SOCS3基因重组腺病毒的构建及其在猪脂肪细胞中的表达

本研究旨在构建细胞因子信号转导抑制因子3(Suppressor of cytokine signaling 3,SOCS3)的重组腺病毒表达载体,获得有感染性的病毒颗粒。以pcDNA3-SOCS3质粒为模板扩增SOCS3基因,将其亚克隆至腺病毒穿梭载体pAdTrack-CMV,经测序验证后,重组的穿梭质粒用PmeI酶切线性化后转化到BJ5183感受态细菌中与其内的骨架载体pAdEasy-1进行同源重组,获得的重组质粒pAd-SOCS3,经PacI线性化后转染至HEK293细胞中进行包装和扩增,纯化后用TCID50法测定病毒滴度。以重组的病毒感染原代培养的猪脂肪细胞后,荧光显微镜下观察报告基因GFP的表达,RT-PCR和Western blotting检测细胞内SOCS3 mRNA和蛋白的表达。重组腺病毒载体pAd-SOCS3经酶切及PCR鉴定正确,病毒滴度为1.2×109PFU/mL;感染原代培养的猪脂肪细胞后,荧光显微镜观察可见报告基因GFP的表达;RT-PCR和Western blotting检测到细胞中SOCS3 mRNA和蛋白的表达显著提高。本研究成功构建了SOCS3基因的重组腺病毒,感染原代培养的猪脂肪细胞可稳定表达SOCS3蛋白,为深入研究SOCS3的功能奠定了基础。     

高糖应激致人PRKCD基因过表达内皮细胞模型构建及鉴定

目的:构建高糖应激下人PRKCD基因过表达内皮细胞模型并鉴定。方法:设计含AgeⅠ和NheⅠ酶切位点的PRKCD基因上下游引物,以含PRKCD基因的原始质粒为模版,PCR扩增获得PRKCD全部序列,经AgeⅠ和NheⅠ酶切后与同样酶切后的真核表达载体pDC316-LacZα重组获得穿梭质粒pDC316-PRKCD,经PCR及酶切、基因测序鉴定后,与腺病毒骨架质粒pB-HGlox△E1,3Cre共转染293细胞获得重组腺病毒Ad5-PRKCD,行PCR鉴定并反复纯化扩增后用TCID50法测定病毒滴度。分组培养人脐静脉内皮细胞,转染重组腺病毒后于高糖(25mmol/L)负荷,并设立空载体对照及渗透压对照组,以免疫荧光法检测PKCδ在细胞中的表达。结果:目的基因成功插入穿梭质粒,基因测序结果与GenBank公布序列一致,重组腺病毒Ad5-PRKCD经PCR鉴定及免疫荧光鉴定成功。测得病毒滴度为1.0×1010IU/ml。激光共焦聚观察高糖负荷下,胞内PRKCD翻译产物PKCδ荧光表达强度明显增强,为正常对照组的1.5倍(P0.05),高糖负荷下内皮细胞感染重组腺病毒后PKCδ荧光强度明显增加,浆/核荧光强度比值较高糖组进一步降低了35%(p0.05),提示核转位明显。结论:成功构建了人重组腺病毒Ad5-PRKCD并有效转染人脐静脉内皮细胞,高糖负荷使PKCδ表达上调并发生核转位激活,为筛选稳定表达PKCδ的内皮细胞株及其蛋白复合体奠定了基础。     

携带siSPK1重组腺病毒的构建及其对N2a细胞凋亡的影响

应用AdMax腺病毒载体系统构建携带siSPK1基因的重组腺病毒,进一步研究SPK1基因对N2a细胞凋亡的影响。设计可形成小发夹结构的SPK1-siRNA模板cDNA序列,克隆至质粒pDC316-siRNA,构建SPK1-siRNA穿梭质粒pDC316-SPK1,经鉴定正确后,将pDC316-SPK1与辅助包装质粒（pBHG loxΔE1,3 Cre）共转染至HEK293细胞,包装纯化并扩增重组腺病毒颗粒,终点稀释法测定病毒滴度;病毒感染N2a细胞,Western blot法检测SPK1蛋白的表达;Hoechst33258染色检测SPK1基因对N2a细胞凋亡的影响。酶切后PCR分析、测序鉴定表明,pDC316-SPK1构建成功,病毒纯化后滴度为2.50E＋10 PFU/mL;重组病毒可在蛋白水平抑制SPK1的表达;SPK1基因抑制后,N2a细胞凋亡增加（P〈0.001）。上述结果表明,含有SPK1-siRNA的重组腺病毒构建成功,且具有抑制SPK1蛋白表达的功能,该基因沉默后,N2a细胞凋亡增加。     

仙台病毒BB1株6个编码基因的克隆及表达

在仙台病毒BB1株全基因组序列测定的基础上,用反转录和PCR方法获得了核蛋白基因(N),磷蛋白基因(P),神经血凝素基因(HN),基质蛋白基因(M)、融合蛋白基因(F)和聚合酶蛋白基因(L)等6个编码基因全长克隆;测序结果表明,其序列与Genbank中登录的序列(DQ219803)完全一致。为了提供仙台病毒基因组载体拯救和包装所需的反式作用蛋白,将N、P、M、F、HN和L分别克隆到腺病毒穿梭表达载体pDC316上,将它们分别与腺病毒基因组质粒pBHGlox△E1,3Cre共转染HEK293细胞,获得了6种复制缺陷性重组腺病毒Ad5-N、Ad5-P、Ad5-M、Ad5-F、Ad5-HN和Ad5-L。酶切结果表明6种重组腺病毒穿梭质粒构建正确;用PCR方法证明所获得的6种重组腺病毒分别携带了上述6个编码基因;用重组腺病毒感染LLC-MK2细胞后用Western blotting和免疫荧光方法检测到了相应仙台病毒编码基因的表达。本研究为仙台病毒BB1株全长基因组的拼接和病毒载体包装系统组建打下了基础。     

表达O型口蹄疫病毒衣壳蛋白的重组腺病毒构建及其免疫原性分析

为构建表达O型口蹄疫病毒（foot-and-mouth disease virus, FMDV）衣壳蛋白的复制缺陷型人5型腺病毒（Ad5）为载体的重组腺病毒,本研究设计、合成特异性引物并扩增出FMDV-OZK93的P12A、3B3C基因,通过融合PCR方法连接2个片段,获得P12A3B3C基因后插入到pDC316-mCMV-EGFP质粒,构建了能够表达FMDV-OZK93株衣壳蛋白前体P12A和3B3C蛋白酶的重组穿梭质粒pDC316-mCMV-EGFP-P12A3B3C。利用AdMaxTM腺病毒包装系统进行重组腺病毒rAdv-P12A3B3C-OZK93的包装、鉴定及扩增;并感染人胚胎肾细胞HEK-293进行表达验证。将鉴定正确且高度纯化后的重组腺病毒肌肉免疫小鼠进行免疫原性分析。结果显示,rAdvP12A3B3C-OZK93在病毒传代过程中目的基因稳定存在,且病毒滴度可达1×10^9.1 TCID₅₀/mL。间接免疫荧光和Western blotting结果均表明rAdv-P12A3B3C-OZK93在HEK-293细胞中表达了FMDV特异...     

腺病毒介导的人巨细胞病毒UL49基因小鼠模型的建立

建立表达HCMV UL49 基因的转基因小鼠,为抗病毒药物研究提供有效的实验动物模型。本实验将UL49-GFP基因插入腺病毒穿梭质粒pDC316中,构建重组质粒pDC316-UL49-GFP,与腺病毒骨架质粒pBHGloxΔE1,3Cre 通过脂质体介导共转染293 细胞,重组产生腺病毒Ad-UL49-GFP, 经PCR和Western Blot鉴定正确后,大量扩增、纯化,制备高滴度重组腺病毒。纯化腺病毒经尾静脉注射感染小鼠,通过荧光定量PCR 和Western blot 方法,检测UL49 基因在小鼠体内组织分布和表达时相。结果显示UL49基因在小鼠的心、肝、脾、肺、肾组织均有表达,并且表达量由高到低顺序依次是：肝、脾、肾、心、肺,在腺病毒感染第3天在各靶器官表达水平较高,此后逐渐下降,第14天时仅存在肝和脾中。表明表达UL49基因的小鼠模型构建成功。小鼠模型的成功建立为下一步筛选以UL49基因为靶的抗病毒药物奠定了基础。     

脂肪储存小滴蛋白5 重组腺病毒的制备

目的:利用AdEasy腺病毒表达系统构建含有小鼠脂肪储存小滴蛋白5(LSDP5)基因的重组腺病毒。方法:从小鼠肝脏cDNA克隆出LSDP5基因全长,克隆至pMD18-T载体中,酶切测序。回收酶切产物,连接到腺病毒穿梭载体pShuttle-CMV,构建pShuttle-CMV-LSDP5重组质粒,经PmeI酶切线性化后转化至含有腺病毒骨架质粒pAdEasy-1的BJ5183中。筛选阳性克隆,提取重组质粒,PacI酶切线性化并转染AD293细胞进行包装,提取病毒DNA,鉴定重组病毒并检测病毒滴度。结果:LSDP5基因克隆经测序证实与Genebank公布一致,双酶切重组pMD18-T载体得到1400 bp左右的片段。重组穿梭载体经Kpn I和Sal I双酶切后得到预期片段。PacI酶切得到30 Kb大片段和4.5 Kb小片段。转染AD293细胞后收集病毒,经PCR鉴定,获得理想的目的片段。取病毒上清反复感染AD293细胞以扩增病毒,最后所得病毒滴度为2.5×109pfu/ml。结论:成功构建了携带脂肪储存小滴蛋白5基因的重组腺病毒载体,为进一步研究LSDP5基因功能奠定基础。     

脂肪储存小滴蛋白5重组腺病毒的制备

目的：利用AdEasy腺病毒表达系统构建含有小鼠脂肪储存小滴蛋白5（LSDP5）基因的重组腺病毒。方法：从小鼠肝脏cDNA克隆出LSDP5基因全长,克隆至pMD18-T载体中,酶切测序。回收酶切产物,连接到腺病毒穿梭载体pShuttle-CMV,构建pShuttle-CMV-LSDP5重组质粒,经PmeI酶切线性化后转化至含有腺病毒骨架质粒pAdEasy-1的BJ5183中。筛选阳性克隆,提取重组质粒,PacI酶切线性化并转染AD293细胞进行包装,提取病毒DNA,鉴定重组病毒并检测病毒滴度。结果：LSDP5基因克隆经测序证实与Genebank公布一致,双酶切重组pMD18-T载体得到1400 bp左右的片段。重组穿梭载体经Kpn I和Sal I双酶切后得到预期片段。PacI酶切得到30 Kb大片段和4.5 Kb小片段。转染AD293细胞后收集病毒,经PCR鉴定,获得理想的目的片段。取病毒上清反复感染AD293细胞以扩增病毒,最后所得病毒滴度为2.5×109pfu/ml。结论：成功构建了携带脂肪储存小滴蛋白5基因的重组腺病毒载体,为进一步研究LSDP5基因功能奠定基础。     

小鼠FAM3C基因重组腺病毒载体的构建及其在肾系膜细胞中的表达

目的:利用Ad Easy TMsystem构建携带小鼠FAM3C基因的重组腺病毒,转染至人胚肾细胞系HEK293细胞,检测外源FAM3C在细胞中的表达。方法:取小鼠肾脏提取RNA,创建c DNA文库,用PCR的方法扩增FAM3C基因,将其克隆到p EASY-1载体中,T3连接酶连接。经Bgl II单酶切后,接入p Ad Tra Ck-CMV穿梭载体,T4连接酶连接,构建重组腺病毒的穿梭质粒p Ad Tra Ck-CMV-FAM3C。将经Pme I线性化的p Ad Track-CMFAM3C电穿孔共转化入BJ5183重组细菌,获取重组腺病毒质粒Ad-FAM3C,再将经Pac I线性化的Ad-FAM3C重组病毒骨架质粒转染人胚胎肾细胞系HEK293细胞,包装并扩增病毒。用Ad-FAM3C感染小鼠肾系膜细胞,Western blot法检测FAM3C在小鼠肾系膜细胞中的表达。结果:DNA序列分析和琼脂糖凝胶电泳结果表明,已构建表达FAM3C基因的重组腺病毒,该腺病毒在体外能有效感染小鼠肾系膜细胞,且高表达FAM3C蛋白。结论:成功地构建了携带小鼠FAM3C基因的重组腺病毒,为进一步阐明FAM3C的功能及作用机制奠定实验基础。     

表达双功能融合蛋白（sCAR-EGF）腺病毒载体的构建及其活性检测

为了提高腺病毒载体用于基因治疗的靶向性,采用PCR和体外连接的方法构建了柯萨奇病毒-腺病毒受体(Coxsackievirus-AdenovirusReceptor)胞外段sCAR和表皮生长因子(Epidermalgrowthfactor)EGF融合基因,然后将此融合基因插入穿梭质粒pDC315。利用Ad-MAX腺病毒系统,将重组质粒pDC315-sCAR-EGF与腺病毒骨架质粒pBHGloxΔE13cre共同转染AD-293细胞,成功包装出一种复制缺陷型腺病毒Ad5-CMV-sCAR-EGF。经PCR鉴定该病毒含有sCAR-EGF融合基因片段,Westernblotting证实该病毒能表达sCAR-EGF融合蛋白。体外试验证实该病毒感染细胞所产生的融合蛋白能够引导携带报告基因的腺病毒Ad5-CMV-luc高水平感染肿瘤细胞,为高水平表达EGFR的肿瘤的靶向性基因治疗提供了新的手段。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

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.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.