VSV糖蛋白酵母双杂交诱饵载体的构建及其自激活作用的检测

构建水泡性口炎病毒糖蛋白(VSV-G)酵母双杂交诱饵载体,检测其在酵母细胞中的表达和自激活作用,为进一步研究酵母双杂交系统筛选与VSV-G相互作用蛋白奠定基础。通过RT-PCR方法从水泡性口炎病毒(VSV)克隆糖蛋白(G)基因,BamH I和Sal I双酶切后连接诱饵载体pSos,获得诱饵质粒pSos-VSV-G,经测序鉴定后pSos-VSV-G转化到酵母菌株cdcH25(α),在营养缺陷培养基中观察pSos-VSV-G的自激活作用和毒性作用,同时利用蛋白印迹法分析诱饵蛋白的表达。成功扩增VSV-G基因,并准确克隆入pSos中,诱饵载体pSos-VSV-G成功转化到酵母菌株cdcH25(α)中,经表型筛选检测无自激活作用和毒性作用,蛋白印迹法检测证实pSos-VSV-G在酵母细胞中表达诱饵蛋白。可以利用酵母双杂交系统筛选与VSV-G相互作的蛋白质。     

应用聚合酶链反应检测口蹄疫病毒的实验研究

聚合酶链反应用于直接检测口蹄疫病毒(FMDV),可快速、灵敏地检出乳鼠组织或细胞繁殖的病毒的核酸。其灵敏度可达0.062pg,整个检测过程可缩短至4～5个小时内完成,比其它检测方法都敏感和快速。本文还探讨了直接用PCR扩增合成生物素化核酸探针检测口蹄疫病毒核酸的存在。     

百合病毒的DNA芯片检测技术研究

根据已知的黄瓜花叶病毒,百合无症病毒、百合斑驳病毒基因核苷酸序列,设计引物和探针,制备寡核苷酸芯片.用Cy3标记核苷酸引物,不对称RT-PCR扩增产物与芯片上的寡核苷酸探针杂交,荧光扫描仪检测并分析信号.研究制备的基因芯片能够检测侵染百合的3种重要病毒核酸的特异性荧光信号,该项技术具有特异、灵敏、快速的优点.     

百合病毒的DNA芯片检测技术研究

根据已知的黄瓜花叶病毒,百合无症病毒、百合斑驳病毒基因核苷酸序列,设计引物和探针,制备寡核苷酸芯片。用Cy3标记核苷酸引物,不对称RT-PCR扩增产物与芯片上的寡核苷酸探针杂交,荧光扫描仪检测并分析信号。研究制备的基因芯片能够检测侵染百合的3种重要病毒核酸的特异性荧光信号,该项技术具有特异、灵敏、快速的优点。     

速成单层法半微量病毒空斑技术的研究

本文报道一种不需预先制备细胞单层的速成单层法半微量病毒空斑技术(简称速成法)及其在空斑中和试验与空斑纯化试验中的应用。作者以脊髓灰质炎病毒为代表,对速成法的实验条件、可靠性、敏感性、重复性等作了系统研究,并以此法对单纯疱疹病毒、呼吸道合胞病毒和水泡性口炎病毒进行了空斑滴定。结果提示速成法简单快速、经济方便、敏感稳定、可靠易行,可能具有较大的实用和推广价值。     

应用RT-PCR技术检测动物组织中的口蹄疫病毒*

通过对影响RT-PER检出率的条件和试剂进行了筛选,确定了提取FMDV RNA的最佳方法和试剂,优选出RT-PCR反应的试剂和最佳反应条件,建立了检测口蹄疫病毒核酸的RT-PER方法。应用所建立的方法检测送检的鲜牛奶、淋巴结、脊髓、牛鼻咽拭子,结果阳性率分别为41.4％(24/58)、13.33％(2/15)、20％(1/5)、37.5％(12/32);检测4个屠宰场送检的组织样品40份,结果阳性率为.10％-70％;检测送检的奶粉(1份)、水泡皮(1份)、老鼠(2份)、患儿口腔棉拭子(4份),阳性率均为     

一种新的病毒空斑技术

本文报道了一种不同于Dulbecco细胞单层法和Cooper琼脂细胞悬浮法的病毒空斑技术——速成单层法病毒空斑试验。用24孔细胞培养板,不经预先制备细胞单层,将Vero细胞和水泡性口炎病毒Indiana株同时加入培养孔内,使细胞在贴壁的同时被病毒吸附,继后在营养凝胶下,细胞形成单层的同时病毒产生空斑。结果表明,此法简单快速,稳定可靠,经济节约,方便易行。用于单纯疱疹病毒分型的鸡胚细胞空斑形成试验,亦获得满意结果。     

基因芯片技术检测肾综合征出血热病毒核酸的研究

肾综合征出血热 (Hemorrhagic Fever with Renal Syndrome,HFRS)是由布尼亚病毒科汉坦病毒属 (Hantavirus,HV)中的病毒引起的急性传染病,该病的病死率较高,早期诊断和治疗尤为重要.     

肾综合征出血热病毒基因检测及分型的研究

根据流行于我国的两型ＨＦＲＳＶ代表株汉滩型７６１１８株及汉城型Ｒ２２株Ｍ节段的核酸序列,设计两型共同引物,建立了逆转录－聚合酶链反应（ＲＴＰＣＲ）方法,检测３９株从不同地区、不同宿主分离的ＨＦＲＳＶ感染鼠脑及细胞培养物;同时还建立了捕捉ＥＬＩＳＡ法（ｃＥＬＩＳＡ）,检测了３９株中的３６株,每份样本设复孔,以Ｐ／Ｎ≥２．１０且Ｐ－Ｎ≥０．１０者判为阳性。ＲＴＰＣＲ及ｃＥＬＩＳＡ两法的检出率分别为９７．６％与８２．４％,二者符合率８４．６％。此外,对ＲＴＰＣＲ产物进行酶切分型,３８份扩增产物中的１５份可被ＡｌｕＩ切开。根据所获酶切图谱的差异,可分为汉滩型及汉城型两型,显示了酶切分型的潜在价值     

携带猪γ干扰素基因逆转录病毒载体的构建及其在猪肾细胞(PK-15)中的表达

将梅山猪γ干扰素基因定向插入逆转录病毒载体pLXSN(neor),构建逆转录病毒重组质粒,利用脂质体介导法将重组质粒转染逆转录病毒包装细胞系PA317,转染细胞经含G418(400μg/mL)培养基筛选一周后获得稳定产毒的PA317细胞系。从细胞培养上清中提取RNA,进行RT-PCR检测,扩增到目的片段;将上清感染猪肾细胞(PK-15),经含G418(400μg/mL、600μg/mL和800μg/mL)的DMEM筛选一周,间接免疫荧光表明表达的猪γ干扰素主要锚定于细胞膜。收取PK-15细胞上清,在牛肾细胞(MDBK)上进行干扰素抗病毒活性检测,结果显示重组病毒表达的猪γ干扰素抗水泡性口炎病毒(VSV)的活性为1200IU/106cells.48h。以表达的干扰素处理PK-15细胞后,经细胞病变抑制法测定,重组猪γ干扰素可以抵抗口蹄疫病毒(FMDV)感染。试验结果表明猪γ干扰素基因已成功插入逆转录病毒基因组并在PK-15细胞中表达,表达的重组猪γ干扰素具有较强的抗病毒生物活性。     

以猪IgG重链恒定区为抗原载体的抗口蹄疫病毒DNA疫苗的研制

口蹄疫(Foot-and-Mouth Disease, FMD)是当今世界上最为严重的家畜传染病之一,主要危害猪、牛、羊等偶蹄动物.FMD的致病原为FMD病毒(FMDV),属小RNA病毒科口蹄疫病毒属,有A、O、C、SATⅠ、SATⅡ、SATⅢ及AsiaⅠ共7个血清型.FMDV结构较简单,完整的病毒颗粒由4种结构蛋白VP1、VP2、VP3及VP4各60个拷贝构成的衣壳包裹一条单股正链RNA组成,其中VP1是主要的抗原蛋白[1].     

膜芯片检测A组轮状病毒的初步研究

建立表面带正电荷的尼龙膜为基片的基因芯片,采用RT-semi-nested PCR方法,对A组轮状病毒RNA实现高度扩增,并通过5'端带地高辛标记的上游引物实现扩增产物的标记.通过同膜芯片上探针的杂交和免疫显色,实现对A组轮状病毒的检测.结果表明,膜芯片对探针的固定效果、杂交吸脱和检测结果可靠,空白对照和阴性对照均为阴性,探针均显示为阳性信号,并且信号强弱与探针浓度关系不大,而主要与探针本身同互补链的结合相关.以上结果说明已经初步建立了快速检测A组轮状病毒的膜基因芯片检测技术.     

基因芯片技术研究柽柳NaHCO3胁迫下基因的表达

运用基因芯片技术研究了NaHCO3胁迫下柽柳(Tamarix androssowii)基因的表达.将Cy5和Cy3两种荧光染料分别标记在NaHCO3处理和对照的柽柳cDNA上,将两种荧光探针混合,与载有柽柳基因的高密度芯片进行杂交并用芯片扫描系统进行扫描,通过Cy5与Cy3信号强度比值的计算研究基因的差异表达.共获得了89个差异表达的基因,其中,27个下调表达,62个上调表达.BlastX分析表明这些基因按功能可以分为光合作用、活性氧清除、渗透调节、信号传导与表达调控、代谢、发育相关、核糖体蛋白、蛋白质的分解与再生、转运类蛋白、水通道蛋白等几大类别.同时,发现了一些与盐胁迫相关的功能未知基因或未有任何功能信息的基因,这些基因可能在柽柳抗盐过程中具有重要作用.揭示了柽柳的抗盐胁迫涉及的几种重要途径,并获得了NaHCO3胁迫前后柽柳基因表达谱.     

Efficient use of lactose for the lac promotercontrolled overexpression of the main antigenic protein of the foot and mouth disease virus in Escherichia coli under fed-batch fermentation conditions

Abstract: Derivatives of the lac promoter (tac, pac, rac) belong to the strongest bacterial promoters which are frequently used for the induced overexpression of foreign genes in Escherichia coli . However, their use in fermentation processes is strongly restricted because of the high cost of the inducer iso-propyl-β-D-thiogalactopyranoside (IPTG). The aim of this work was to investigate the possibility of using lac-derived promoters in high cell density processes resulting in a high yield of the induced recombinant protein if glucose is the main carbon and energy source. Lactose is tested as inducer of the main antigenic coat protein (VP1) of the foot and mouth disease (FMD) virus in a T7-RNA polymerase expression system. It was shown that lactose is able to induce the expression of the recombinant gene to an amount of the VP1 protein corresponding to 20% of the total cell protein.     

Effect of temperature on the transmission of orbiviruses by the biting midge,Culicoides sonorensis

The influence of temperature on the likelihood of Culicoides sonorensis Wirth & Jones (Diptera: Ceratopogonidae) transmitting African horse sickness virus (AHSV) serotypes 4 and 6, bluetongue virus (BTV) serotypes 10 and 16 and epizootic haemorrhagic disease of deer virus (EHDV) serotype 1 was investigated. Extrinsic incubation periods (EIP), vector competence and vector survival were determined at 15, 20, 25 and 30 degrees C. The effect of humidity on vector survival was also investigated by maintaining adult C. sonorensis at 40, 75 and 85% r.h. at each temperature. Higher temperatures were associated with a shorter EIP for all virus serotypes except AHSV6, to which C. sonorensis was orally refractory, increased vector competence for AHSV4 and EHDV1, but not for BTV10 or BTV16, and a reduction in vector survival. Humidity interacted with temperature in influencing vector survival, such that at low temperatures, lower humidity (40 and 75% r.h.) was detrimental for survival (up to 18% reduction in longevity), whereas at high temperatures, high humidity (85% r.h.) was detrimental (up to 36% reduction in longevity). In general, the transmission potential of C. sonorensis for AHSV4, EHDV1, BTV10 and BTV16 was greater at higher temperatures, because although vector survival was reduced, this was more than compensated for by the accompanying decrease in duration of the EIP.     

Advances in novel vaccines for foot and mouth disease: focus on recombinant empty capsids

Foot and mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals, which causes severe economic losses in the livestock industry. Currently available vaccines are based on inactivated FMD virus (FMDV). Although inactivated virus vaccines have proved to be effective in FMD control, they have a number of disadvantages, including the need for high bio-containment production facilities and the lack of induction of immunological memory. Novel FMD vaccines based on the use of recombinant empty capsids have shown promising results. These recombinant empty capsids are attractive candidates because they avoid the use of virus in the production facilities but conserve its complete repertoire of conformational epitopes. However, many of these recombinant empty capsids require time-consuming procedures that are difficult to scale up. Achieving production of a novel and efficient FMD vaccine requires not only immunogenic antigens, but also industrially relevant processes. This review intends to summarize and compare the different strategies already published for the production of FMDV recombinant empty capsids, focusing on large-scale production.     

Gene co-expression network analysis for identifying genetic markers in Parkinson's disease - a three-way comparative approach

Parkinson's disease (PD) is a neurodegenerative disorder involving progressive deterioration of dopaminergic neurons. Although few genetic markers for familial PD are known, the etiology of sporadic PD remains poorly understood. Microarray data was analysed for induced pluripotent stem cells (iPSCs) derived from PD patients and mature neuronal cells (mDA) differentiated from these iPSCs. Combining expression and semantic similarity, a highly-correlated PD interactome was constructed that included interactions of established Parkinson's disease marker genes. A novel three-way comparative approach was employed, delineating topologically and functionally important genes. These genes showed involvement in pathways like Parkin-ubiquitin proteosomal system (UPS), immune associated biological processes and apoptosis. Of interest are three genes, eEF1A1, CASK, and PSMD6 that are linked to PARK2 activity in the cell and thereby form attractive candidate genes for understanding PD. Network biology approach delineated in this study can be applied to other neurodegenerative disorders for identification of important genetic regulators.     

Where all the Roads Meet? A Crossover Perspective on Host Factors Regulating SARS-CoV-2 infection

COVID-19 caused by SARS-CoV-2 is the latest pandemic which has thrown the world into an unprecedented social and economic uncertainties along with huge loss to humanity. Identification of the host factors regulating the replication of SARS-CoV-2 in human host may help in the development of novel anti-viral therapies to combat the viral infection and spread. Recently, some research groups used genome-wide CRISPR/Cas screening to identify the host factors critical for the SARS-CoV-2 replication and infection. A comparative analysis of these significant host factors (p < 0.05) identified fifteen proteins common in these studies. Apart from ACE2 (receptor for SARS-CoV-2 attachment), other common host factors were CSNK2B, GDI2, SLC35B2, DDX51, VPS26A, ARPP-19, C1QTNF7, ALG6, LIMA1, COG3, COG8, BCOR, LRRN2 and TLR9. Additionally, viral interactome of these host factors revealed that many of them were associated with several SARS-CoV-2 proteins as well. Interestingly, some of these host factors have already been shown to be critical for the pathogenesis of other viruses suggesting their crucial role in virus-host interactions. Here, we review the functions of these host factors and their role in other diseases with special emphasis on viral diseases.     

Diabetes as a risk factor for Alzheimer’s disease in the Middle East and its shared pathological mediators

The incidence of Alzheimer’s disease (AD) has risen exponentially worldwide over the past decade. A growing body of research indicates that AD is linked to diabetes mellitus (DM) and suggests that impaired insulin signaling acts as a crucial risk factor in determining the progression of this devastating disease. Many studies suggest people with diabetes, especially type 2 diabetes, are at higher risk of eventually developing Alzheimer's dementia or other dementias. Despite nationwide efforts to increase awareness, the prevalence of Diabetes Mellitus (DM) has risen significantly in the Middle East and North African (MENA) region which might be due to rapid urbanization, lifestyle changes, lack of physical activity and rise in obesity. Growing body of evidence indicates that DM and AD are linked because both conditions involve impaired glucose homeostasis and altered brain function. Current theories and hypothesis clearly implicate that defective insulin signaling in the brain contributes to synaptic dysfunction and cognitive deficits in AD. In the periphery, low-grade chronic inflammation leads to insulin resistance followed by tissue deterioration. Thus insulin resistance acts as a bridge between DM and AD. There is pressing need to understand on how DM increases the risk of AD as well as the underlying mechanisms, due to the projected increase in age related disorders. Here we aim to review the incidence of AD and DM in the Middle East and the possible link between insulin signaling and ApoE carrier status on Aβ aggregation, tau hyperphosphorylation, inflammation, oxidative stress and mitochondrial dysfunction in AD. We also critically reviewed mutation studies in Arab population which might influence DM induced AD. In addition, recent clinical trials and animal studies conducted to evaluate the efficiency of anti-diabetic drugs have been reviewed.