杆状病毒囊膜糖蛋白gp64基因启动子活性分析

GP64是杆状病毒在其发育循环第一时相所产生的芽生型病毒粒子（budded voirus,BV)的特异性囊膜糖蛋白。它在病毒入侵细胞过程中具有重要作用。为了探明杆状病毒gp64基因的表达调控。从所克隆的BmNPV和AcMNPVgp64基因ATG上游437-439bp启动子的序列分析发现。该启动子同时具有早期和晚期转录模体,将所构建的该启动子控制下荧光素酶报告基因（Luc)的非融合表达质粒分别转染Bm-N和Sf-21细胞进行瞬间表达分析,BmNPVgp64启动子能被允许宿主Bm-N细胞RNA聚合酶Ⅱ所识别,AcMNPVgp64启动子既能被允许宿主Sf-21又能被非允许宿主Bm-N细胞RNA聚合酶Ⅱ所识别,同时,这两个启动子的转录活性在各自的允许宿主细胞中被相应的病毒因子所反式激活2.4-4倍。将家吞核多角体病毒同源重复序列3（BmNPV homologous region-3,hr3)克隆到该启动子控制下的Luc报告基因下游,用所构建的质粒分别转染细胞,瞬间表达分析结果表达,BmNPVhr3能分别增强该启动子在Bm-N和Sf-21细胞中的转录活性13-22倍和7000-14000倍以上,同时,相应的病毒因子能反式激活插入了hr3的gp64启动子在各自允许宿主细胞中转录活性73-78倍,这暗示着BmNPVhr3除了具有病毒DNA复制原点和增强子的功能外,它在病毒的反式激活过程中起着重要的作用。     

本氏烟Ⅰ型启动子的克隆及其转录起始位点分析

启动子是转录水平上一个重要的调控元件,其决定着基因的表达模式和表达强度。Ⅰ型启动子具有高转录活性和种属间特异性等特点。如将其应用于植物RNA病毒载体表达系统,有利于提高表达系统的表达效率和生物安全性。本氏烟(Nicotiana benthaminana)是一种被广泛地应用于植物生物反应器和植物病理学的模式生物,但是现有核酸数据库中尚没有其Ⅰ型启动子的相关信息。因此,克隆本氏烟Ⅰ型启动子并分析其转录起始位点就具有重要的应用价值。通过半巢式PCR获得了514 bp的本氏烟Ⅰ型启动子序列(KC352713);生物信息学分析初步预测其转录起始位点位于其核心序列TATA(G)TA(N)GGGGG中的第3位A处;通过植物RNA病毒表达载体和5'RACE技术在体内验证本氏烟Ⅰ型启动子转录起始位点与生物信息学预测结果一致。研究结果为深入研究Ⅰ型启动子和构建Ⅰ型启动子介导转录的植物RNA病毒载体表达系统奠定了基础。     

小鼠精胺氧化酶基因启动子结构和功能研究

目的:克隆和鉴定小鼠精胺氧化酶(mSMO)启动子DNA序列.方法:用Trizol试剂法从小鼠成纤维细胞中提取总RNA,应用5'-RACE法获得mSMO的转录起始位点;巢式PCR方法克隆含有mSMO启动子的DNA序列,并由此构建5'端系列截短的mSMO启动子荧光素酶报告质粒;将报告质粒瞬时转染Cos7细胞后,用荧光素酶分析法测定启动子活性.结果:确定mSMO基因至少有6个转录起始位点(+1,+4,+27,+31,+51,和+63),且均定位于外显子1中.克隆获得mSMO基因转录起始位点上游大约2.1kb的DNA片断,以此片段为基础构建了11个5'端系列截短的启动子报告质粒.报告基因分析证实,该上游DNA片段具有启动子活性,截短至-615和-176bp时,获得2个启动子活性峰值,截短至-373bp时启动子活性最低.结论:mSMO基因含有多个转录起始位点,其上游-176～+124bp为mSMO核心启动子区,-615～-176bp区为重要的转录调控区.     

香蕉果实特异性ACC合酶基因启动子区的克隆及其功能初探

根据本实验室所获得的香蕉果实特异性ACC合酶cDNA序列,以改进的接头连接PCR方法通过两次步行从香蕉基因组中分别扩增并克隆了其基因5′旁侧区近端1.2kb和远端1.6kb的片段。通过拼接,构建出含有2505bp启动子区和转录起始位点下游86bp的共2591bp的基因5′旁侧区片段;其启发性动子区中34至28为推测的TATA盒序列,158至146为推测的CCAAT盒,与其它植物基因启动子结构相类似。将2.5kb启动子片段与β-葡糖苷酸酶(GUS)基因编码序列融合,用基因枪法将构建的嵌合基因转入香蕉叶、根和果实的细胞后,只在果实细胞中观察到报告基因的瞬时表达,从功能上证明了此25kb的启动子片段具有指导报告基因在香蕉果实中特异性表达的作用。同时构建5个含不同5′端缺失启动子与GUS融合基因的表达载体。瞬时表达结果表明可能负责果实特异性表达的调控区存在于转录起始位点至-1111的启动子区中,而在-1111至-608区间可能存在一个正控制区。     

人淋巴细胞CD2基因5’侧翼序列的克隆和鉴定

本文报告以ＣＤ２ｃＤＮＡ５’端的片段作探针,从人Ｔ淋巴细胞基因组文库筛选阳性重组克隆,经限制性内切酶降解和Ｓｏｕｔｈｅｒｎ杂交分析,证明其中一个阳性克隆的插入片段中含ＣＤ２基因５’侧翼顺序。经插入片段的亚克隆、限制性内切酶图谱及ＤＮＡ序列分析,鉴定出一含转录起始点及其上游序列的４．０ｋｂ片段。将此片段中含转录起始点和两个ＤＮ（ａｓｅ）Ⅰ高敏感位点的２．５ｋｂ片段定向克隆到以虫萤光素酶为报告基因的表达载体ｐＭＧ３中,并用限制性内切酶对此２．５ｋｂ片段作不同程度缺失,构成一系列突变子。这些重组的表达质粒转染人ＪｕｒｋａｔＴ细胞后,以瞬时表达实验分析各突变子驱动虫萤光素酶基因的表达,结果发现在ＣＤ２基因５’上游具有很弱的启动子活性,初步测定该启动子位于－１．２ｋｂ～－９８ｂｐ域。ＣＤ２基因具有弱启动子、强增强子的特点与Ｔ细胞表面其它抗原分子基因是相似的。     

小鼠白蛋白启动子的组织特异性功能研究

以绿色荧光蛋白(GFP)基因作为报告基因,通过对比小鼠白蛋白启动子在不同来源细胞系中启动HGFP基因的转录活性,对小鼠白蛋白启动子的组织特异性进行了研究。结果发现,小鼠白蛋白启动子在小鼠肝癌细胞系Hepa 1—6和人肝癌细胞系：HepG2均有很强的转录起始功能,荧光显微镜下可以观察到IGFP表达。Hepa 1—6细胞在转染早期的48h内,CMV的启动子和增强子序列是小鼠白蛋白启动子转录活性的4倍。G418加压筛选2周后,CMV的启动子的转录活性下降到只有小鼠白蛋白启动子活性的1／2。转染人肝癌细胞系HepG2 2周后,荧光显微镜下可以观察到GFP表达。其他的细胞如中华仓鼠卵巢细胞系CHO和人肺癌细胞系PLA 801中转染的小鼠白蛋白启动子不能启动GFP的表达,而对照CMV启动子控制下的GFP基因可在CHO和PLA 801中表达。以上结果说明,小鼠白蛋白启动子仅在肝脏来源的细胞中可以起始下游基因的转录,在其他组织来源的细胞中不能起始转录,这表明小鼠白蛋白启动子具有肝脏组织特异的转录活性,但没有种属特异性。     

LRP16基因启动子克隆及特征分析

克隆LRP16基因启动子分子,并对启动子特征进行分析,预测启动子区调控元件,为深入研究LRP16基因的表达调控机制奠定基础。在NCBI的人类基因组数据库中截取并下载LRP16基因转录起始位点5′侧翼区2.7kb的基因组序列,设计PCR引物,从健康外周血单个核细胞中扩增,利用Genomatix程序对5′侧翼区近1000bp进行启动子特征分析,获得了与GenBank序列一致,长度为2.7kb的LRP16基因启动子DNA序列,该序列具有典型的真核生物RNA聚合酶Ⅱ启动子特征及多个核受体结合位点,如α视黄酸受体及RAR相关孤生受体。     

含VEGF启动子的报告基因的构建及雌激素受体对其活性的影响

目的：构建含血管内皮生长因子（VEGF）基因启动子的荧光素酶报告基因载体,并检测其在雌激素受体作用下的转录活性。方法：以乳腺癌细胞系MCF-7基因组为模板,扩增VEGF启动子片段,克隆到荧光素酶报告基因载体pGL3-basic中。用脂质体介导的基因瞬时转染法,将重组正确的报告基因载体转染293T细胞,检测重组质粒中荧光素酶报告基因的表达。结果：酶切鉴定和DNA序列分析表明构建了正确的pGL3-basic—VEGF报告基因载体;转录活性实验表明构建的报告基因载体具有启动子活性,雌激素受体α（ERα）能以剂量依赖的方式升高VEGF启动子调控下的报告基因的转录。结论：克隆了VEGF启动子,为ERα共调节子的功能研究奠定了基础。     

家蚕丝素重链启动子驱动DsRed的瞬时分泌表达

根据家蚕丝蛋白基因的启动子活性高、丝蛋白具有高效分泌的特性,克隆了家蚕丝素重链基因(Fib-H)启动子及其下游的信号肽序列(FibHS),将DsRed基因与信号肽序列融合构建了分泌型瞬时表达载体;转染细胞实验显示,该载体能在家蚕BmN细胞中瞬时表达DsRed;家蚕注射载体后,可在丝腺腔中检测到红色荧光,表明瞬时表达的DsRed分泌到丝腺腔,推测所克隆的序列具有信号肽的功能。此外,本研究为家蚕丝腺生物反应器分泌表达外源基因的研究奠定了基础。     

甘蔗ScMOC1基因启动子的克隆与瞬时表达分析

MOC1属于植物特有的GRAS家族蛋白基因,是调控植物腋芽形成发育的关键基因。启动子对基因转录效率起直接调控作用,其功能分析可以精确定位基因的表达部位、发育阶段和调控机制,克隆甘蔗腋芽形成发育关键基因ScMOC1的启动子序列,研究其功能对该基因表达调控机制具有重要意义。本研究以我国主栽甘蔗品种新台糖22号(ROC22)的基因组DNA为模板,通过基因组步移和巢式PCR技术克隆到ScMOC1起始密码子ATG上游1874 bp的启动子序列。PlantCARE在线分析预测表明,该序列包含多个真核生物启动子必需的核心元件TATA-box、CAAT-box以及与光响应、激素响应和分生组织表达等相关的顺式作用元件,推测ScMOC1启动子可通过激素诱导调控ScMOC1表达,且该启动子可能通过分生组织表达顺式调控元件CAT-box参与ScMOC1对甘蔗分蘖的调控。将获得的启动子序列替换pBI121质粒中的CaMV35S启动子驱动下游GUS基因表达进行活性分析,结果表明:本研究克隆的启动子片段能驱动GUS基因在甘蔗嫩叶中瞬时表达。5′缺失分析表明该启动子的基础启动子序列在起始密码子ATG上游350～500 bp之间。该结果为后续ScMOC1的调控机制研究奠定了良好的基础。     

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.