棉铃虫单核衣壳核多角体病毒组织蛋白酶基因的序列分析和进化研究

对棉铃虫单核衣壳核多角体病毒（HaSNPV)的组织蛋白酶（v-cath）基因进行了序列分析,此基因编码区长1098bp,预防编码一个366个氨基酸的蛋白产物,序列分析表明,HaSNPV的V－CATH蛋白与共它杆状病毒的同源蛋白具有相似的保守结构并保留有相同的酶活性位点,根据已知的杆状病毒组织蛋白酶序列构建了v-cath基因的进化树,发现HaSNPV的v-cath位于NPV组中一个单独的分枝,结合v-cath基因在棉铃虫病毒基因组中的位置与其它NPV有较大不同,推测HaSNPV的v-cath基因可能拥有特殊的进化历程。     

东方粘虫颗粒体病毒超氧化物歧化酶基因的克隆与分析

为获得东方粘虫颗粒体病毒(Pseudelatia separata granulovirus,PsGV)基因组序列,采用随机克隆方法,建立PsGV的质粒基因文库,并通过对插入片段进行克隆鉴定和序列分析,获得编码超氧化物歧化酶蛋白的基因(PsGV-sod)。该基因阅读框为462bp,共编码153个氨基酸。核苷酸和氨基酸同源性比较结果表明该基因与其他颗粒体病毒同源性较高,通过保守基序分析,认为其为铜锌超氧化物歧化酶。     

马尾松毛虫质型多角体病毒多角体蛋白基因的cDNA克隆及序列分析

通过对马尾松毛虫质型多角体病毒的增殖、纯化,获得一株单一类型的质型多角体病毒.提纯的病毒粒子经SDS-酚抽提,琼脂糖凝胶电泳分离基因组dsRNDA,回收纯化第十片段S10.S10经DMSO变性,逆转录合成cDNA第一链,PCR扩增后,克隆在pGEM-T载体上.对重组子进行限制性内切酶分析及序列测定,结果表明,克隆片段全长763bp,起始密码AUG位于3～5残基,终止密码UGA位于747～749残基.推测DpGPV多角体蛋白基因编码248个氨基酸的多肽,分子量28kD.和家蚕质型多角体病毒(BmCPV)多角体蛋白基因相比较,核苷酸和编码氨基酸序列同源性分别为89.3%和97.6%.     

马尾松毛虫CPV基因组第5片段的cDNA克隆及其序列分析

通过差速离心从感染的马尾松毛虫幼虫虫体中提取质型多角体病毒。碱解法提纯病毒粒子,1％琼脂糖凝胶分离基因组dsRNA,回收纯化第五片段(S5)。根据同源性设计五对引物,经RT-PCR,最终获得五个亚克隆片断,测序拼接后,得到S5全长。片断全长2851个核苷酸,包括一个2643个核苷酸的开放阅读框。推测DpCPVS5基因编码了881个氨基酸长的多肽,分子量为100．3kDa,与舞毒蛾质型多角体病毒(LACPV—1)和家蚕质型多角体病毒(BmCPV)比较,核苷酸和氨基酸的同源性都很高。进一步分析,利用几种CPV序列绘制了系统进化树,对病毒的分类和进化做了探讨研究。     

马尾松毛虫CPV基因组第5片段的cDNA克隆及其序列分析

通过差速离心从感染的马尾松毛虫幼虫虫体中提取质型多角体病毒.碱解法提纯病毒粒子,1%琼脂糖凝胶分离基因组dsRNA,回收纯化第五片段(S5).根据同源性设计五对引物,经RT-PCR,最终获得五个亚克隆片断,测序拼接后,得到S5全长.片断全长2851个核苷酸,包括一个2643个核苷酸的开放阅读框.推测DpCPV S5基因编码了881个氨基酸长的多肽,分子量为100.3kDa,与舞毒蛾质型多角体病毒(LdCPV-1)和家蚕质型多角体病毒(BmCPV)比较,核苷酸和氨基酸的同源性都很高.进一步分析,利用几种CPV序列绘制了系统进化树,对病毒的分类和进化做了探讨研究.     

棉铃虫多核型多角体病毒38k基因的克隆、序列分析及其蛋白高级结构的预测

采用半补齐方法建立棉铃虫多核衣壳型多角体病毒基因组文库,通过对插入片段进行克隆鉴定和序列分析,获得了38k基因.该基因上游具有晚期调控保守序列TTAAG,是一个晚期表达基因,基因阅读框为903 bp,共编码300个氨基酸,氨基酸序列同源性分析结果表明其与α类杆状病毒的同源性较高,有较近的亲缘关系.氨基酸高级结构的分析表明其与与磷酸酶结构相似性达到95%,与病毒核衣壳的组装有关.     

马尾松毛虫质型多角体病毒NS5蛋白基因的cDNA克隆及序列分析

通过对马尾松毛虫质型多角体病毒的增殖、纯化,获得一株单一类型的质型多角体病毒.提纯的病毒粒子经SDS-热酚法抽提,在使用低熔点琼脂糖凝胶电泳分离基因组dsRNA,回收纯化第九片段S9.SgRNA双链经高温变性,使用正反两种引物逆转录合成cDNA双链,使用同样的引物经PCR扩增后,克隆在pMD18-T载体上.利用两种引物组合,获得了大小两个亚克隆片段.序列测定结果表明,较小亚克隆片段长405bp,较大亚克隆片段长677bp,经过序列拼接,得到一个977bp的序列,其中包含一个963bp大的开放阅读框(ORF).推测DpCPVS9基因编码一个320个氨基酸的多肽,分子质量35.66kDa.和家蚕1型质型多角体病毒的I株(BmCPV-II strain)位于第九片段的NS5蛋白基因相比较,核苷酸和编码氨基酸序列同源性分别为86.0%和93.4%.     

马尾松毛虫质型多角体病毒多角体蛋白基因的cDNA克隆及序列分析

通过对马尾松毛虫质型多角体病毒的增殖、纯化,获得一株单一类型的质型多角体病毒。提纯的病毒粒子经SDS－酚抽提,琼脂糖凝胶电泳分离基因组dsRNDA,回收纯化第十片段S10。S10经DMSO变性,逆转录合成cDNA第一链,PCR扩增后,克隆在pGEM-T载体上,对重组子进行限制性内切酶分析及序列测定。结果表明,克隆片段全长763bp,起始密码AUG位于3－5残基,终止密码UGA位于747－749残基。推测DpCPV多角体蛋白基因编码248个氨基酸的多肽,分子量28kD。和家蚕质型多角体病毒（BmCPV）多角体蛋白基因相比较,核苷酸和编码氨基酸序列同源性分别为89.3%和97.6%。     

马尾松毛虫质型多角体病毒NS5蛋白基因的cDNA克隆及序列分析

通过对马尾松毛虫质型多角体病毒的增殖,纯化,获得一株单一质型多角体病毒。提纯的病毒粒子经SDS－热酚法抽提,在使用低熔点琼脂凝胶电泳分离基因组dsRNA,回收纯化第九片段S9。SgRNA双链经高温变性,使用正反两种引物逆转录合成cDNA双链,使用同样的引物经PCR扩增后,克隆在pMD18-T载体上。利用两种引物组合,获得了大小两个亚克隆片段。序列测定结果表明,较小亚克隆片段长405bp,较大亚克隆片段长677bp,经过序列拼接,得到一个977bp的序列,其中包含一个963bp大的开放阅读框（ORF）。推测DpCPVS9基因编码一个320个氨基酸的多肽,分子质量35.66kDa。和家蚕1型质型多角体病毒的I株（BmCPV-I Istrain)位于第九片段的NS5蛋白基因相比较,核苷酸和编码氨基酸序列同源性分别为86．0％和93．4％。     

棉铃虫单粒包埋型核型多角体病毒极早期基因(ie-1)分析(英文)

克隆了棉铃虫Helicoverpaarmigera单粒包埋型核型多角体病毒 (HaSNPV)C1株基因组DNA ,并通过随机测序的方法测定了经XbaI酶切后的H片段的核苷酸全序列。序列比较和分析发现该片段中ORF1 3与苜蓿丫纹夜蛾Autographacalifornica多粒包埋型核型多角体病毒 (AcMNPV)基因组ORF1 47(ie 1 )同源。ie 1基因编码区全长 1 986bp ,根据推测的氨基酸序列 ,可编码 6 6 1个氨基酸残基组成的多肽 ,预计分子量为 76 .5kD。将所推导的HaSNPVIE 1氨基酸序列与其它已知的杆状病毒IE 1氨基酸序列进行比较 ,结果表明 ,HaSNPV和谷实夜蛾H .zea单粒包埋型核型多角体病毒IE 1氨基酸序列最为相似 ,同源性高达 98%。与AcMNPV、家蚕Bombyxmori核型多角体病毒 (BmNPV)、云杉卷叶蛾Choristoneurafu miferana多粒包埋型核型多角体病毒 (CfMNPV)、舞毒蛾Lymantriadispar多粒包埋型核型多角体病毒(LdMNPV)、黄杉毒蛾Orgyiapseudotsugata多粒包埋型核型多角体病毒 (OpMNPV)、甜菜夜蛾Spodopteraex igua多粒包埋型核型多角体病毒 (SeMNPV)、小菜蛾Plutellaxylostella颗粒体病毒 (PxGV)和Xestiac ni grum颗粒体病毒 (XcGV)的IE 1氨基酸序列同源性较低 ,分别为 2 3 %、2 3 %、2 3 %、2 5 %、2 3 %、1 4%、2 7%和 7%。根据氨基酸序列由GENETYX     

嗜水气单胞菌气溶素基因的克隆与序列分析

以嗜水气单胞菌BZ和NK分离株的DNA为模板, 采用PCR技术, 扩增气溶素基因(aerA)的DNA片段, 将其克隆到pMD18-T载体上。通过序列测定, 分析结果表明：所克隆的1393 bp片段为aerA部分序列, 编码产生464个氨基酸。BZ与NK之间aerA核苷酸同源性为97.6%, 氨基酸同源性为98.3%, 与其它分离物核苷酸同源性为71.6%~97.5%, 氨基酸同源性为68.0%~98.9%。利用邻接法构建了aerA分子树状图, 树状图分析表明：气单胞菌属各分离物聚为三支, 其中嗜水气单胞菌各菌株之间关系密切, 被聚类为同一支。     

cDNA cloning and expression of Bauhinia purpurea lectin.

Bauhinia purpurea lectin (BPA) was purified from seeds of B. purpurea alba. The purified lectin was digested with an endoproteinase, Asp-N, or trypsin and then the amino acid sequences of the resultant fragments were analyzed. Furthermore, a cDNA library for BPA was constructed using RNA isolated from germinated Bauhinia purpurea seeds. By gene cloning, the nucleotide sequence of BPA cDNA and its deduced amino acid sequence were analyzed. The cloned BPA cDNA comprised 1,152 nucleotides and the open reading frame of the cDNA encodes a polypeptide of 290 amino acids including a signal peptide composed of 28 amino acids. BPA expressed in Escherichia coli showed a relative molecular mass of 29 kDa on sodium dodecyl sulfate-polyacrylamide gel. On comparison of its sequence with those of other leguminous seed lectins, BPA showed high homology to the others.     

Genomic sequencing and analyses of HearMNPV-a new Multinucleocapsid nucleopolyhedrovirus isolated from Helicoverpa armigera

ABSTRACT: BACKGROUND: HearMNPV, a nucleopolyhedrovirus (NPV), which infects the cotton bollworm, Helicoverpa armigera, comprises multiple rod-shaped nucleocapsids in virion(as detected by electron microscopy). HearMNPV shows a different host range compared with H. armigera single-nucleocapsid NPV (HearSNPV). To better understand HearMNPV, the HearMNPV genome was sequenced and analyzed. METHODS: The morphology of HearMNPV was observed by electron microscope. The qPCR was used to determine the replication kinetics of HearMNPV infectious for H. armigera in vivo. A random genomic library of HearMNPV was constructed according to the "partial filling-in" method, the sequence and organization of the HearMNPV genome was analyzed and compared with sequence data from other baculoviruses. RESULTS: Real time qPCR showed that HearMNPV DNA replication included a decreasing phase, latent phase, exponential phase, and a stationary phase during infection of H. armigera. The HearMNPV genome consists of 154,196 base pairs, with a G + C content of 40.07%. 162 putative ORFs were detected in the HearMNPV genome, which represented 90.16% of the genome. The remaining 9.84% constitute four homologous regions and other non-coding regions. The gene content and gene arrangement in HearMNPV were most similar to those of Mamestra configurata NPV-B (MacoNPV-B), but was different to HearSNPV. Comparison of the genome of HearMNPV and MacoNPV-B suggested that HearMNPV has a deletion of a 5.4-kb fragment containing five ORFs. In addition, HearMNPV orf66, bro genes, and hrs are different to the corresponding parts of the MacoNPV-B genome. CONCLUSIONS: HearMNPV can replicate in vivo in H. armigera and in vitro, and is a new NPV isolate distinguished from HearSNPV. HearMNPV is most closely related to MacoNPV-B, but has a distinct genomic structure, content, and organization.     

嗜水气单胞菌气溶素基因的克隆与序列分析

以嗜水气单胞菌BZ和NK分离株的DNA为模板,采用PCR技术,扩增气溶素基因(aerA)的DNA片段,将其克隆到pMDl8-T载体上.通过序列测定,分析结果表明:所克隆的1393 bp片段为aerA部分序列,编码产生464个氨基酸.BZ与NK之间aerA核苷酸同源性为97.6%.氨基酸同源性为98.3%,与其它分离物核苷酸同源性为71.6%～97.5%,氨基酸同源性为68.0%～98.9%.利用邻接法构建了aerA分子树状图,树状图分析表明:气单胞菌属各分离物聚为三支,其中嗜水气单胞菌各菌株之间关系密切,被聚类为同一支.     

麻疹病毒S191疫苗株N基因稳定性研究及免疫细胞表位分析

研究麻疹病毒(Measles virus,MeV)疫苗株S191毒种和传代病毒核蛋白(Nucleoprotein,N)基因稳定性及其遗传与变异特点;对该序列一些重要位点的氨基酸进行比较,探讨其功能结构及生物学活性变化以及S191疫苗株的保护效果。利用RT-PCR方法扩增S191减毒株23、26、27、29、32、37不同代次N基因,测序进行比对分析。S191传代病毒N基因序列之间核苷酸同源性99.7%~100%,氨基酸同源性为99.6%~100%;S191株与7个疫苗株之间核苷酸序列同源性达99.1%~99.4%;S191和中国流行代表株序列同源性在95.0%~95.4%;S191与世界流行代表株同源性达94.7%~99.4%;S191疫苗株和中国流行代表株CHN93/7(H1a)的4个重要T细胞表位氨基酸保持一致。S191各传代病毒基因具有较高稳定性,该疫苗有一定的保护作用。     

The sequence of human retinal S-antigen reveals similarities with alpha-transducin

The complete amino acid sequence of human retinal S-antigen (48 kDa protein), a retinal protein involved in the visual process has been determined by cDNA sequencing. The largest cDNA was 1590 base pairs (bp) and it contained an entire coding sequence. The similarity of nucleotide sequence between the human and bovine is approximately 80%. The predicted amino acid sequence indicates that human S-antigen has 405 residues and its molecular mass is 45050 Da. The amino acid sequence homology between human and bovine is 81%. There is no overall sequence similarity between S-antigen and other proteins listed in the National Biomedical Research Foundation (NBRF) protein data base. However, local regions of sequence homology with alpha-transducin (T alpha) are apparent including the putative rhodopsin binding and phosphoryl binding sites. In addition, human S-antigen has sequences identical to bovine uveitopathogenic sites, indicating that some types of human uveitis may in part be related to the animal model of experimental autoimmune uveitis (EAU).     

Sequence of heat-labile enterotoxin of Escherichia coli pathogenic for humans.

We determined the complete nucleotide sequence of the toxB gene (375 base pairs in length), which encodes the B subunit of heat-labile enterotoxin produced from Escherichia coli pathogenic for humans (hLT). The amino acid sequence of the B subunit of hLT was deduced from the nucleotide sequence. Consequently, it has become possible to study the homology between the B subunits of three similar toxins: hLT, LT produced from E. coli pathogenic for piglets (pLT), and cholera toxin (the latter two sequences have been reported by others). The three B subunits are all 103 amino acids in length. A comparison of the toxB gene and the eltB gene, which encodes the B subunit of pLT, showed a 98% homology at the nucleotide level and a 95% homology at the amino acid (of a precursor) level, indicating the possibility that the two genes share a common ancestor. With respect to the B-subunit sequences, the homologies between hLT and pLT, between hLT and cholera toxin, and between pLT and cholera toxin were 96, 81, and 79%, respectively. Several large common sequences are conserved by the three peptides. In contrast, no sequences are present in both pLT and cholera toxin but missing in hLT.     

Nucleotide sequence encoding the precursor of the small subunit of ribulose 1,5-bisphosphate carboxylase from Lemna gibba L.G-3

We have sequenced a cDNA clone, pLgSSU, which encodes the small subunit of ribulose 1,5-bisphosphate carboxylase of Lemna gibba L.G-3 a monocot plant. This clone contains a 832 basepair insert which encodes the entire 120 amino acids of the mature small subunit polypeptide (Mr = 14,127). In addition this clone encodes 53 amino acids of the amino terminal transit peptide of the precursor polypeptide and 242 nucleotides of the 3' non-coding region. Comparison of the nucleotide sequence of pLgSSU with Lemna gibba genomic sequences homologous to the 5' end of the cDNA clone suggests that nucleotides encoding four amino-terminal amino acids of the transit peptide are not included in the cDNA clone. The deduced amino acid sequence of the Lemna gibba mature small subunit polypeptide shows 70-75% homology to the reported sequences of other species. The transit peptide amino acid sequence shows less homology to other species. There is 50% homology to the reported soybean sequence and only 25% homology to the transit sequence of another monocot, wheat.     

我国肾综合征出血热疫苗生产株LR1株的全基因组序列分析

为测定我国肾综合征出血热疫苗生产株LR1株的全基因组序列 ,了解该株分子基础 ,从提取的细胞总RNA逆转录PCR扩增 ,产物纯化后克隆T载体纯化后测序 ,结果证明 ,LR1株全基因组序列由L6 5 33、M36 16、S片段的16 92个核苷酸组成 ,依各自读码框架分别编码 2 15 1、1135、42 9个氨基酸。序列同源比较分析表明 ,LR1毒株与国外HTN型毒株高度同源 ,属同一亚型 ,尤其与HTN代表株 76 - 1183个片段同源率高达 99 3%～ 99 8% ,而与国内的HTN型病毒差异较大 ,同源率仅为 79 4%～ 84 6 %。氨基酸比较也显示了同样的结果。