云南五个不同地区烟草花叶病毒外壳蛋白基因的序列比较

Five Tobacco mosaic virus isolates, obtained from tobacco leaves showing typical symptom in Qujing, Honghe, Dali, Chuxiong and Yuxi in Yunnan province, were selected and studied from 637 TMV samples. Using a pair of primers specific for TMV-U1 strain, a specific fragment of 530bp including the TMV coat protein gene was amplified using IC-PCR. The products of PCR were cloned and sequenced. The nucleotide and amino acid sequences of the coat protein of the five isolates were found to be very similar each other and have more than 90% sequence identity with TMV-U1, TMV-B,TMV-P, TMV-FUJIAN, although minor differences existed among them. The results showed that the five isolates from Yunnan province belong toTMV-U1 strain.     

野猪及其与家猪杂种猪Myostatin基因3'编码区的克隆和序列分析

Myostatin, which is a highly conservative gene among breeds, is a negative regulator of muscle. The 3' coding region of wild boar and crossbred pig myostatin was cloned by RT - PCR and sequenced. Compared with that of GenBank, the homology of the nucleotide sequence between wild bear and crossbred pig is identical in this region indicating that domestic pigs were evolved from wild boar and there was not changed in this region during the evolution processes.     

Genome organization and variation in the 3′-partial sequence of garlic latent virus in China

Ten different isolates of a carlavirus were detected by degenerate PCR from 12 garlic samples collected from 6 provinces in China, and the complete genome sequence of the Zhejiang isolate ZJ1 and 3'-terminal sequences of 9 other isolates were determined. The RNA genome of isolate ZJ1 consisted of 8363nts excluding the 3'-poly (A) tail, and the genome organization was similar to other carlaviruses with 6 open reading frames encoding a replicase, TGB1, TGB2, TGB3, CP and NABP respectively. Sequence comparisons showed that all 10 isolates were Garlic latent virus (GarLV). The variations in the TGB2, TGB3 and NABP were more significant than those in the CP. High homology was also detected between those isolates and Shallot latent virus (ShLV). Phylogenetic analysis suggested that GarLV isolates from garlic can be divided into 4 main groups and Chinese isolates belonged to each group. This is the first reported molecular analysis of members of the genus Carlavirus in China.     

伪狂犬病病毒湖北株糖蛋白gD基因的克隆及序列测定

According to the sequence of gD gene of PRV Rice strain, the primers of 22bp were designed.Using PRV genomic DNA of Hubei and Shuangcheng virus strains which infected BHK 21 cell separately as template, the gD gene of PRV was amplified sucessfully by PCR and cloned into pGEM T vector. Restriction enzyme analysis showed that the cloned gD gene at SmaⅠ,SalⅠ,KpnⅠ,PvuⅡ sites was the same as that of PRV Rice strain. The gD gene consisted of 1,263 nucleotides including an open reading frame spanning 1,197 nucleotieds which could encode a protein of 398 amino acids. The ORF didn′t include an amino acid sequence directing N linked glycosylation (NXT or NXS). Comparison of our complete Hubei strain gD gene sequence with the Rice strain gD gene sequence showed that the nucleotide and deduced amino acid homology were about 97% and there was an 12 basepair deletion in 835 846 nucleotide sites that coded Arg Pro Arg Pro. A region of the amino acid sequence and the positions of the cysteine residues of PRV HB gD were homologous to HSV I glycoprotein D. This work laid foundation for PRV gene immunization and studying PRV sub unit vaccine.     

耐辐射奇球菌超氧化物歧化酶基因的克隆与序列分析

By using a 453 bp length gene fragment of superoxide dismutase(SOD)as a probe,which was firstly amplified from Deinococcus radiodurans genomic DNA by PCR with degenerate oligonucleotide primers corresponding to the conservative regions of known SODs,a putative SOD gene was identified from the database of D.radiodurans whole genome.Its 636 bp length open reading frame and 5′ and 3′ flanking sequence was determined.The conventional E.coli ribosomal and RNA polymerase binding sites were found upstream from SOD encoding region and an inverted repeat sequence downstream of the termination codon.The deduced 211 amino acid sequence of the structural gene showed a high similarity to other manganese and iron containing SODs in normally conserve regions.     

A Pair of Novel Primers for Universal Detection of the NS1 Gene from Various Bluetongue Virus Serotypes

Twenty five serotypes of Bluetongue virus （BTV） have been identified worldwide. Rapid and reliable methods of virus universal detection are essential for fighting against bluetongue （BT）. We have therefore developed and evaluated a pair of primers which can detect various serotypes of BTV by RT-PCR. Analysis of the viral protein 7 （VP7） and the non-structural protein （NS1） gene from different serotypes of BTV by DNAstar showed that the 5＇end of the NS1 gene is the most conserved region. The primer pairs （P1 and P2） were designed based on the highly conserved region of NS 1. The novel primers were evaluated by detecting BTV serotypes 1, 3, 5, 8, 10, 11, 21 and 22. The specificity of the primers was estimated by comparing to gene sequences of viruses published in GenBank, and further assessed by detecting BTV serotype 1-12 and Epizootic hemorrhagic disease virus （EHDV） serotype 1-4. The sensitivity and repeatability of PCR with the novel primers were evaluated by successfully detecting the recombinant plasmid pGEM-T121 containing the diagnosed nucleotide sequence. Our results suggest that these unique primers can be used in high throughout and universal detection of the NS1 gene from various BTV serotypes     

蜘蛛基因组DNA Cosmid文库构建和拖丝蛋白基因的克隆

The genomic DNA Cosmid library was constructed from Nephila clavipes spider muscle using SuperCos 1 Cosmid as a vector.The title of library was >5×10 4 cfu/μg ligated DNA.On the basis of published sequence from a partial cDNA sequence of the 3′end of the dragline silk gene, we designed and synthesized 3 oligonucleotides.Oligonucleotides were labeled with non radioactive digoxigenin dUTP and detected with chemilluminescent substrate.56 positive recombinants were screen from the Cosmid library using DIG Oligo 2 as a probe.DNA dot hybridization using DIG Oligo 1 and DIG Oligo 3 as the probes, respectively, 3 positive signals were identified from 56 colonies.They were appeared the same pattern when DNA from the colones digested by restriction enzymes.The spider dragline silk gene was confirmed again by Southern blot hybridization.     

Variation in the Coat Protein Gene of Papaya ringspot Virus Isolates from Multiple Locations of China

The potyvirus Papaya ringspot virus （PRSV） is an important pathogen of papaya that causes severe losses in economic crops for papaya production globally. The coat protein （CP） genes of five PRSV isolates originating from different locations in China were cloned and sequenced. The CP-coding region varied in size from 864-873 nucleotides, encoding proteins of 288-291 amino acids. The five Chinese isolates of PRSV have been characterized as papaya-infecting （PRSV-P）. The CP sequences of the Chinese isolates were compared with those of previously published PRSV isolates originating from different countries at amino acid levels. A number of KE repeat boxes in the N terminus of the PRSV-CP were found in all Chinese isolates. The phylogenetic branching pattern revealed that there was certain extended grouping between geographic locations, and the Asian type probably represents the oldest population of PRSV. The information of CP genes will be useful in designing and developing durable virus resistant-PRSV transgenic papaya in China. Meanwhile broad-spectrum-virus resistant, strongly resistant-PRSV and good safe papaya lines are required.     

Molecular Epidemiological Investigation of Infectious Hypodermal and Hematopoietic Necrosis Virus and Taura Syndrome Virus in Penaeus Vannamei Cultured in China

The Infectious hypodermal and hematopoietic necrosis virus(IHHNV) and Taura syndrome virus(TSV) are two important shrimp viruses in cultured shrimp in America.These two viruses were transmitted to China at the beginning of the 21st century.In this study,214 shrimp samples of Penaeus vannamei were collected from seven different areas of China and tested by PCR for IHHNV and TSV infection.The results showed that there were a high prevalence of IHHNV(65.42%) and low prevalence of TSV(3.27%) in the tested samples.Several samples were found to be co-infected with these two viruses.A 3 kb fragment of 7 positive IHHNV samples and a structure protein region(ORF2) of three TSV positive samples were amplified and sequenced.The sequence comparison indicated that both IHHNV and TSV sequenced in China have a low genetic variations compared with the prototype IHHNV and TSV from Hawaii.Phylogenetic analysis showed that TSV isolates were clustered into two groups,Asia and America group,which was genetically correlated to geographic distribution.     

登革病毒NS3全基因的扩增与克隆

登革热(DF)、登革出血热及登革休克综合征(DHF/DSS)是由登革病毒所致的两种不同临床类型的急性传染病,广泛流行于全球热带及亚热带地区.DHF/DSS以高热、出血、休克、高病死率为主要特征,近年来其发病率有迅速增加的趋势,已成为严重影响人类健康的公共卫生问题.     

登革病毒NS3全基因的扩增与克隆

登革热(ＤＦ)、登革出血热及登革休克综合征(ＤＨＦ/ＤＳＳ)是由登革病毒所致的两种不同临床类型的急性传染病,广泛流行于全球热带及亚热带地区。ＤＨＦ/ＤＳＳ以高热、出血、休克、高病死率为主要特征,近年来其发病率有迅速增加的趋势,已成为严重影响人类健康的公共卫生问题。迄今,ＤＨＦ/ＤＳＳ的发病机制仍不清楚,亦无有效的特异性预防方法[1]。登革病毒属于黄病毒科的黄病毒属,有Ⅰ、Ⅱ、Ⅲ、Ⅳ四个血清型,基因组为单股正链ＲＮＡ,全长约11ｋｂ,编码三种结构蛋白和七种非结构蛋白。基因组顺序为5′ＣＰｒＭＥＮＳ1ＮＳ2ａＮＳ2ｂＮＳ3Ｎ…     

丙型肝炎病毒Core与NS3基因的不同融合方式及其表达

丙型肝炎病毒(简称ＨＣＶ)是危害人类健康的传染性疾病,预防该病的传播主要借助于ＨＣＶ血清学诊断来筛选献血员和检测临床标本。从国内外已分离到的ＨＣＶ株得知,ＨＣＶ核心蛋白(Ｃｏｒｅ)和非结构蛋白ＮＳ3含有较强的优势抗原表位,其相应的抗体出现早,阳性率高,已成为目前第二、第三代ＨＣＶ免疫诊断试剂的重要成份[1,2]。通常Ｃｏｒｅ蛋白和ＮＳ3蛋白分别用基因工程方法表达,然后作为固定化抗原,检测人体内ＨＣＶ的抗体。我们对Ｃｏｒｅ和ＮＳ3的两种不同组合方式及其表达的研究,对于发展新的ＨＣＶ诊断试剂很有帮助,现将结果报道如下。1…     

口蹄疫病毒非结构蛋白P3区的基因序列及分析

以口蹄疫Akesu/ 5 8分离株的 5 3代牛舌皮病料为材料 ,采用RT PCR法 ,扩增和克隆了两个约 1.5kb的DNA片段。核酸序列测得结果对接后 ,涵盖了全部P3区的基因序列。口蹄疫Akesu/ 5 8分离株基因组P3区的核酸序列共计 2 ,72 4nt,包括一个终止密码子TAA ,共编码 90 7个氨基酸 ;其中非结构蛋白 3A的基因是 45 9nt,编码 15 3个氨基酸 ;3个 3B(VPg)基因分别是 6 9、72和 72nt,氨基酸分别为 2 3、2 4和 2 4;3C是 6 39nt,2 13个氨基酸 ;3D是1,413nt ,471个氨基酸。各蛋白间由Glu/Gly(Ser)连接。序列比较显示 :3A的C端易变 ,其它区的变易呈零星散在     

甲3型流感病毒M2基因在痘苗病毒天坛株中的表达

为获得表达甲3型流感病毒(H3N2)M2蛋白的重组天坛株痘苗病毒RVJ1175M2,使用PCR方法扩增流感病毒全长M2基因,将其克隆到天坛株痘苗病毒同源重组质粒pJSC1175中,获得重组质粒pJSC1175M2,通过与痘苗病毒载体同源重组,构建了含流感病毒M2基因的重组痘苗病毒株RVJ1175M2。PCR检测结果证明,流感病毒(H3N2)M2蛋白基因准确插入到天坛株痘苗病毒TK区;Western blot、免疫荧光和流式细胞计数表明重组病毒RVJ1175M2可以有效地表达M2蛋白,表达的M2蛋白有两条带,分别为15kD和13kD,与相关文献报道一致;M2蛋白可有效分布在感染细胞的细胞膜上。这些结果表明重组痘苗病毒株RVJ1175M2可以有效地表达流感病毒M2蛋白,为使用表达M2蛋白的不同类型疫苗进行广谱流感疫苗效果的比较研究奠定了基础。     

登革3型病毒prM-E基因重组质粒DNA的构建和真核表达

构建登革 3型病毒 prM E基因的真核表达重组质粒 ,并进行体外表达 ,为登革DNA疫苗的研究奠定基础。用RT -PCR法获得 prM -E基因片段 ,然后将其克隆到真核表达载体中。用电穿孔法将重组质粒DNA转入BHK细胞 ,通过免疫荧光法检测外源基因在真核细胞中的表达。结果 ,通过酶切和序列测定证实了构建的重组质粒DNA含序列正确的 prM- E基因。用免疫荧光法检测到转染了重组质粒DNA的BHK细胞的胞浆中有登革 3型病毒特异蛋白的表达。说明含有登革 3型病毒prM -E基因的真核表达重组质粒可以在BHK细胞中表达 ,该结果为观察该重组质粒的免疫原性奠定了基础。     

三种丙型肝炎病毒转基因小鼠系的同时制备

为研究丙型肝炎病毒的致病致瘤机理及结构基因与非结构基因3区(NS3)的功能及其在HCV感染致病中的作用,建立一个HCV分子治疗的动物模型,构建了含金属硫蛋白启动子和HCV结构基因或NS3基因的质粒,将两者等量混合后用显微注射法接种于昆明白小鼠受精卵内制备转基因小鼠.通过PCR筛选获得三种整合HCV结构基因或/和NS3基因的首建鼠.结果表明:a.注射后卵存活率与仔鼠出生率分别为81%、30%;b.检测60只G0代小鼠,结构基因整合鼠6只(10%),NS3基因整合鼠4只(6.7%),双基因整合鼠9只(15%),总整合率为31.7%;c.RT-PCR法检测阳性鼠肝中有靶基因mRNA的转录;d.4只首建鼠与正常鼠回交获得38只G1小鼠,其中20只为整合鼠,整合率为52.6%;e.转基因鼠表型迄今无明显异常.表明一次显微注射同时获得了三种整合HCV结构基因或/和NS3基因的转基因小鼠.     

表达鸡新城疫病毒F、HN基因和传染性喉气管炎病毒gB基因的重组鸡痘病毒的构建及其特性研究

利用同源重组将新城疫病毒(NDV)的F和HN基因、传染性喉气管炎病毒(ILTV)的gB基因以及报告基因LacZ插入鸡痘病毒(FPV)的017株的复制非必需区,其中NDV的F、HN基因、ILTV的gB基因以及报告基因LacZ是在早晚期启动子LP2EP2的控制下,大肠杆菌报告基因LacZ在晚期启动子P11的控制下。经过10轮蓝斑纯化获得了包含了NDV的F和HN基因、ILTV的gB基因以及报告基因LacZ的重组鸡痘病毒,称为rFPV-F/HN/gB/LacZ。经PCR方法证明rFPV-F/HN/gB/LacZ基因组中含有NDV的F基因、HN基因和ILTVgB基因;间接免疫荧光试验和Western-blot试验表明NDV的F、HN蛋白和ILTVgB蛋白在rFPV-F/HN/gB/LacZ感染的CEF细胞中获得表达。与亲本毒相比,重组病毒在病毒的复制和致鸡胚成纤维细胞的病变方面无显著不同。这证明了在鸡痘病毒载体的一个复制非必需区可以同时插入多个禽类病原的多个外源基因,为制备多价基因工程疫苗奠定了基础。     

庚型肝炎病毒—中国河北分离株部分NS3区基因序列分析

在急慢性输血后肝炎、散发性肝炎及爆发型肝炎中,大约１０～２０％的病人不属于已发现的Ａ～Ｅ型肝炎,提示存在新型肝炎病毒。在美国１９９５年第４６届医学年会上,已有了一些有关庚型肝炎病毒（ＨＧＶ）分子生物学及庚型肝炎血清学方面的摘要报道。１９９６年初Ｌｉｎ...     

口蹄疫病毒NSP 3ABC基因在昆虫细胞中的分泌表达及其活性检测

用设计的特异引物,扩增得到了N-端带有6×His编码序列的口蹄疫病毒完整3ABC基因序列,并将其亚克隆入带有蜂毒溶血肽序列的穿梭质粒pMelBac-B中,构建了重组质粒pMel-3ABC。将该重组质粒与杆状病毒骨架DNABac-N-BlueTM共转染Sf9昆虫细胞,通过噬斑筛选和PCR鉴定,获得了含有目的基因的重组杆状病毒。重组病毒感染Sf9昆虫细胞,采用通过SDS-PAGE和Western blot检测,证明目的基因在昆虫细胞中得到了正确的表达,表达产物分泌至细胞培养上清中,并具有良好的生物活性。表达的目的蛋白经过镍柱亲和层析法纯化后,用间接ELISA方法检测与口蹄疫病毒感染动物血清的反应性,证明表达目的蛋白与感染动物血清有很好的反应性而与正常动物以及免疫动物血清不发生反应。该研究为建立一种更加敏感和特异的口蹄疫病毒感染动物与疫苗免疫动物的鉴别诊断方法奠定了基础。