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2003年18卷6期

Research Article

细胞内表达的小干扰RNA靶向丙肝病毒5 保守区的研究

吴叔文, 方 骢, 潘纪安, 田 波, 郭德银

2003, 18(6): 518

摘要:将丙型肝炎病毒(HCV)基因组的5 非编码区(5 UTR)插入到报告基因绿色荧光蛋白(eGFP)和荧光 素酶(1uciferase)的上游,并构建基于III型启动子的表达载体,这种载体能产生针对HCV5 UTR的小干扰RNA。 然后将含有HCV 5 UTR的eGFP/luciferase和能产生小干扰RNA的质粒共转染入Hela细胞,通过测定细胞发出 的荧光和化学发光强弱来观测抑制效果。实验结果表明,与HCV 5 UTR特异性小干扰RNA表达质粒共转染的 细胞无论从定性还是从定量上所测得的荧光和化学发光强度都明显低于阴性对照,且细胞密度经核染色与对照组 无明显区别。这揭示了小干扰RNA确实能引起HCV特异基因如5 UTR的沉默,且转染进去的小干扰RNA表 达质粒对细胞没有毒害作用。这一工作是通过载体直接在细胞内表达小干扰RNA(siRNA)而不是化学合成的,可 以使小干扰RNA在细胞内得到稳定表达,因此本研究设计的siRNA表达载体不仅可以有效沉默HCV 5 UTR,而 且该系统可以灵敏地筛选更有效的针对HCV的siRNA,因而这⋯结果为研究利用RNA干扰进行基因治疗HCV 感染做了初步探索。 Abstract:In this study,we inserted the 5 untranslated region(UTR)of Hepatitis C virus(HCV) genome into the upstream of the reporter genes of enhanced green fluorescent protein (eGFP)and luciferase.an d we also constructed the expression vector that Can express the short interfering RNAs (siRNA) against the HCV 5 .UTR.The 5 .UTR.eGFP/luciferase an d the siRNA-prod ucing plasmid were cotran sfected into Hela cells,an d the inhibition efect was detected by the intensity of the fluorescence an d luminescence.Th e vesults showed that the light from the cotransfected cells was obviously weaker than the negative control both in quality an d in quan tities,while density of the cotran sfected cells had no diference with the control plasmid as detected by nucleus staining.TlliS work demonstratedthat certain siRNA Can targetthe 5 UTR ofHCV whilenotoxic efecthadbeenobserved in the cells.TlliS work is the basis for future research in which RNAi activity is supposed to be utilized in the gene therapy with the the HCV infection.The siRNA is expressed intracellularly by vectors instead of chemical synthesis,an d a new method Can be used as a model to quickly an d safely screen effcctive siI AstargetingHCV

巨细胞病毒感染对多向造血祖细胞体外增殖的影响

刘文君, 郭渠莲, 邓正华

2003, 18(6): 519

探讨人巨细胞病毒(Human cytomegalovirus,HCMV)对脐血多向造血祖细胞(CFU-Mix)体外增殖的抑制 作用,采用造血祖细胞体外半固体培养技术,培养、观察、计数HCMV-ADl69株对脐血CF -Mix集落产率、抑 制率、集落峰值时间和集落维持时间;用聚合酶链反应(PCR)技术检测集落细胞内HCMV-DNA。结果发现 HCMV.AD169株感染的CFU-Mix集落产率较对照组明显减少(P0.01),集落产率随病毒感染滴度的增高而减少, 抑制率随病毒滴度的增高而逐渐增加;各组CFU.Mix集落峰值时间为10~12d(P0.05),不同滴度病毒感染组集落 维持时间(15~l8d)较对照组(22●24d)明显宿短(P0.01);经PCR检测病毒感染组,发现CFU-Mix集落细胞内有 HCMV-ADI69 DNA存在。 因此认为多向造血祖细胞是HCMV的宿主细胞之一,HCMV-AD169能直接感染多向 造血祖细胞,抑制多向造血祖细胞的增殖和分化,此可能是临床HCMV感染患儿出现粒细胞减少、血小板减少和 贫血的主要原因。 Abstract:To investigate the suppression effect of Human cytomegalovirus(HCMV)on multipotential hematopoietic progenitors(CFU—Mix)in vitro,colony forming unit—assay was applied to observe the effect of HCM V—AD169 strain on CFU—M ix of cord blood.1[11e technique of PCR Was used to demonstrate the existence of HCM V-DNA in the colony cells of cultured CFU-M ix.The numbers of CFU—M ix colonies in the groups infected with HCM V were significan tly less than that in control group, an d it showed an increase tendency with the decrease of HCMV concentration.1[11e suppression efect showed a dose—dependent fashion:the higher the HCM V.At)169 conce ntration.the more the CFU—Mix colonies decreased .111e pcak of CFU—M ix colonies in control groups an d groups infected with HCM V appeared on the same time(10~12th day),the lasting time of the CFU—Mix colonies in groups infected with HCM V Was significan tly shorter than that in control groups.HCNⅣ 一DNA Was positively detected in the colony cells of viral infected groups by PCR.while negative in the control groups.HC Ⅳ 一AD169 strain inhibited the diferentiation and proliferation of CFU—Mix by infec ting the hematopo ietic progenitors.HCMV may cause the suppression of hematopoiesis by direct infection,which may be the main reason of HCMV infection associated with anemia,neutropenia an d thrombocytope nia.

利用基因芯片分析拉米夫定治疗过程中HBV DNA的基因变异

高英堂, 陈瑞阳, 宋文芹, 陈成彬, 齐之丽, 景丽, 孙金英, 钱绍诚

2003, 18(6): 523

摘要:依据乙型肝炎病毒(Hepatitis B virus;HBV)聚合酶基因序列研制HBV基因芯片,此芯片可分析HBV的 7个基因型、4种血清型和HBV聚合酶基因rtV173、rtL180、rtM204和rtV207位点的突变。利用此芯片对A、B 两组共计45例拉米夫定治疗12个月的患者进行服药前和服药后3、6、9、12个月的动态检测,其中C基因型 39例,且血清型均为adr;B基因型6例,其血清型均为adw。在完成全程检测的38例患者中,17例 升高 的A组出现1例拉米夫定耐药变异株,而21例 正常的B组出现4例变异株,且所有变异株均为rtM204 V/rtL180M,其中2例野生株和变异株共存。rtM204V变异最早在服药6个月时出现,随后出现rtL180M 变异。 lO份PCR产物测序分析表明,芯片检测结果与测序结果基本一致,仅在rtL173位点出现1例差异。进一步分析 HBV DNA变异与I-IBV DNA含量、ALT水平和HBeAg血清转换率的相关性,初步结果表明变异株的出现与治 疗过程中的DNA反弹呈正相关,而与起始HBV DNA水平、ALT值无关联。HBV基因芯片可初步用于HBV DNA 检测,可能是临床追踪评价抗病毒治疗效果的较好方法之一。 Abstract:The Hepatitis B virus(HBV)oligochip was made according to the sequence of HBVpolymerase gene.7 genotypes and 4 sero—subtypes of HBV,as well as position rtV173,~.L180,rtlVl21M,rtV207 in the reverse transcriptase(rt)domain of HBV polymerase,were detected with the chip.45patients were divided into A an d B groups according to their ALT levels.Serum samples for chipanalysis were obtained at 0,3,6,9,12 months of treatment.Among 45 patients,39 were genotype C andsubtype adr,6 were genotype B an d subtype adw.Among 38 patients whom were treated continuouslyfor 12 months,1 lamivudine resistant mutan t was discovered in 17 of A group with high ATL level,4varian ts were momtored in 21 of B group with normal ALT leve1.All varian ts were rtM 204V an drtL180M ,2 of them were mixed with HBV wild type .Th e rtM 204V mutan t was found at 6 months ofthempy,the~L180M mutant was detected afterward.Th e results obtained by sequencing ofthe 10 PCRproducts an d chip arraying were almost the same,the only diferent was that 1 varian t at position rtV173Was not detected by the gene chip.Further an alysing HBV DNA values,ALT levels an d HBeAgseroconversion in relation to HBV mutants,the results showed that a more rapid occurrence of varian tWas assoc iated with HBV DNA re—elevation.whereas not associated with HBV DNA values an d ALTlevels of pretreatm ent.Th e HBV gene chip could monitor genetic variability of HBV,it is a promi singmethod forevaluating effects oflamivudine therapy.

中国人血清中丙型肝炎病毒聚合酶全长基因的克隆表达及鉴定

杨振, 蒋建东, 祁自柏, 陈鸿珊, 张华远, 李河民

2003, 18(6): 530

摘要:构建中国人丙型肝炎病毒(HCV)复制的RNA聚合酶原核表达载体pET30aNS5b,并在大肠杆菌中获得NS5B 聚合酶蛋白的高效表达,为建立HCV NS5b聚合酶细胞外分子复制模型的方法创造条件。使用高保真Pfu DNA 聚合酶进行反转录及套式PCR扩增,从我国HCV RNA阳性血清中扩增出HCV NS5b RNA多聚酶全基因序列, 经BamHI和Sa/l酶切。将其克隆至同样酶切的pET 30a载体中;转化大肠杆菌BL21,IFrG诱导表达。用抗HCV NS5b单克隆抗体做Western Blot进行鉴定。结果表明构建了原核表达载体,pET30aNS5bpET30aNS5b明显表达出 12 His NS5b聚合酶蛋白。测序结果表明,与已发表的相关HCV NS5b RNA聚合酶序列比较,其核苷酸和氨基酸 的同源性分别在69%~92.7%及88.8%~96.8%之间。在最佳表达条件下,可高效诱导表达融合蛋白(65kDa),最 高表达量占菌体蛋白18.9%。Western Blot结果显示表达蛋白为HCV NS5b酶。HCV聚合酶蛋白全长基因可以成 功地克隆在pET-30a载体上并有效表达出目的蛋白,为研究建立HCV NS5b聚合酶细胞外分子复制模型奠定了基 础。 Abstract:To ampUfy Hepatitis C virus(HCV)polymerase sequence and express HCV NS5b protein inorder to,in the next,establish a molecular model of HCV replication.RT—PCR method was used toamplify HCV NS5b polymerase gene from a Chinese HCV(+1 patient’S serum. e polymerasefragment Was constructed in the plasmid pET一30a an d expressed in E coli.HCV NS5b protein Wasidentified by W lestern blot with a monoclonal an tibody against HCV NS5b.Sequence analysis showedtllat the isolate had 69% 一95% an d 89%一97% homologies to the reported HCV sequences in respect tonucleotide an d amino acid respectively.HCV NS5b gene was expressed at a high level an d themolecular weight ofthe expressed prod uct was 65kDa.which was within the ran ge of expectation.Thisprotein Was confirmed to be HCV NS5b by Western blot.HCV NS5b from a Chinese patient Can beSuccess—fully cloned an d expressed with pET一30a plasmid an d E coli. is protein could be used toestablish a molecular mod el of HCV replication.

清热消炎复方制剂抗流感病毒作用的研究

杨占秋, 文利, 王征

2003, 18(6): 534

为评价清热消炎复方制剂(简称AI)的抗流感病毒活性,我们以病毒唑为对照,通过在体外观察病毒致细胞 病变效应(CPE)、MTT细胞染色检查病毒抑制率和检测病毒血凝滴度;在体内观察其对染毒小鼠的死亡保护作 用,对小鼠流感病毒性肺炎的抑制作用,以及对小鼠肺内病毒增殖的影响,从而判定其抗流感病毒作用。结果发 现AI在160ug/mL时能完全抑制流感病毒在MDCK细胞内的增殖复制作用。体内实验中0.1 gg,O.5gg,1.2g/kg 3个剂量均能明显降低染毒小鼠的致死率,延长平均存活时间;降低肺炎小鼠的肺指数和血凝滴度(P0.01)。其 作用与病毒唑相当。结论认为清热消炎复方制剂是一种有效的体内、体外抗流感病毒中药复方制剂。 Abstract:To observe the antiviral activity of Algefacient and anti-inflammatory(AI)against influenzavirus,cell culture technique was used in M DCK cells to get virus inhibitory rate an d virus tiler by MTTassay an d hemagglitination test.Influenza virus infection model was established in mice,to observe theprotection efect to mi ce death ,the inhibitory efect to pulmonary index and pulmonary virus titer.Theresults showed that 160ug/mL Algefacient an d anti-inflammatory Can completely inhibit theproliferation of influenza virus in MDCK cel1.0.1 gg, 0.5g/kg.1.2g,/kg dosage med cme orally givenfor 5 days could significan tly decrease the mortality rate,prolong the living time of infec ted mi ce,decrease the pulmonary index and pulmonary virus hemagglitination titer(PO.oi).This efect issimilar to that of virazole at the same dosage leve1.W e conclude that AI is an efective anti-influenz avirus.compound/n vitro and in vivo.

HCV全长基因真核表达载体的构建及其细胞内表达

焦成松, 王升启, 张贺秋, 凌世淦

2003, 18(6): 538

本实验成功构建了HCV全长基因的真核表达载体pCI·HCVFL,转染HepG2细胞后经免疫荧光和免疫组化 法分别检测到结构基因区(c)和非结构基因区(NS3)病毒蛋白的表达,该栽体可用于建立HCV转基因细胞模 型以及进一步开展有关HCV复制与表达的深入研究。

SARS—CoV感染Vero E6细胞诱导细胞凋亡

鄢 然, 沈 超, 雷 磊, 郑从义, 肖庚富, 郭德银, 朱 应, 鄢慧民

2003, 18(6): 541

摘要:为了确定SARS冠状病毒(SARS—CoV)感染Vero E6是否引起细胞凋亡,我们利用细胞DNA琼脂糖电泳, 感染细胞的间接荧光染色和Hoechst 33258细胞核染色,以及流式细胞仪分析等方法证明了SARS.CoV感染的Vexo E6具有典型的凋亡细胞学和生物化学特征。实验证明具有细胞凋亡特征的所有细胞均为处于感染晚期的细胞。表 现明显细胞病变(CPE)的细胞大多已经出现核质凝缩或形成凋亡小体进入细胞凋亡的过程。可以断定SARS.CoV 感染VeroE6细胞诱发了细胞凋亡。 Abstract:To determine if SARS—associated coronavirus(SARS—CoV)induces apoptosis.we studied the SARS—CoV infected Vero E6 cells by agarose gel electrophoresis.indirect fluorescent staining for infection an d Hoechst 33528 staining for nuclei,as well as by flow cytometry.It was found that SARS—C0V infected Vero E6 cells showed typical apoptosis characteristics.Au the typical apoptotic ceHs were in late—infection period.Most of the cells with cytopathic effect(CPE)showed nuclei witll condensed chromatin or fragmented into apoptotic bodies.It was suggested that SARS—CoV infection induces apoptosis of Vero E6.

SARS冠状病毒的分离培养与鉴定

江丽芳, 赵卫, 晏辉钧, 方丹云, 周经姣, 龙北国, 吴义芳, 周俊梅, 张文炳, 郭辉玉

2003, 18(6): 544

摘要:采集急性期病人的咽拭子或漱口液,用Vero、Vero E6、MDCK、Hela、Hep.2等传代细胞,人胚肺二倍 体细胞(髓L)和人胚肺(1iP)细胞分离培养严重急性呼吸系统综合症(SARS)的病原体。结果用Vero、Vero E6、 MDCK和HP细胞从标本中分离到一株病毒。间接免疫荧光试验发现,恢复期病人血清可与所分离的病毒起反应, 在胞膜和胞浆中出现翠绿色荧光;中和试验结果表明,恢复期病人血清能中和病毒对细胞的致细胞病变作用;电镜 下可观察到冠状病毒样颗粒;RT-PCR 法可扩增到冠状病毒特异性基因片段,且其核苷酸序列与国内外发表的 SARS冠状病毒(SARS。Coy)相应的基因序列相符,同源性达到100%。从传染性非典型肺炎病人的漱口液中分 离到SARS冠状病毒,这种病毒与传染性非典型肺炎密切相关。

猪繁殖与呼吸综合征病毒Ⅳ基因和流感病毒HA基因的联合表达及应用

许立华, 苏小运, 苏鑫铭, 任雪枫, 苏春霞, 陈溥言

2003, 18(6): 548

摘要:参照已公布的流感病毒血凝素基因(HA基因)及猪繁殖与呼吸综合征病毒(PRRSV)基因组序列,设计并合成 一对引物Pl、P2,以RT.PCR方法扩增出PRRSV的ORF7片段(约410bp),其中含HA基因主要核苷酸序~J(33bp)。 用BamH I、Xho 1分别对扩增出的片段及pET32a质粒进行酶切,连接后构建了重组质粒pETHN并转化到BL21 (DE3)宿主菌中诱导表达。用纯化后的表达产物与流感病毒血凝素单抗及乳胶建立了诊断猪繁殖与呼吸综合征 (PRRS)的乳胶凝集试验。检测结果显示:该方法有良好的特异性及敏感性,与IDEXX公司—1~—JRA检测试剂盒符合率 达93。8%。 Abstract:From to the nucleotide sequences of haemoagglutinin gene of Influenza virus and Porcine R rD c and Respiratory Syndrome Virus(PRRSV)VR2332 in GenBank,a pair of primers which includes the main sequences of haemoagglutinin(HA)gene(33bp)was designed to amplify N gene (ORF7)of PRRSV by RT—PCR.The amplified fragment and pET一32a plasmid were digested by BamHI and XhoI.A recombinant plasmid nam ed pETHN was constructed and transformed into BL21(DE3). Consequently,the target protein was expressed by IPTG induction,and the purified fusion protein was obtained by His—binding purification kit.As a result latex—agglutination test Was set up an d plenty of serum sam ples were tested by the method.Th e results showed the method had same sensitivity and specificity and had 93.8% accord rate compared with ELISA(IDEXX)diagnosis kit.

猪流感病毒分离鉴定

伍锐, 金梅林, 陈焕春, 王贵华, 蒋增海

2003, 18(6): 553

用非免疫鸡胚从我国上海、福建、湖北分离到3株猪流感病毒,分另lJ命名为SSHI、SFJI和SHBI。SIV 分离株第5代病毒液对0.7%豚鼠红细胞的血凝活性分别1:2 、1:2 和1:2。,与H 亚型标准血清血凝抑制价1: 2。、1:2’和1:2 ,但不能被鸡新城疫阳性血清所抑制。纯化的病毒在电镜下观察,可见到猪流感典型的病毒粒 子。病毒液接种于小自鼠,可表现出临床症状,剖杀后可观察到病毒性肺炎。猪体回归试验,可表现出临床症状 和病理变化,从肺脏中可分离到病毒并且RT-PCR也可检测到病毒相应的基因片段。从纯化的病毒中提取RNA, 进行RT.PCR,可扩增出预期的条带。 Abstract:Three H3 subtype strains of Swine influenza virus(SlV),which were named SSH1,SFJ1 andSHB1,were isolated from Shanghai,Fujian and Hubei.These isolates could grow and replicate inchicken emb~o,and the hemagglutinin(HA)values were 1:2 ,1:2 and 1:2 ,respectively.Thehemagglutinin inhibitor(HI)titers of H subtype antiserum were 1:2 ,1:2 and 1:2。,respectively,whereas they were negative in HI assays with Newcastle disease an tiserum.Under electron micrograph,the virions showed various shapes.The mice infected witl1 allantoic fluid could arise syndromes andpathology;SIV could be isolated again and amplified by RT-PCR from the lung.RNA Was extractedfrom SIV which Was purified from infected allantoic fluid,an d expected ban d Was observed viaRT—PCR.

兔出血症病毒NJ85株衣壳蛋白变异性分析及立体结构预测

王永山, 陆承平, 周宗安

2003, 18(6): 557

摘要:用分子克隆技术从兔出血症病毒(RHDV)中国早期流行株NJ85中成功克隆出vp60基因,序列分析表明 基因长度为1740nt,编码579aa。利用GenBank数据库,NJ85与WX84、TP二个RHDV中国毒株vp60基因DNA 序列的同源性分别为92.7%和97.2%,氨基酸序列的同源性分别为96.1%和98.6%,与其他国家l6个毒株的vp60 基因DNA序列的同源性在83.7%~97.0%之间,氨基酸序列的同源性在90.5%~99.0%之间,具有高度的同源性。进一步分析vp6O基因的六个分区,A、B、D、F四个区变异率较低,C、E二个区变异率较高。在遗传进化上,历年来的RHDV毒株在氨基酸水平上分析可分为三个支谱系,在核苷酸水平上趋向四个支谱系,谱系没有呈现地域或时间特征。三个中国毒株分布在二个不同的支谱系中。与RHDV 同为兔病毒属的欧洲野兔综合征病毒(EBHSV)组成了另一个谱系。运用生物信息学方法分析了NJ85 VP60蛋白的分子量、等电点、疏水性和二级结构,根据同源模型预测分析T-级结构。NJ85 VP60的二级结构以B片层为主,三级结构稳定。病毒衣壳表面有32个杯状凹陷, 由9o个二聚体组成,二聚体由VP60单体以A/B5和C/C2两种方式构成,单体在二聚体中的构象有A、B、C三种形式。单体含有S、P两个结构域,两者通过柔性绞链连接,P结构域由VP60的C端部分形成,P分为P1和P2二个亚结构域,P2位于病毒衣壳表面,含有病毒株特异性抗原表位和红细胞结合位点。

鼠痘病毒的分离鉴定及感染性研究

安学芳, 刘峰松, 方明刚, 朱幼玲, 王汉中

2003, 18(6): 563

分别从自然感染死亡的小鼠不同病理组织内分离到一种大小为250~300nmX160~190nm 的卵圆型病毒粒 子。以腹腔注射的方式将纯化的病毒粒子回复接种到小鼠体内,能引起小鼠死亡,从不同的病理组织内分离到与 接种病毒粒子相同的病毒粒子。病理组织的超薄切片电镜观察结果表明在肝、肺、脾、肠等组织的细胞质内均能 观察到病毒粒子的存在,表明病毒的复制和装配是在细胞质内完成的,该病毒应归类于痘病毒科。

牛泡沫病毒中国株感染兔的研究

余 荭, 孔晓红, 宣成昊, 王金忠, 陈启民, 耿运琪

2003, 18(6): 566

将牛泡沫病毒(BFV3026)感染的细胞经耳缘静脉注射兔子,并以正常细胞注射的兔为对照。1年后处死, 病毒挽救实验及PCR检测显示:兔经一次注射即可被BFV3026感染,病毒广泛分布于感染兔的多种脏器中,通 过共培养可从感染兔血、肝、脾、肺、肾中拯救出相应感染性病毒颗粒,并在脑、骨髓、心、胰、肠系膜中检到 高拷贝BFV原病毒DNA存在。同时,血清学检测表明:感染兔在接受注射一个月后即产生高滴度抗病毒蛋白抗 体,并维持该滴度水平直至实验终止,兔未表现任何可观病变。 Abstract:Rabbits were injected with a single intravenation of Bovine foamy virus 3026(BFV3026)· infected cells in the ear and monitored for sera-conversion serologically up t0 l year.Results of the serological an d virus-rescue assays indicated that all animals were persistently infected with BFV3026 an d showed sustained BFV3O26.spec ific humoral immune response.BFv3026 Was rescued by co-cultivation with fetal bovine lung(FBL)cells from the spleen,kidney,lung,liver and peripheral blood leukocytes of the infected an imals.Virus sequence in poZ was、recovered an d amplified from the tissues where the virus Was rescued from,such as from the heart,brain,bone marrow,mesentery an d the pan creas.In addition.BFv transcripts could be detected in PBL by RT.PCR. S animal model might be usefulfor studyingtheinteractionbe tweenBFv an dBIV

甜菜夜蛾核多角体病毒sod基因的克隆及原核表达

张海元, 牛国栋, 洪靖君, 张忠信

2003, 18(6): 576

甜菜夜蛾核型多角体病毒中国株(Spotoptera exigua MNPV—z)超氧化物歧化酶基因(sod)业已被克隆及 在大肠杆菌中进行了表达,证明了SeMNPV—Z的sod基因产物确有SOD活性,其活力单位约为291.1913/mL培 养液。DNA测序结果表明SeMNPV.Z的sod 基因编码151个氨基酸,与人的sodl基因的核苷酸的同源性为50%, 与LdNPV、HaSNPV、HcNPV、AcNPV和BmNPV的sod 基因的同源性分别为64%、63%、63%、65%、63%。 Abstract:Superoxide dismutases scavenge superoxide radicals and protect cells from oxidative stresssod gene of Spotoptera exigua Multi Nucleopoleohedrovirus(SeMNPV)from China has been clonedan d expressed in E.coli with the activity of SOD being 29 1.1 9U,mL.DNA sequence an alysis showedmat sod gene of SeMNPv encoded 1 5 1 amino acids.The sequences homology between SeMNPv sodgene an d human sodl gene was 50% ,an d 64% between SeM NPV an d LdNPV,63% be tw e n SeMan d HaSNPV,HcNPV,BmNPV,65% between SeMNPv and AcNPV

马尾松毛虫CPV基因组S7的序列分析及部分序列的原核表达

洪靖君, 张海元, 赵淑玲, 段家龙, 彭辉银

2003, 18(6): 581

马尾松毛虫质多角体病毒(湖南株)基因组s7节段(AYI80908)cDNA克隆及序列分析结果表明:S7由 1501个碱基组成,编码由448个氨基酸组成的分子量为49.8 kDa的多肽P50。5 末端和3 末端具有5 -AGTAA-3 和5 -GTrAGCC-3 末端保守序列。该基因组与舞毒蛾质多角体病毒l型和家蚕质多角体病毒1型s7节段有很高的 同源性,核苷酸序列同源性分别为97.2%和87.0%,氨基酸序列同源性分别为98.7%和92.8%。P50多肽与人型支 原体的DnaK样蛋白在C.末端有相似性。本文报道了编码P50 C259的cDNA序列的克隆并作了原核表达,当用 1.0 mmol/L皿P,I’G 诱导2h,分子量约为37_3 kDa的融合蛋白在大肠杆菌BL21中得到大量表达。

SeMNPV抑制HaSNPV诱导的Se—UCR细胞凋亡

梁昌镛, 胡志红, 陈新文

2003, 18(6): 587

摘要:在采用共感染和共转染的方法构建扩大杀虫范围的重组病毒的研究过程中发现棉铃虫单核衣壳核多角体 病毒(Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus。HaSNPV)能诱导甜菜夜蛾细胞Se-UCR发生典 型凋亡,但不能诱导另一株甜菜夜蛾细胞Se.301产生凋亡。以5 MOI的HaSNPV感染Se-UCR。在12h左右可 以观测到少量细胞凋亡。24h能观察到明显的凋亡,凋亡细胞数量随时间不断增加,到72h基本上所有的细胞均 发生凋亡,成为凋亡小体,基因组DNA片段化。同时发现HaSNPV诱导的甜菜夜蛾Se-UCR细胞凋亡能够被甜 菜夜蛾多核衣壳核多角体病毒(Spodoptera exigua multicapsid nucleoplyhedrovirus,SeMNPV)所抑制, 进一步点杂 交试验发现SeMNPV 和HaSNPV共同感染Se.UCR获得了HaSNPV在该细胞中的复制。 Abstract:Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus(HaSNPV)and Spodopteraexigua muUicapsid nucleopolyhedrovirus(SeMNPV)are commercially produced as pesticides.Aspe sticides,both viruses have very narrow host range and Can not make cross infection.Trying tOgenerate a rec ombinant baculovirus with broader host range by CO—infection and co-transfection,wefound that HaSNPV could trigger apoptosis of a exigua cell line Se·UCR.which could be blocked bySeM NPV,but apoptosis did not occur in Se一30 1,a cell line also from exigua.W hen infected Se-UCRwith 5M OI,theapoptoticbodywasIn'stfound at 12hpi,obvious apoptosis couldbe observedat 24hpi,at the end almost all ceHs were dead at 72 hpi.Those observations were confu-med by genome DNAfragmen-ration analysis.Dot blot an alysis indicated that SeMNPV could also help HaSNPV genomeDNA an d then virus repficafion in Se-UCR cells,but not in Se-301.

亚洲小车蝗痘病毒球状体蛋白基因的克隆与序列分析

赵朝阳, 王丽英, 李永丹, 贪桂玲

2003, 18(6): 593

摘要:通过PCR扩增,获得OaEPV球状体蛋白基因编码区序列,并进行克隆、测序。分析结果显示,OaEPV球 状体蛋白基因编码区全长为2967bp,编码分子量为l1lkDa的球状体蛋白。同源性分析表明,OaEPV球状体蛋白 基因与直翅目昆虫痘病毒关系最近,与鳞翅目和鞘翅目昆虫痘病毒关系较远。对已知的几种昆虫痘病毒球状体蛋 白基因做系统进化树,结果显示,以病毒寄主的昆虫分类目作为昆虫痘病毒的分属依据,更符合分子水平的分析 结果,也与近年来许多学者所提出的“将鳞翅目与直翅目昆虫痘病毒分为2个不同的属”的观点相一致。对这些 球状体蛋白氨基酸序列的疏水性进行分析,表明球状体蛋白的疏水性随寄主昆虫所处的目不同而表现出较大差 异,这可能是因为病毒与寄主长期的协同进化的结果。 Abstract:TIle coding region of Oedaleus asiaticus EPV spheroidin gene was obtained by PCR.and thisfragment Was subsequently cloned,seq uenced an d an alyzed.Tlle data shows OaEPV sph coding re onis 2967bp in size which encodes a ll l l(Da protein.Homology an alysis demonstrated that 0aEPV iscloser to Orthoptcran EPVS than Lepidopteran an d Coleopteran EPVS.Phylogenetic tree derived fromall known EPVS spheroidin gene indicates that the order of the EPV host is in relationship with thegenus of corresponding EPV.which agres wit|l a recent point that “Lepidopteran EPVs an dOrthopteran EPVS should be divided into two distinct genus”.Hydrophobicity plots of these kn ownsperoidin show that the more distan t relationship of the orders the viruses hOsts be long to.the morediverse of the property of EPV spheroidin to each other.It may be explained as the result ofCO.evolutionbe tweentheEPVSan dtheirhos

棉铃虫单核衣壳核多角体病毒3个bro基因的原核表达与抗体制备

陈明明, 吴东, 袁 丽, 陈新文, 胡志红料

2003, 18(6): 597

摘要:根据棉铃虫单核衣壳核多角体病毒(HaSNPV)基因组全序列,设计引物,用PCR的方法扩增得到bro-a、bro-b 和bro.C三个全长基因。将这三个基因的片段分别克隆至原核表达载体pProExHTb,经IFIX3诱导,在E.coliDH50 菌株中得到了目的基因的高效表达。表达的目的蛋白大小分别为32kDa、64kDa和58kDa,经SDS-PAGE分离纯 化,免疫新西兰大白兔制备了多克隆抗体。抗体经1:2500倍稀释后用于WestemBlot分析,获得特异性显色信 号,所制备的三种抗体适合用作bro基因的功能的进一步研究。 Abstract:Three baculovirus repeated open reading frame(bro)genes were amplified by PCR from thegenome DNA of HaSNPV.The PCR products were cloned into pBluescript KS(+)plasmid.The geneswere introd uced into the expression vector pProExHTb.After IFFG induction.the E coli DH5a straincontaining each of the recombinant plasmids expressed proteins with molecular weights of 32 kDa、64kDa an d 58 kDa,respectively,which were in agreement with the prospeetation.Th e purifiedrecombinant proteins were used to immune the rabbits separately.Western blot an alysis using themulticlonal an tibodies derived from the rabbits indicated that these an tibodies could react specificallywith thetargetproteinsan dwere suitableto beusedforfurtherfunctional an alysisofthebrogenes.

感染HaSNPV棉铃虫幼虫的组织病理和免疫组化研究

欧阳志荃, 孙修炼, 邓 菲, 袁 丽, 胡志红

2003, 18(6): 603

摘要:本文报道了棉铃虫单核衣壳核多角体病毒(Helicoverpa axmigera single nucleocapsid nucleopolyhedrovims, HaSNI’V)感染棉铃虫幼虫的病理时相及HaSNPV 多角体蛋白在幼虫组织中表达的免疫组化研究。以5xl03pFu 的HaSNPV出芽病毒粒子(BV)注射4龄初的棉铃虫幼虫,石蜡切片的H-E染色表明,在感染后24h,病理变 化不明显;48h后脂肪体、气管组织的细胞核开始肿大,细胞开始变形;72h后脂肪体、气管、真皮细胞核肿大 十分明显,组织结构松散;但中肠和肌肉组织未见明显病变;96h后,脂肪体、气管、真皮组织结构完全被破坏, 肌肉组织变疏松。免疫组化结果表明,感染后24h,未能检测到多角体蛋白在幼虫组织中的表达;感染后48h, 多角体蛋白可在部分脂肪体、气管组织、血细胞和真皮组织的细胞核中表达;72h后,被感染的脂肪体、气管组 织、和真皮组织的细胞数比48h多; 96h后,多角体蛋白可以在脂肪体、气管、真皮组织中大量表达,48h到96h 间,被感染的血细胞数目基本不变,中肠组织和肌肉都未检测到多角体蛋白的表达;幼虫死亡后,可在中肠上皮 细胞的基底膜间隙中检测到多角体蛋白的表达,肌肉组织中未见表达信号,其它组织全部被感染并且组织结构被 破坏。H.E的结果与免疫组化的结果基本相符。 Abstract:Here we report the pathogensis process of Helicoverpa armigera larvae infected with Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus(HaSNPV)using H.E staining and immunohistoc hemistry with an tibody against polyhedrin.The fourth instar Helicoverpa armigera larvae were injected with 5×l PFU HaSNPV per larva.H.E staining showed that no apparent pathogensis could be detected in all tissues at 24 hours post infection(hpi).But at 48 hpi,the nucleus ofthe fat body an d trachea epidermis became swollen,an d the shape ofthe cells in these tissues were chan g~ .At 72 h P i,significant hispathogensis were observed in fat body,trachea epidermis an d the epidermis,while no chan ges were found in midgut an d muscles.At 96 hpi,the constitution of the fat body,trachea and epidermis were destroyed.Th e immunohistoc hemistry results indicated the po lyhedrin protein could not be detected at 24 hpi.At 48 hpi,it was found partially expressed in the tissues of fat body,trachea epidermis an d epidermis.From 72 to 96 hpi,the po lyhedrin was found heavily expressed in fat body, trachea epidermis and epidermis.No po lyhedrin was found expressed in midgut untill larvae dead an d themusculewasnotbe infected allthetime.

Harpin的表达及其诱导抗烟草花叶病毒感染的活性

徐进平, 孟小林, 王健, 鲁伟, 张俊杰

2003, 18(6): 607

摘要:采用人工分段合成梨火疫病细菌(Erwinia amylovora)Harpin基因A、B、C、D片段,然后连接构建全长 harpin基因,克隆到pET-23(+)表达载体中,在E.coliBL21中表达Harpin蛋白。通过His BindResin Ni” 层析柱 纯化,获得Harpin蛋白。经SDS.PAGE和Westernblot分析,该蛋白分子量为45 kDa。采用叶片穿刺法,5pL3 edrnL Harpin能诱发烟草叶片的过敏反应。采用叶脉注射法,5pL 300g/mL Harpin能诱发辣椒叶片的过敏反应。将烟草 和辣椒用3 g/mE Harpin喷雾处理5d后,接种烟草花叶病毒(Tobacco mosaic virus,TMV),观察植株病症,并 采用ELISA检测TMV含量。结果表明经Harpin处理的烟草和辣椒对TMV侵染有一定的抑制作用。
Contents

含生物型增效剂棉铃虫病毒悬浮剂的应用效果研究

张忠信, 张海元, 牛国栋, 袁克静, 段智勇, 张秋红

2003, 18(6): 571

摘要:本文报道含生物型增效剂棉铃虫核多角体病毒(Helicoverpa armigera nucleopolyhedrovirus,HaNPV)悬浮剂 对害虫的防治效果及其对天敌数量的影响。作者筛选灭幼脲类似物作为棉铃虫核多角体病毒的生物型增效剂, HaNPV+4.2ppm 氟啶脲(chlorfluazuron)感染3龄初棉铃虫幼虫,感染幼虫的半致死时间(U『50)为2.24d,比 单用HaNPV感染幼虫的U’50缩短2.66d。含生物型增效剂的棉铃虫核多角体病毒悬浮剂在田问应用,施药后5d, 对2代和4代棉铃虫的防治效果分别为81.4%-85.2%和70.7%,-82.6%,施药后7d,对2代和4代的防治效果分别 为85.2%,-,86.3%和69.6%,-,82.9%。棉铃虫病毒悬浮剂中的生物型增效剂对天敌数量仅有轻微影响。含生物型增效 剂棉铃虫核多角体病毒悬浮剂不仅能有效控制害虫,而且对天敌有很好的保护作用。 Abstract:The chlorfluazuron,a chitinase inhibitor acting on pest like a biological agent was selectedfor enhance Helicoverpa口, era nucleopolyhedrovirus(HaNPV)pesticide.The third instar larvaewere infected with the virus mixed witI1 4.2ppm chlofluazuron,the LT50 value Was reduced to 2.44 days,while the larvae infected only with the virus,the LTso was 4.9O days,and only 4.2 ppm chlofluazumdid not cause the larva death.TIle H V suspension formulation pesticide.which containingchlorfluazuron,Was produced in the factory.In field test,the 2nd or 4th generation larvae Was controlledby the viral pesticide,7 days after spaying the pesticide,the efi ciency were 85.2%,-,86.3% an d69.6% —暑2.9% respectively,only slight efec t of the viral pe sticide on the nature enemi es Was observed .
Brief Reports

河北省丙型肝炎病毒基因分型研究

牛建章, 许若丹, 白瑞婷, 张立平, 孟宗达

2003, 18(6): 611

丙型肝炎病毒(Hepatitis C virus,HCV)感染是 输血后肝炎的主要原因,主要通过输血或使用污 染的血制品传播【2】,且与肝硬化和肝细胞癌的发生 有密切关系。1985年河北省在国内首先报告了丙型 肝炎的爆发流行【2】,此后,在HCV的病源学【3】、流 行病学【4】和检测技术[51等方面进行了较深入的研 究,取得了一批重要的研究成果。1991年美、日学 者先后报告了HCV的全基因序列,为基因分型研究 奠定了重要基础。Okamoto等[6.71根据HCV核心区 基因序列的不同,设计型特异性引物,用巢式 RT.PCR方法,将HCV分为I(1a)、II(1b)、III(2a)、 IV(2b)4型。为了探讨河北省HCV基因型分布,为病 毒性肝炎的防治提供科学依据,本文对河北省HCV 基因型分布进行了研究。

猪繁殖与呼吸综合征与伪狂犬病混合感染的分子生物学快速诊断

许立华, 苏鑫铭, 王志亮, 吴延功, 陈溥言

2003, 18(6): 614

猪繁殖与呼吸综合征自1987年发现至今,已 在世界大多数养猪国家和地区普遍存在【1.2】。而伪狂 犬病作为仅次于口蹄疫、猪瘟的危害养猪业的重要 疫病,一直引起人们的高度重视【3.4l。这两种传染病 在猪群中发生以后,同样出现妊娠母猪流产、产木 乃伊胎等繁殖障碍,从流行病学、临床症状等方面 也难以做出判断。 Abstract:According to the nucleotide sequences of Porcine Reproductive and Respiratory Synd romeV/ms VR2332 strain and Pseudorabies virus shan ghai strain provided in GenBank, two pairs ofprimersweredesignedto amplifytheN geneofPRRSV byRT—PCR an dTkgeneofPRV byPCR. Asa result, a rapid molecular diagn osis with high specificity an d accuracy was set up.The resultindicated that PRRSV Was detected from 11 out of 33 samples, PRV was detected from 9 out of 33sam ples, an d CO—infec tion by PRRSV an d PRV was confirmed in 8 samples, the rate of co-infectionWas at24.2% .
Review

肥胖可能与病毒感染有关

李爱春

2003, 18(6): 616

随着生活水平的提高,肥胖已呈现为全球性问 题。当今已将肥胖与艾滋病、癌症并称为21世纪 威胁人类健康的三大疾病。而其中肥胖症则是人类 健康的最大威胁。当前人们普遍认为肥胖是由于遗 传、过食、运动不足、代谢异常、内分泌异常、脑 疾患等因素引起。但是这些均不能完满地解释肥胖 的成因。为此,美国科学家Dhurandhar等提出了一 种新的解释。他们认为,肥胖可能与病毒感染有关⋯。 并已研究发现了第1株可引起动物肥胖的人腺病毒 血清36型(adenouirus Ad-36),这是迄今所发现的 第1株与肥胖有关的人腺病毒【2】。证据表明,该病 毒感染在动物肥胖发病中起着重要作用【2 。是否 能导致人类肥胖尚不得而知。但是,有关肥胖的病毒 发现使肥胖成因的研究有了新的思考,为人类进一 步了解肥胖问题的根源,以及肥胖的防治开辟了新 的领域,展现出新的希望。至于病毒导致动物肥胖 的具体机理以及人类肥胖是否与病毒感染有关至 今尚不明确,有待深入研究。