCR方法,检测BHV-1感染EBTr细胞6、12、24、48、72、96、120、144h后病毒的增殖规律,并观察对应时间点的细胞病变(CPE)。结果显示,用100TCID_(50)的BHV-1感染EBTr细胞,48h后开始出现细胞病变,72h细胞病变明显,120h细胞大部分变圆,开始脱落,144h细胞大面积脱落、崩解。实时荧光定量PCR检测结果表明,BHV-1感染EBTr在6h~24h内,病毒缓慢增殖,48h~96h,病毒增殖速度加快,拷贝数呈对数增长,120h~144h,BHV-1的含量仍然呈现升高的趋势,但增长速度变慢。结果证明,BHV-1能够在EBTr中产生CPE,而且CPE程度与病毒DNA增殖规律一致,该结果可以为深入研究BHV-1对EBTr细胞的致病机理提供基础资料。     

实时荧光定量PCR筛选牛源整联蛋白αv基因的siRNA片段

设计并合成三条针对牛口蹄疫病毒(FMDV)整联蛋白受体αv亚基基因的特异性干扰小RNA(SiRNA)片段,将其转染至本身表达整联蛋白受体αv亚基基因的MDBK细胞.分别在转染后36 h和48 h收集细胞,提取细胞总RNA并反转录为cDNA.应用SYBR GreenI实时荧光定量RT-PCR的方法检测3个siRNA片段的...     

蒙古马不同组织器官TLR1、TLR2、TLR4和TLR6 mRNA转录水平研究

根据GenBank中马Toll样受体基因序列设计特异性引物,建立检测马Toll样受体（TLRs）mRNA转录水平的SYBR GreenⅠ实时荧光定量PCR方法,检测TLR4、TLR2、TLR1和TLR6在蒙古马不同组织器官中的转录水平。4种TLRs在心脏、肝脏、脾脏、肺脏、肾脏、胃、十二指肠、空肠、盲肠和骨髓中均有转录。其中,TLR4mRNA除在空肠和肝脏外,在其他组织器官中的表达水平均高于TLR2、TLR1、TLR6。免疫器官中,TLR4、TLR1mRNA在骨髓中表达量高于脾脏,而TLR2、TLR6mRNA在脾脏中表达量高于骨髓。各肠段,TLR4、TLR2、TLR1、TLR6mRNA表达水平之间在空肠的差异不是很大,而在十二指肠和盲肠中差异很大。结果表明,TLRs mRNA在马各组织器官转录水平差异较大,可能与其对病原体的识别和抵抗能力有关。     

BHV-1感染牛巨噬细胞对CD300a转录的影响

为探究抑制性受体CD300a在牛Ⅰ型疱疹病毒(bovine herpesvirus 1,BHV-1)免疫调控机制中的作用,分离鉴定了1株BHV-1内蒙古株,并建立CD300a基因的荧光定量PCR方法,检测BHV-1感染牛单核巨噬细胞后0,6,12,24,48,72 h 6个时间点以及空白对照组CD300a基因的转录变化。将β-actin基因作为内参,使用双标准曲线法计算攻毒组与对照组CD300a基因各时间段的相对表达量,分析CD300a在攻毒前、后的表达差异。结果显示,BHV-1分离株在MDBK细胞上出现典型细胞病变效应,测得其病毒滴度为10^7.23 TCID₅₀/mL。本研究建立的CD300a基因荧光定量PCR检测方法,CD300a基因和β-actin基因标准品的Ct值与模板浓度之间均具有良好的线性关系,溶解曲线均为特异单峰,最低检测限均为10拷贝/μL,具有良好的灵敏性,可以准确检测出BHV-1攻毒后巨噬细胞内的CD300a基因表达量。经计算,攻毒组CD300a表达量均高于对照组,其中,6,12,24 h的表达差异倍数显著高于0,72 h...     

AP-1及其siRNA对心肌细胞中TGF-β_1的影响

采用RNA干扰方法研究转录因子AP-1对TGF-β1的转录调控影响及其对心肌细胞的影响。设计合成两对AP-1基因的siRNA-59和siRNA-60,同时合成与AP-1基因序列无关的siRNA-58,试验设置阴性对照、阳性对照组及干扰组(AP-1-58、AP-1-59和AP-1-60)共5组,使用LipofectantineTM2000转染AP-1siRNA入培养的大鼠心肌细胞,倒置显微镜观察细胞形态;采用实时荧光定量PCR法检测AP-1和TGF-β1在心肌细胞中mRNA水平表达量的变化。结果显示,siRNA-58对AP-1的基因沉默(表达下调)无显著差异(P0.05),siRNA-59和siRNA-60对AP-1的基因沉默(表达下调)有显著差异(P0.05),同时对应siRNA-59和siRNA-60组的TGF-β1基因有显著性下调(P0.05),说明AP-1和TGF-β1的基因表达存在正相关。     

靶向狂犬病病毒N基因shRNA抑制狂犬病病毒复制的研究

为探讨小干扰RNA(siRNA)对狂犬病病毒(RV)复制的干扰作用,以RVN基因为靶向目标,设计合成4对编码siRNA的DNA序列,然后分别连接到载体pRNATU6.3-Hygro上,转染BHK细胞,在潮霉素-B抗性压力下筛选出4个阳性细胞株(BHK-N1、BHK-N2、BHK-N3和BHK-N4)。用100TCID50CVS-11毒分别感染24孔板内的上述细胞,利用Real-time RT-PCR、半数细胞培养物感染量(TCID50)、直接免疫荧光和Western blot检测抑制效率。接毒后48 h,在BHK-N1、BHK-N2、BHK-N3、BHK-N4细胞株中,Real-time RT-PCR检测到各细胞株均在不同程度上抑制了RV的增殖,其中BHK-N2、BHK-N1抑制效率高,分别为99%、67.72%,而BHK-N3、BHK-N4仅为38.59%和11.33%,抑制效果较弱;TCID50检测病毒数量比空载体表达细胞毒价分别低24、96.2、2.14和1.1倍;直接免疫荧光检测表明,4株细胞中RV含量为病毒对照的31%、1%、54%、82%,即BHK-N1、BHK-N2对病毒的沉默作用较强;Western blot结果显示BHK-N1、BHK-N2细胞株中RV N蛋白条带明显减弱。4种检测结果均表明BHK-N1和BHK-N2能有效干扰RV的复制。本研究在细胞水平筛选出具有高效抑制RV复制的2个靶位点N-489和N-701,为RV的基因功能研究、抗病毒药物的开发奠定了基础。     

雏鸡不同组织TLR1、TLR2、TLR4、TLR5和TLR15 mRNA转录水平相对定量研究

参考GenBank登录的ChTLRs基因序列设计实时定量PCR特异性引物,建立检测鸡Toll样受体(ChTLR)mRNA相对转录水平的实时定量PCR方法,分析ChTLR1、ChTLR2、ChTLR4、ChTLR5和ChTLR15在雏鸡不同器官组织中的转录水平。结果显示5种ChTLRs在脾脏、法氏囊、胸腺和各段肠道组织中均有转录。其中,ChTLR1 mRNA在法氏囊、肾脏和盲肠组织中转录水平较高;ChTLR2 mRNA在脾脏、法氏囊和肝脏等组织中转录水平较高,在肾脏、肺脏和皮肤未检测到转录;ChTLR4 mRNA在所检测组织中转录水平差异较小,在脾脏、十二指肠和胸腺转录水平较高;ChTLR5 mRNA在肾脏、脾脏和空肠中的转录水平较高;ChTLR15 mRNA在法氏囊中转录水平最高,其次为脾脏和盲肠。本研究建立了检测ChTLRs mRNA在不同器官组织中表达水平的实时定量PCR方法,ChTLRs mRNA在雏鸡各器官组织中转录水平差异较大,可能与雏鸡各器官组织对病原的识别和抵抗能力有关。     

酿酒酵母细胞壁可通过TLR2受体调控绵羊瘤胃上皮细胞表达SBD-1

旨在研究酿酒酵母细胞壁对体外培养绵羊瘤胃上皮细胞β-防御素-1(sheep beta-defensin-1,SBD-1)表达的影响及其调控途径,为揭示酿酒酵母细胞壁对机体免疫的调控机制提供理论依据。本研究将不同质量浓度的酿酒酵母细胞壁提取物(0,25,50,100,200,400mg/L)作用于绵羊瘤胃上皮细胞不同时间(0,2,4,8,12,24h)后,检测上皮细胞SBD-1和Toll样受体-2(TLR2)mRNA表达水平,并进一步通过TLR2阻断试验来证实TLR2是否介导酵母细胞壁促进SBD-1表达。结果表明,不同浓度的酵母细胞壁刺激瘤胃上皮细胞时,随着细胞壁浓度的增加SBD-1mRNA表达量呈先升高后降低的趋势,且酿酒酵母细胞壁质量浓度为200mg/L刺激12h时,SBD-1 mRNA表达量达到峰值,与对照组和其他时间组相比差异显著(P0.05);用质量浓度为200mg/L的酿酒酵母细胞壁刺激绵羊瘤胃上皮细胞不同时间,同样在12h时,TLR2的mRNA表达量达到最大;阻断试验表明TLR2可介导SBD-1的表达。本研究结果表明酿酒酵母细胞壁可促进绵羊瘤胃上皮细胞SBD-1的表达,且质量浓度200mg/L时,刺激瘤胃上皮细胞12h时SBD-1的表达量达到最高,而TLR2参与该调控过程。     

DF-1细胞中TLR7编码基因的敲除及其抗FAdV-4感染的天然免疫应答

旨在构建高质量的禽源细胞系,应用CRISPR/Cas9技术实现鸡胚成纤维细胞(DF-1)的Toll样受体7(TLR7)编码基因的敲除,构建稳定的细胞培养禽腺病毒4型(FAdV-4)增殖体系。采用荧光定量PCR的方法对FAdV-4感染DF-1细胞后先天性免疫信号通路中效应分子进行检测,选择转录水平显著上调的基因TLR7作为研究对象;构建sgRNA载体与Cas9表达载体共转染DF-1细胞,经绿色荧光蛋白(GFP)阳性特征分选单克隆,PCR验证及增毒试验筛选后获得TLR7编码序列敲除的DF-1-TLR7-KO#3单克隆细胞系,命名为DF-1-TLR7-KO细胞。对敲除前后DF-1细胞的天然免疫系统对FAdV-4感染的拮抗效应和病毒在不同细胞中增殖效率的差异进行了研究。结果表明：成功构建的TLR7编码序列敲除的DF-1细胞系,与原始细胞相比病毒增殖能力提高。试验对细胞的天然免疫系统与FAdV-4感染的互作机理进行了初步探索,为更好地研究宿主对病毒入侵的应答机制,提高疫苗生产效能提供了研究基础和生物材料储备。     

Establishment of the Quantitative Real-time PCR Method for PRRSV Nsp9 Gene Rapid Detection and Its Expression in PRRSV Infected Cells

The study was conducted to establish a quantitative Real-time PCR method for PRRSV Nsp9 gene rapid detection and study its expression in PRRSV infected cells.A pair of specific primers targeted to Nsp9 of PRRSV was designed and a Real-time PCR method based on SYBR Green Ⅰ fluorescent was developed for the quantization of PRRSV.The melting curve analysis using SYBR Green Ⅰ dye showed one specific peak,and no primer dimers peak was observed.No amplification was detected from HEV,SIV and PRV samples by this method.There was good reproducibility and low variation coefficient.The quantitative Real-time PCR method developed in this study would be useful for rapid laboratory diagnosis and epidemiology investigation of PRRSV.During the process of virus infecting cells,the expression level of Nsp9 increased gradually,and it got the highest at 36 h.This study laid the theoretical basis to explore the law of virus replication and clinical vaccine production.     

Nsp9基因实时荧光定量PCR检测方法的建立及其在感染细胞过程中表达量的变化

试验旨在建立猪繁殖与呼吸综合征病毒（porcine reproductive and respiratory syndrome virus,PRRSV）Nsp9基因的实时荧光定量PCR检测方法,并探究其在感染细胞过程中表达量的变化。根据PPRSVNsp9基因序列设计引物,建立基于SYBR Green Ⅰ检测模式的实时荧光定量PCR。结果显示,PRRSV Nsp9基因在1×10⁴~1×10⁸拷贝/μL范围内有很好的线性关系,扩增产物的熔解曲线只有1个单特异峰,无引物二聚体。该方法与猪戊肝病毒（HEV）、猪流感病毒（SIV）、伪狂犬病病毒（PRV）基因组均无交叉反应,可重复性好,组内变异系数小,可用于临床PRRSV检测。在病毒感染细胞过程中,Nsp9基因表达量逐步升高,36 h达到最高。本研究为探究病毒复制规律与临床疫苗生产奠定了理论基础。     

Toll样受体7基因敲除对水疱性口炎病毒增殖的影响

【目的】 探究敲除Toll样受体7（Toll-like receptor 7,TLR7）基因对水疱性口炎病毒（Vesicular stomatitis virus,VSV）复制的影响。【方法】 利用慢病毒介导的CRISPR/Cas9基因编辑技术构建TLR7基因敲除的稳定猪肾上皮细胞（porcine renal epithelial cells,PK15）系。通过构建pTLR7-sgRNA重组质粒并转染至HEK293T/17细胞,收获慢病毒并感染PK15细胞,经嘌呤霉素筛选后得到PK15-TLR7^-/-多克隆细胞,并在Western blotting鉴定后通过有限稀释法获得PK15-TLR7^-/-单克隆稳定细胞系。为验证敲除TLR7基因稳定细胞系是否构建成功,分别利用光学显微镜和细胞毒性检测（cell counting kit 8,CCK-8）观察并检测PK15、PK15-TLR7^-/-细胞的形态与活力;利用荧光倒置显微镜和流式细胞术观察VSV-GFP感染PK15、PK15-TLR7^-/-细胞后病毒增殖情况;利用Western blotting和实时荧光定量PCR分别检测VSV-GFP感染PK15、PK15-TLR7^-/-细胞后GFP蛋白和VSV-N基因mRNA水平的表达情况;利用病毒滴度检测VSV-GFP感染PK15、PK15-TLR7^-/-细胞后子代病毒产生情况。【结果】 采用CRISPR/Cas9基因编辑技术构建的3种sgRNA均对TLR7进行了有效的编辑,其中sgRNA2的基因编辑效率最高,但敲除TLR7基因并不影响PK15细胞的形态及活力;当感染VSV-GFP后,通过荧光显微镜观察发现,荧光强度随着时间逐次增加,且PK15-TLR7^-/-细胞的GFP荧光强度强于PK15细胞。流式细胞术检测结果显示,相同时间点的PK15-TLR7^-/-细胞感染VSV-GFP的比例显著或极显著高于PK15细胞（P<0.05;P<0.01）;实时荧光定量PCR结果表明,感染4~36 h VSV-N基因mRNA相对表达量随着时间逐渐增加,且PK15细胞中VSV-N基因的mRNA相对表达量显著或极显著低于PK15-TLR7^-/-细胞（P<0.05;P<0.01）;Western blotting结果表明,PK15与PK15-TLR7^-/-细胞中的VSV-GFP GFP蛋白均从6 h开始表达,表达量随时间逐渐增多,且在PK15-TLR7^-/-细胞中VSV-GFP GFP蛋白含量比PK15细胞更高;感染VSV-GFP 6 h后子代病毒开始释放,此时PK15细胞中VSV-GFP子代病毒滴度低于PK15-TLR7^-/-细胞（P>0.05）,但PK15细胞中VSV-GFP子代病毒滴度随着感染时间的增加显著或极显著低于PK15-TLR7^-/-细胞（P<0.05;P<0.01）。【结论】 TLR7基因的敲除可以促进VSV在PK15细胞中的复制,初步验证了TLR7在天然免疫中的作用,为VSV等RNA病毒性疾病的防控提供新思路。     

猪TLR4基因在F18大肠杆菌抗性型和敏感型资源群体间的差异表达分析

本研究旨在检测TLR4基因mRNA在猪各个组织的分布以及在F18大肠杆菌抗性型和敏感型群体间差异表达水平,为探讨该基因在免疫识别和F18大肠杆菌抗性中发挥的作用提供理论依据。本试验运用SYBR GreenⅠ实时荧光定量PCR技术进行定量测定。首先,各取8头35日龄F18大肠杆菌抗性型和敏感型苏太仔猪组织样,包括心脏、肝脏、脾脏等11个组织,提取总RNA,以GAPDH基因为内参基因,进行实时荧光定量PCR。对TLR4基因mR-NA进行均一化处理,利用荧光阈值(Ct值)计算TLR4基因mRNA在各个组织以及F18大肠杆菌抗性型和敏感型群体间表达量。结果显示,TLR4 mRNA在猪各种组织中广泛表达,在肺、淋巴结、肾脏和脾脏等免疫器官中表达量较高,F18大肠杆菌敏感型的TLR4基因表达量普遍高于抗性型个体的表达量,在各个组织中(除胃外)TLR4表达量差异倍数从1.083到2.980不等;在肺、淋巴结、肾脏和胸腺组织中,F18大肠杆菌敏感型的TLR4基因表达量显著高于抗性型个体的表达量(P<0.05)。本研究结果表明,TLR4基因通过天然免疫识别机制对猪F18大肠杆菌病等革兰氏阴性疾病有一定的影响,TLR4基因表达量...     

GDF9对绵羊卵丘细胞增殖的影响

本试验旨在探究生长分化因子9（GDF9）对卵丘细胞扩展相关基因和激素受体基因表达量及激素分泌的影响,为GDF9在绵羊卵泡发育中的作用提供依据。以绵羊卵丘细胞为研究对象,通过在低血清细胞培养液中添加不同浓度（0、50、100、200、400 ng/mL）的GDF9,培养绵羊卵丘细胞48 h后,提取细胞总RNA,利用实时荧光定量PCR技术,以β-actin为内参基因,检测卵丘细胞扩展相关基因透明质酸合酶2（HAS2）、前列腺素内过氧化物合酶2（PTGS2）、穿透素3（PTX3）及激素受体相关基因卵泡刺激素受体（FSHR）、促黄体生成素受体（LHR）和雌激素受体（E₂R）的mRNA相对表达量;利用酶联免疫吸附法（ELISA）测定培养液中卵丘细胞分泌的雌二醇（E₂）和孕酮（P₄）含量。结果显示：在细胞培养液中添加200 ng/mL GDF9时,HAS2、PTX3、FSHR、E₂R和LHR的mRNA相对表达量极显著高于对照组与其他处理组（P<0.01）;PTGS2 mRNA相对表达量极显著高于对照组、50和400 ng/mL GDF9组（P<0.01）,显著高于100 ng/mL GDF9组（P<0.05）。当添加400 ng/mL GDF9时,各基因mRNA相对表达量均极显著低于200 ng/mL GDF9组（P<0.01）;E₂分泌量极显著高于对照组与50 ng/mL GDF9组（P<0.01）,显著高于100 ng/mL GDF9组,与200 ng/mL GDF9组差异不显著（P>0.05）。100、200和400 ng/mL GDF9组P₄分泌量显著高于对照组（P<0.05）,与50 ng/mL GDF9组没有显著差异（P>0.05）,且3组之间差异不显著（P>0.05）。综上所述,GDF9能够促进绵羊卵丘细胞扩展,并参与绵羊卵丘细胞激素分泌的调控。     

Effect of GDF9 on Sheep Cumulus Cell Proliferation

The purpose of this study was to investigate the effect of growth differentiation factor 9 (GDF9) on the gene expression of cumulus cells expansion and hormone receptors as well as hormone secretion,in order to provide evidence for the role of GDF9 in the development of sheep cumulus cells.Sheep cumulus cells were used as the research object in this study,and were cultured for 48 h by adding different concentrations (0,50,100,200,400 ng/mL) GDF9 to low serum cell culture medium.Total RNA were extracted from the cells,using β-actin as the reference gene,Real-time quantitative PCR technology were used to detect the cumulus cells expansion related genes hyaluronic acid synthase gene 2 (HAS2),prostaglandin lead oxide synthase 2 (PTGS2),pentraxin 3 (PTX3) and hormone receptor genes follicle-stimulating hormone receptor (FSHR),luteinizing hormone receptor (LHR) and estrogen receptors (E₂R).Using the enzyme-linked immunosorbent assay (ELISA) method to test the content of E₂ and P₄.The results showed that HAS2,PTX3,FSHR,E₂R and LHR mRNA relative expression of 200 ng/mL GDF9 group was extremely significantly higher than the control group and other GDF9 groups (P<0.01),PTGS2 mRNA relative expression was extremely significantly higher than the control group and 50,400 ng/mL GDF9 groups (P<0.01),and significantly higher than 100 ng/mL GDF9 group (P<0.05).When added 400 ng/mL GDF9,the relative mRNA expression of all the mentioned-above genes were all extremely significantly lower than that of the 200 ng/mL GDF9 group.Moreover,the E₂ secretion level was extremely significantly higher than that of the control group and 50 ng/mL GDF9 group (P<0.01),significantly higher than that of the 100 ng/mL GDF9 group(P<0.05),while had no significant difference from the 200 ng/mL GDF9 group (P>0.05).When added 100,200 and 400 ng/mL GDF9,the concentration of P₄ was significantly higher than the control group (P<0.05),and there was no significant difference from the 50 ng/mL group (P>0.05),and there was no significant difference between 100,200 and 400 ng/mL GDF9 groups (P>0.05).To sum up,GDF9 could promote the expansion of sheep cumulus cells and participated in the regulation of hormone secretion of sheep cumulus cells.     

Bovine herpesvirus type 1 (BHV-1) and bovine viral diarrhoea virus (BVDV) infections in dairy herds: self clearance and the detection of seroconversions against a new atypical pestivirus

The epidemiology of bovine herpesvirus type 1 (BHV-1) and bovine viral diarrhoea virus (BVDV) was studied in a population of small dairy herds that had not been vaccinated. Bulk tank milk samples of 186 herds in Thailand were collected four times between 2002 and 2004. Serum samples from individual animals in 11 herds were also taken on three occasions. The prevalence of BHV-1 in the 186 herds was 61% in 2002, decreasing to 48% in 2004 and for BVDV was 91% in 2002, decreasing to 72% in 2004. A BVDV antigen-positive calf was found in one of the 11 herds, and animals in this herd and three other herds seroconverted to a recently described atypical BVDV strain (HoBi). This study showed a significantly decreasing prevalence for both BHV-1 and BVDV due to a self-clearance process. Further studies are needed to find out how the atypical BVDV strain entered the cattle population.