小分子G蛋白Rap1 shRNA表达载体的构建和鉴定

应用RNA干扰（RNA interference,RNAi）技术抑制Rap1基因的表达,构建RaplshRNA（small hairpin RNA．shRNA）表达载体,观察其对小鼠肝脏细胞中RaplmRNA和蛋白表达的干扰作用．根据小鼠RaplmRNA的全序列．设计了3种Rap1 siRNA序列（Rap1 siRNA1、Rap1 siRNA2、Rap1 siRNA3）和阴性对照序列（HK）;采用克隆技术,将其插入带有报告基因绿色荧光（EGFP）的pGenesi1－3载体,构建RaplshRNA表达载体：经双酶切和测序证实Rap1 siRNA表达载体克隆构建成功,插入片段测序结果与合成的siRNA结果一致：昆明小鼠40只,体重18～20g,随机分成4组：I组（转染HK组）、Ⅱ组（转染RaplshRNAl组）、Ⅲ组（转染RaplshRNA2组）、Ⅳ组（转染Rap1 shRNA3组）．于0、16、24h腹腔内注射Rap1 shRNA2．0－2．5mg／kg（用PBS稀释至1mL）：48h后收集小鼠肝脏．用显微荧光、定量RT—PCT、免疫组化检测小鼠肝细胞中Rap1 shRNA的转染率、Rap1基因表达以及蛋白质表达水平．I组、Ⅱ组、Ⅲ组、Ⅳ组小鼠肝脏细胞体内转染率均大于60％．Ⅱ组、m组、Ⅳ组的RaMmRNA表达、Rap1蛋白表达均降低．其中Rao1 shRNA1干扰效果最佳．     

Smad4基因siRNA表达载体的构建与鉴定

目的:以Smad4基因为靶标构建小干扰RNA(siRNA)真核表达载体.方法:根据GenBank公布的人Smad4核酸序列及SiRNA设计原则,用AmbionRNAi在线软件,筛选得到2个19 bp片段为靶序列,合成两端带有Bam H Ⅰ、HindⅢ酶切位点的发夹结构寡核苷酸序列,经过退火,将此序列克隆到真核表达质粒pSilencerTM3.1-H1 neo vector中,构建成重组质粒,酶切及测序验证.脂质体介导转染人原代增生性瘢痕成纤维细胞,经G418筛选细胞克隆,并用RT-PCR检测Smad4基因的表达.结果:成功构建了pSilencerTM3.1.H1 neo-Smad4 shRNA表达载体克隆,插入片段测序结果与合成的siRNA序列一致.RT-PCR结果显示转染shRNA1、shRNA2重组质粒的成纤维细胞内Smad4 mRNA水平均降低,其中以pSilencerTM3.1-H1-Smad4 shRNA1更为明显(P＜0.01).结论:构建P-Smad4 shRNA袁达载体成功,为进一步研究Smad4基因的RNA干扰奠定了基础.     

靶向FTL基因的shRNA真核表达载体的构建及鉴定

目的:为了构建铁蛋白轻链(ferritin light chain,FTL)的短发夹小干扰RNA表达载体,并提供进一步研究FTL功能的平台。方法:针对小鼠FTL基因的特异性位点设计并合成特异性长度约65bp的短发夹状小干涉RNA(shRNA)寡核苷酸序列,退火形成双链后将其插入真核表达载体pRNA-U6.1/neo的限制性内切酶HindⅢ和Bam HⅠ之间中,构建成FTL基因的干扰载体(FTL shRNA)。经测序鉴定正确后,用lipofectamineTM2000将FTL shRNA及其对照空载体pRNA-U6.1/neo分别转染到小鼠腹腔单核巨噬细胞(RAW264.7)中,提取蛋白后利用Western blot技术检测细胞中FTL蛋白的表达。结果:得到与预期目的片段大小相似的重组载体,测序结果显示所构建的小鼠FTL shRNA质粒序列正确。细胞内的FTL表达被FTL shRNA成功干扰,效率高达80%。结论:实验成功构建了小鼠FTL基因的短发夹小RNA干扰表达载体FTL shRNA。     

LOX-1特异性小发夹RNA 表达载体的构建及其对巨噬细胞源性泡沫细胞形成的影响

[摘　要]　目的：靶向血凝素样氧化型低密度脂蛋白受体-1基因的发卡样siRNA(shRNA)表达载体及其对巨噬细胞源性泡沫细胞形成的影响。方法：(1)采用DNA重组技术,将LOX-1 shRNA双链与线性化pGenesil-1质粒表达载体连接,脂质体法转染小鼠单核巨噬细胞（RAW264.7）,半定量逆转录聚合酶链反应法检测LOX-1 mRNA的表达,Western blot法检测LOX-1蛋白的表达。(2) Ox-LDL诱导巨噬细胞建立泡沫细胞模型, LOX-1-shRNA进行干预,干预组使用脂质体法进行细胞转染,转染24小时后,再加入Ox-LDL作用24小时,用油红O染色法及细胞内游离胆固醇及总胆固醇测定法观察对泡沫细胞形成的影响,倒置荧光显微镜观察转染LOX-1 shRNA后RAW264.7细胞对Dil-ox-LDL的摄取率。结果：测序鉴定发现插入的发卡样序列正确,成功合成了发卡样LOX-1基因RNA干扰表达载体;靶向LOX-1基因的发卡样shRNA表达载体转染RAW264.7细胞后,其LOX-1基因和蛋白表达显著下调, 同时可抑制巨噬细胞源性泡沫细胞形成及对Dil-ox-LDL的摄取。结论：成功构建了能有效抑制LOX-1 mRNA表达的发卡样LOX-1基因RNA干扰表达载体,并在一定程度上能抑制巨噬细胞源性泡沫细胞的形成,为进一步利用RNA干扰技术防治动脉粥样硬化提供理论基础。     

小鼠Dppa2基因shRNA载体的构建及干扰效果的检测

目的:构建有效的针对小鼠Dppa2基因的shRNA(short hairpin RNA)干扰载体。方法:设计合成2对针对小鼠Dppa2基因的shRNA序列以及1对与哺乳动物基因组无同源性的shRNA序列作为对照,构建pSUPER.Retro.puro干扰载体并进行PCR,酶切和测序验证。进一步将各干扰载体分别转染小鼠胚胎干细胞(embryonic stem cells,ESCs),RT-PCR检测干扰效率。结果:PCR,酶切和测序验证均表明各shRNA载体构建成功。将空载体及各重组载体分别转染小鼠ESCs发现,干扰组Dppa2基因表达水平相对于空载体对照组和阴性shRNA载体对照组明显下调。结论:成功构建了有效的针对小鼠Dppa2基因的shRNA干扰载体,为进一步研究Dppa2基因在维持小鼠ESCs不分化过程中的作用提供了基础。     

泛素C端水解酶L1的RNA干扰载体的构建及效果评价

目的:获得抑制效果好的泛素 C 端水解酶 L1(UCH-L1)基小干扰 RNA(siRNA)干扰载体.方法:根据 Gen?Bank 中大鼠 UCH-L1基序列设计并合成4对 siRNA 寡核苷酸序列,将4对寡核苷酸序列退火成双链后分别插入siRNA 表达载体 pcDNA6.2-GW/EmGFP-miR 中构建4个 siRNA 表达质粒,测序鉴定后将4个 siRNA 表达质粒分别转染 HEK293细胞,利用 Western 印迹和 qPCR 方法检测干扰效果;将干扰效果最好的质粒包装成腺病毒,感染大鼠血管平滑肌细胞(VSMC),并采用 TNFα干预诱导 UCH-L1表达升高,Western 印迹验证干扰效果.结果:测序分析证实4对 siRNA 寡核苷酸序列分别插入 siRNA 表达载体 pcDNA6.2-GW/EmGFP-miR;qPCR 检测与 Western 印迹均表明第3号 siRNA 表达载体对 UCH-L1表达的抑制程度最高,将其包装成腺病毒并转染 VSMC 能显著抑制 TNFα诱导的UCH-L1表达升高.结论:构建并筛选出干扰效果好的 UCH-L1 siRNA 干扰载体.     

靶向Smad4基因shRNA质粒表达载体的构建及筛选

目的:构建编码Smad4mRNA的shRNA真核表达载体,并筛选出基因沉默效果最明显的shRNA质粒表达载体.方法:根据GenBank提供的人Smad4基因的mRNA序列构建2个shRNA质粒表达栽体和1个阴性对照质粒表达载体,并通过基因测序进行鉴定.经鉴定后转染体外培养人成纤维细胞,western-blot检测Smad4蛋白水平抑制表达效果.结果:构建质粒表达载体测序结果显示,插入片段测序结果与合成的shRNA序列一致;靶向Smad4基因shRNA质粒表达栽体对所转染的人成纤维细胞中Smad4蛋白水平表达均有抑制作用,其中shRNA1最为明显.结论:成功构建了靶向Smad4基因shRNA质粒表达栽体,其中抑制Smad4基因表达效果最为明显的是shRNA1,为进一步研究Srnad4基因的RNA干扰奠定了基础.     

靶向人CHOP基因shRNA真核表达载体的构建

[目的]构建靶向干扰内质网应激标志性因子CHOP的shRNA真核表达载体,并检测其对CHOP的干扰效率。[方法]查询Gen Bank数据库,获取人源CHOP基因mRNA的序列,按照小干扰RNA(siRNA)靶序列的设计原则,设计并构建靶向CHOP基因mRNA的4个特异性shRNA真核表达载体(shRNA-1、shRNA-2、shRNA-3、shRNA-4)和1个无同源性的阴性对照载体(shRNA-NC),经PCR和测序鉴定确认shRNA载体构建成功后,脂质体转染人正常肝细胞系(L-02),Western Blot法检测CHOP蛋白的表达,筛选出干扰效果最好的表达载体。[结果]PCR和测序结果显示,5个shRNA表达载体均构建成功。Western Blot结果显示,0.06 g/L衣霉素损伤24h后,与内质网应激模型组相比,shRNA-1、shRNA-2、shRNA-3、shRNA-4组的CHOP蛋白表达水平均明显降低(P0.01),其中shRNA-1和shRNA-4组CHOP干扰效果最明显。[结论]构建了并成功筛选出靶向干扰CHOP基因的真核表达载体,为深入研究CHOP介导内质网应激所致细胞凋亡的信号通路奠定了实验基础。     

针对核因子-kB P65基因的短发夹干扰RNA逆转录病毒载体构建与鉴定

目的:构建针对人核因子kB亚基P65基因mRNA的短发夹干扰RNA(shRNA)逆转录病毒表达载体,并探讨小干扰RNA(siRNA)靶向抑制NF-kB P65基因表达的作用.方法:根据shRNA设计原则,在人NF-kB P65全长序列中选取合19个核苷酸靶序列,设计形成siRNA的DNA模板并克隆到shRNA表达载体pSUPER.retro.neo中,构建针对NF-kB P65基因的shRNA表达载体.经293A细胞包装,并感染NIH3T3细胞进行病毒滴度测定后,感染THP-1细胞.分别采用RT-PCR和Western blot从mRNA和蛋白水平检测干扰效果.结果:限制性酶切和基因测序证实针对人NF-kB P65亚基的shRNA表达逆转录病毒载体成功构建;其感染THP-1细胞后,NF-kB P65的mRNA和蛋白表达明显抑制.结论:成功构建了NF-kB P65 shRNA逆转录病毒表达载体,该载体能高效感染THP-1并明显抑制NF-kB P65的表达.     

大鼠晚期糖基化终产物受体基因特异性小干扰RNA表达载体的构建及其活性鉴定

应用两个RNA干扰实验技术网络的RNA设计软件,模拟SD大鼠晚期糖基化终产物受体(RAGE) mRNA的二级结构,设计针对RAGE mRNA417、221和534位点的3对小干扰RNA(siRNA)序列(21nt),再转化为能表达其小发夹结构RNA(shRNA)的寡核苷酸序列,分别将其克隆于pGCsi-U6/Neo/GFP载体,利用酶切和序列分析鉴定克隆的正确性。将RAGE特异性siRNA表达克隆转染肝星状细胞(HSC)-T6细胞系,以空白及转染非特异性siRNA表达克隆作为对照,分别以实时荧光定量PCR和Western印迹法检测各组HSC-T6细胞RAGE基因和蛋白质的表达。结果显示:成功构建了RAGE特异性siRNA重组表达载体pGCsi-R1、pGCsi-R2、pGCsi-R3和非特异性siRNA重组表达载体pGCsi-C,与对照组相比,转染特异性siRNA重组表达载体的HSC-T6细胞的RAGE mRNA表达受到明显抑制,在0.25～1.0nmol/L范围内,RAGE mRNA下调幅度呈浓度依赖性增加,以1.0nmol/LpGCsi-R1最显著,转染pGCsi-C的HSC-T6细胞的RAGE mRNA表达水平无明显变化。转染pGCsi-R1的HSC-T6细胞24、48和72h表达的RAGE mRNA分别较空白对照组下调(79.65±8.88)%、(78.96±7.94)%和(73.11±6.89)%(F=71.397,61.824,61.98,P均<0.01),在72h范围内,RAGE mRNA下调幅度呈时间依赖性降低。同时,转染pGCsi-R1的HSC-T6细胞50kDa和46kDa的RAGE、α-平滑肌肌动蛋白(α-SMA)mRNA及蛋白质的表达也明显下调,分别为空白对照组的(43.91±1.18)%(F=386.19,P<0.01)、(36.33±0.78)%(F=386.07,P<0.01)、(57.53±3.25)%(F=20.91,P<0.05)和(58.48±3.08)%(F=56.59,P<0.05)。结果表明:pGCsi-R1表达的特异性siRNA可高效抑制HSC-T6细胞中RAGE基因和蛋白质的表达及肝星状细胞的激活。     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

Enterovirus 71 3C proteolytically processes the histone H3 N-terminal tail during infection

Highlights
1. The N-terminal tail of histone H3 is specifically cleaved during EV71 infection.
2. Viral protease 3C is identified as a protease responsible for proteolytically processing the N-terminal H3 tail.
3. Our finding reveals a new epigenetic regulatory mechanism for Enterovirus 71 in virus-host interactions.     

Detection of EBV and HHV6 in the Brain Tissue of Patients with Rasmussen’s Encephalitis

Rasmussen’s encephalitis (RE) is a rare pediatric neurological disorder, and the exact etiology is not clear. Viral infection may be involved in the pathogenesis of RE, but conflicting results have reported. In this study, we evaluated the expression of both Epstein-Barr virus (EBV) and human herpes virus (HHV) 6 antigens in brain sections from 30 patients with RE and 16 control individuals by immunohistochemistry. In the RE group, EBV and HHV6 antigens were detected in 56.7% (17/30) and 50% (15/30) of individuals, respectively. In contrast, no detectable EBV and HHV6 antigen expression was found in brain tissues of the control group. The co-expression of EBV and HHV6 was detected in 20.0% (6/30) of individuals. In particular, a 4-year-old boy had a typical clinical course, including a medical history of viral encephalitis, intractable epilepsy, and hemispheric atrophy. The co-expression of EBV and HHV6 was detected in neurons and astrocytes in the brain tissue, accompanied by a high frequency of CD8⁺ T cells. Our results suggest that EBV and HHV6 infection and the activation of CD8⁺ T cells are involved in the pathogenesis of RE.     

Perinatal Vertical Transmission of Chikungunya Virus in Ruili,a Town on the Border between China and Myanmar

正Dear Editor,Chikungunya virus (CHIKV), an arbovirus in the family of Togaviridae, genus Alphavirus, is transmitted by the A.aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia (Silva and Dermody 2017; Suhrbier 2019). It was first reported in 1953 in Tanzania, and caused only a few outbreaks and sporadic cases in Africa and Asia in last century. However, in the epidemic in 2004, CHIKV acquired mutations that conferred enhanced transmission by the A. albopictus mosquito(Schuffenecker et al. 2006). Since then, it has successively caused outbreaks in Africa, the Indian Ocean, South East Asia, the South America, and Europe (Zeller et al. 2016).     

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.