Biological and molecular characterization of Tomato spotted wilt Virus in Israel

Received April 24, 1997; received in final form June 29, 1997. Symptoms resembling tomato spotted wilt virus (TSWV) infections were documented among ornamental and vegetable crops in commercial greenhouses and open fields in Israel. Plants exhibiting these symptoms were collected from January 1992 to December 1996. Among cultivated plants analyzed for TSWV by enzyme-linked immunosorbent assay (ELISA), 19 species representing five families were found to be infected; natural infection was also recorded in six plant species of weeds. Virus identity was characterized by host range, serology and electron microscopy. Serological reaction with the isolates, found in Israel, using antisera from different sources as well as the sequence analysis of the nucleocapsid gene, demonstrated that the Israeli isolates of TSWV are a member of tospovirus serogroup I, type I (BR-01 strain). No virus transmission was found in seeds collected from virus-infected vegetable and ornamental crops. A non-radioactive molecular probe derived from the cloned nucleocapsid isolate enables specific detection of the virus in crude sap from infected plants. The detection of TSWV in Israel constitutes a severe potential threat to the ornamental and vegetable industry.     

猪大肠杆菌水肿毒素SLT-IIeA基因的克隆和序列分析

研究以本地猪水肿病大肠杆分离物ED1株为材料 ,利用 PCR克隆了含猪水肿病大肠杆菌 (VTEC) slt-IIe A基因 987bp的片段 ,并测定了含该克隆片段的 Bam HI、Hind 酶切片段的核苷酸序列。结果表明 ,slt-IIe A基因的编码区全长 960 bp,编码 3 1 9个氨基酸的蛋白质 ,序列与国外报导的 S1 1 79株进行比较发现其核苷酸同源性为 98.9%。经推导的氨基酸序列的同源性为 99.7%。这为进一步研究 slt-IIe A的生物学特性、水肿病的分子诊断及其防制打下基础     

Cortical gene expression pattern in rat focal cerebral ischemia

AIM: To investigate the genes differential expression in cortex during rat focal cerebral ischemia.METHODS: cDNA microarray chips containing numerous cDNAs were used to investigate the gene expression pattern between samples of focal cerebral ischemia and sham-control operation rats. RESULTS: Two hundred and eleven genes differentially expressed were screened out, among these genes, up-and down-regulated genes were 199 and 12, respectively. CONCLUSIONS: The analysis of gene expression pattern of focal cerebral ischemia based on cDNA microarray can realize high-throughput screening of the genes associated with the focal cerebral ischemia. The differential expression of genes may be related to the pathogenesis of focal cerebral ischemic diseases.     

Effects of blocking the expression of PKARⅠβ gene with antisense nucleotide on Shuang long-Jie gu Pill (SLJGP) containing serum osteoblast proliferation

AIM: To further investigate the role of PKARⅠβ in the growth-promoting effects of shuang long Jiegu pill (SLJGP), a Chinese medicine, on cultured osteoblasts. METHODS: pcDNA- antiPKARⅠβ, a recombinant expressing the antisense sequence of PKARⅠβ, was constructed and transformed HFOB1.19 by lipofectin. MTT was undertaken to assess the cell growth with the treatment of high dosage of SLJGP containing serum. RESULTS: Antisense gene blocked the growth-promoting effects of SLJGP containing serum on HFOB1.19. CONCLUSION: The function of SLJGP is closely related to cAMP-dependent protein kinase A.     

丝状霉形体丝状亚种SC型脂蛋白LppQ N末端基因克隆与序列分析

脂蛋白LPPQ是丝状霉形体丝状亚种SC型(MmmSC)非洲株、欧洲株和疫苗株所特有的。LPPQ N末端域具有良好的免疫原性，在牛体内可诱导产生强大、特异、早期、持续的免疫反应。本研究根据已发表的LPPQ基因序列设计引物，用Pyrobest^TM高保真DNA聚合酶从MmmSC HVRI X株中扩增出了LPPQ N末端基因序列，并进行了克隆与序列测定。核苷酸序列比较结果显示，HVRI X株的LPPQ N末端基因序列与国外发表的序列同源性为99．7％，由其推导的氨基酸序列同源性为99，1％，为脂蛋白LPPQ N末端基因体外表达奠定了基础。     

禽大肠杆菌外膜蛋白基因C(ompC)的序列分析

根据 Gen Bank中人源大肠杆菌 K- 12外膜蛋白基因 C(omp C)的核苷酸序列设计引物 ,应用 PCR方法从禽大肠杆菌 O2 、O78株及它们的融合双价弱毒菌株 O2 ,78(Norr Chlr)中分别扩增得到 omp C基因 ,序列测定及分析比较发现 ,3个菌株的 om p C基因均由 170 2 nt组成 ,核苷酸序列完全相同 ,只有 1个大的开放性阅读框 (ORF) ,长 10 92 bp,编码由 36 3个氨基酸组成的前 Om p C蛋白 ,前 2 1个氨基酸残基组成信号肽 ,成熟的 Omp C蛋白由 342个氨基酸残基组成 ,Mr为 4 0 0 0 0。其氨基酸序列也完全相同。从基因水平上证明了禽大肠杆菌 O2 、O78株及融合双价弱毒菌株 O2 ,78(NorrChlr)存在相同的外膜蛋白 C抗原 ,从而为进一步研究 Omp C蛋白的免疫原性奠定了基础     

猫细小病毒NS部分基因的克隆及序列分析

将疫苗用猫细小病毒(FPV)株提取基因组DNA,针对NS特定片段设计引物,利用PCR扩增出了部分基因并将该基因克隆到pMD18-T Vector中测序.结果表明,该基因长613bp,编码204个氨基酸.分离株基因与犬的细小病毒(CPV)、貂的细小病毒(MEV)毒株核苷酸同源性均为99%.氨基酸同源性达到97%、98%以上,与犬、貂的肠炎病毒有密切的亲缘关系.     

土耳其斯坦东毕吸虫肌动蛋白基因的克隆及其序列分析

目的利用RT-PCR和RACE技术克隆土耳其斯坦东毕吸虫肌动蛋白基因全长序列并进行序列分析.方法根据日本血吸虫和曼氏血吸虫肌动蛋白基因的保守区设计引物,利用RT-PCR扩增出土耳其斯坦东毕吸虫的肌动蛋白基因的大片段,再结合RACE技术分别得到肌动蛋白的3＇端和5＇端,将三部分序列拼接后获得肌动蛋白基因的全长cDNA序列并进行序列分析.结果成功克隆了土耳其斯坦东毕吸虫肌动蛋白的全长cDNA并提交GenBank,登录号为DQ017265,核酸序列比对表明东毕吸虫肌动蛋白基因的核苷酸序列和日本血吸虫、曼氏血吸虫的同源性分别为90%和91%.结论土耳其斯坦东毕吸虫肌动蛋白的全长cDNA的克隆为进一步表达及其生物学性能的分析提供了理论基础.     

H1N1猪流感广东株血凝素基因的克隆与序列分析

用RT-PCR方法扩增H1N1亚型猪流感病毒广东分离株A／Swine／GuangDong／711／2001HA基因，对其进行了克隆和测序。结果显示，HA基因全长1771bp．共编码579个氨基酸。A／Swine／Guang Dong／711／2001HA基因编码的氨基酸序列中有8个糖基化位点，5个位于HAl基因的第27、28、40、104和287位点，3个位于HA2基因的21、153和213位点。与H1N1亚型猪流感经典毒株比较后发现，血凝素糖基化位点并不是高度保守的。将A／Swine／Guang Dong／711／2001与自Gen Bank读取的1918年人流感毒株A／South Carolina／1／18、1991年人流感毒株A／MD／12／91和1997年猪流感毒株A／Swine／Wisconsin／238／97等进行核苷酸同源性比较分析，A／Swine／Guang Dong／711／2001与A／SouthCarolina／1／18、A／MD／12／91和A／Swine／Wisconsin／238／97等毒株的核苷酸同源性分别为93．9％、94．7％和93．9％。从系统发生树来看，A／Swine／Guang Dong／711／2001与A／South Carolina／1／18和A／Swine／Wisconsin／238／97的亲缘关系相近。     

一株鸭源禽流感病毒（H9N2）全基因序列分析及其排毒规律的研究

为研究一株鸭源H9N2亚型禽流感病毒(JN1株)全基因组的分子特征和遗传进化情况以及感染雏鸭后的排毒规律,对其生物学特性、全基因组序列以及遗传进化进行了分析,通过点眼滴鼻方式感染3周龄健康雏鸭,利用建立的实时荧光定量PCR(RT-PCR)检测鸭的排毒规律,并测定血清抗体效价。JNI株的鸡胚半数感染量为10~(-7.54)/0.2mL,鸡胚最小致死量的平均死亡时间为72h,静脉致病指数为0.266,抗原相关系数为0.47。系统发育分析表明,JN1株的HA与CK/HK/G9/97和DK/HK/Y280/97在同一分支上,NA、M、NP、PA、PB1、PB2基因与SH/F/98同源性较高,NS基因则与H5N1亚型禽流感病毒进化关系较近。HA的裂解位点为PSRSSR↓GL,存在6个潜在糖基化位点,在234位的氨基酸残基为L,NA基因颈部存在缺失。雏鸭在感染该病毒后,饮食正常,精神良好,3~17d均有排毒,咽拭子和泄殖腔棉拭子排毒规律基本一致,分别在第3天和第13天出现排毒高峰。该H9N2亚型禽流感病毒属欧亚大陆分支,为低致病性禽流感,病毒可通过呼吸道和泄殖腔向外界排毒,排毒时间较长。