舞毒蛾核型多角体病毒多角体蛋白基因的克隆、测序和表达

从东北林业大学实验林场采集并纯化了舞毒蛾核型多角体病毒（LdMNPV-NEFU)。用蛋白酶K消化,提取了病毒基因组DNA。用PCR方法,克隆出了该病毒的多角体蛋白（polyhedrin)基因,并对该基因进行了序列测定。结果显示,该基因序列是一个含有735个碱基对的开放阅读框（ORF）,该阅读框编码245个氨基酸。有5对碱基与加拿大病毒株LdMNPV-G的多角体蛋白基因序列存在差异。LdMNPV-NEFU分离株的多角体蛋白基因的第54,109,379,508和701位（从起始密码子中的A开始）分别是C,G,T,C和G,而LdMNPV-G分离株的多角体蛋白基因（ORF）相应位置上的碱基分别是G,C,C,T和T,两个ORF编码的对应位置的氨基酸绝大多数相同,只有一对不同,即由LdMNPV-NEFU编码的天冬氨酸和由LdMNPV-G编码的对应位置的组氨酸。以质粒pT-7-7为载体,多角体蛋白基因在大肠杆菌BL21（DE3）菌株中进行了原核表达。     

繁缕核糖体失活蛋白基因克隆及序列分析

采用同源克隆结合RACE法,克隆了繁缕核糖体失活蛋白的全长cDNA,命名为q3(GenBank accession GQ870262)。序列分析结果表明,q3的开放阅读框(ORF)长780 bp,编码259个氨基酸。序列G+C含量为41.5%,与大部分Ⅰ型RIP基因相近。q3编码的蛋白质命名为Q3,理论分子量为28.16 kD,pI为9.44,均与Ⅰ型核糖体失活蛋白相近;包含由23个氨基酸组成的信号肽。功能结构域分析发现,该蛋白含有3个蛋白激酶磷酸化位点、4个络氨酸蛋白激酶磷酸化位点和7个N-肉豆蔻酰化位点。三级结构预测发现,有35.52%的氨基酸残基参与了α螺旋,24.32%的氨基酸残基组成延伸链,40.15%的氨基酸残基随机缠绕其中。基于繁缕及其近缘种核糖体失活蛋白的氨基酸序列构建的系统发育树显示,其结构与经典分类结果基本一致。     

甜菜坏死黄脉病毒内蒙分离物自然缺失突变体缺失区域的定位分析

以甜菜坏死黄脉病毒内蒙分离物（ＢＮＹＶＶ ＮＭ）总ＲＮＡ为模板,经ＲＴ－ＰＣＲ扩增,分别获得ＲＮＡ２、ＲＮＡ３和ＲＮＡ４自然缺失突变体ｃＤＮＡ克隆。序列分析结果表明,ＲＮＡ２自然缺失突变体在７５ｋＤ通读蛋白编码区Ｃ端缺失３４８个核苷酸（缺失位置ｎｔ１４８８￣ｎｔ１８３５）。ＲＮＡ３在其２５ｋＤ蛋白编码区内缺失３６０个核苷酸（缺失位置ｎｔ７２９￣ｎｔ１０８８）。ＲＮＡ４的自然缺失区域位于３１ｋＤ蛋白     

鹅副粘病毒WF（01）G株F基因的测序与蛋白特性分析

用鹅副粘病毒WF01G分离株进行9-10日龄SPF鸡胚尿囊腔接种,成功增殖了该病毒,采用一步法RT-PCR技术扩增WF01G病毒的F基因,获得了1条长约1.7 kb的特异性条带。对PCR扩增产物测序。结果表明,扩增片段大小为1782 bp,含有1个1662 bp的开放性阅读框,编码554个氨基酸。核苷酸同源性分析表明：WF01G株与其他7株鹅副粘病毒的同源性为84.8%-98.8%,与国内外其他NDV F基因的同源性为84.7%-93.8%,其中与国内标准强毒株F48E9的同源性为86.8%,说明WF01G与国内外的传统毒株有较大变异。与Tai-wan95株的同源性为93.8%,说明WF01G与Taiwan95株亲缘关系较近,具有较高的相似性。F蛋白裂解位点的氨基酸顺序为112Arg-Arg-G ln-Lys-Arg-Phe117,表明为副粘病毒强毒株。蛋白疏水性和抗原性分析表明与标准强毒F48E9株相比没有太大的变异。     

Characterization of the full length uracil-DNA glycosylase in the extreme thermophile Thermotoga maritima

A full length (192 amino acids) uracil-DNA glycosylase (TMUDG) has been expressed and purified from the extreme thermophile Thermotoga maritima. This protein is active up to 85 degrees C. The enzyme is product inhibited by abasic sites in DNA and weakly inhibited by uracil. TMUDG was originally cloned from an ORF which encoded a protein of 185 amino acids. This shorter protein was stable up to 70-75 degrees C and it seemed unusual that this enzyme had an optimal activity temperature below the growth temperature of the organism (80-90 degrees C). Following the publication of the complete genomic sequence of T. maritima, it was shown that the gene contains an additional seven amino acids (LYTREEL) at the N-terminal end of the protein. It is suggested that these seven residues are important in maintaining proper protein folding that results in increased temperature stability. We have also demonstrated that TMUDG can substitute for the Escherichia coli uracil-DNA glycosylase and initiate base excision repair using a closed circular DNA substrate containing a unique U:G base pair.     

Nucleotide sequence of Lymantria dispar nuclear polyhedrosis virus polyhedrin gene

The polyhedrin gene of the nuclear polyhedrosis virus of the gypsy moth (Lymantria dispar) (LdMNPV) was cloned and sequenced. A polyhedrin open reading frame of 735 nucleotides (nt) was identified which can code for a protein of 245 amino acids that demonstrates a high degree of similarity to other polyhedrins. The protein predicted from the nucleotide sequence shows differences in several regions to that previously sequenced from the LdMNPV polyhedrin protein. The consensus sequence AATAAGTATTTT found at the mRNA start site of baculovirus hyperexpressed genes was located 55 nt upstream from the translational start site.     

Primary amino acid and DNA sequences of gassericin T, a lactacin F-family bacteriocin produced by Lactobacillus gasseri SBT2055

A broad-spectral bacteriocin, named gassericin T, produced by Lactobacillus gasseri SBT 2055 (from human feces) was isolated to homogeneity from the culture supernatant by hydrophobic chromatography. By SDS-PAGE and in situ activity assay, the purified gassericin T migrated as a single band with bacteriocin activity and molecular size of 5,400. A 2.9-kbp HindIII-HindIII fragment of chromosome DNA was hybridized with the oligonucleotide probe designed from the partial N-terminal amino acid sequence of gassericin T and was cloned. Six ORFs including the structural gene of gassericin T were deduced by computer analysis and the data bases. The structural gene of gassericin T (gatA) was identified as the fourth ORF, which encoded a protein composed of 75 amino acids that included the GG motif of the cleavage site. Chemical sequencing analysis of the complete amino acid sequence showed that gassericin T (57 amino acids) had a disulfide bond in the molecule and no modified amino acid residues, making it a class II bacteriocin. The gassericin T had 60% sequence similarity to mature LafA (57 amino acids, lactacin F, bacteriocins produced by L. johnsonii VPI11088), and the sequences around the processing site and C-terminal area were well conserved. The fifth ORF was designated as gatX, encoded as a peptide composed of 65 amino acids containing the GG motif of the putative cleavage site, however mature GatX and its antibacterial activity were not detected in the culture supernatant. GatX has higher similarity with LafX than with lactobin A (50 amino acids) belonging to the first lactacin F-family. These results indicated that gassericin T belongs to the hydrophobic class II bacteriocins and the most vicinal lactacin F-family.     

黄河裸裂尻鱼细胞色素C氧化酶Ⅰ、Ⅱ和Ⅲ亚基基因的克隆及序列特征分析

采用RT-PCR技术克隆获得了黄河裸裂尻鱼(Schizopygopsis pylzovi)CO Ⅰ、Ⅱ、Ⅲ基因的编码序列,并对此进行了初步分析.结果表明,黄河裸裂尻鱼CO I基因全长为1 551 bp,开放阅读框(ORF)由基因全长组成,编码516个氨基酸;COⅡ基因全长为691 bp,开放阅读框为690 bp,编码2...     

邻单胞菌邻苯二酚1,2-双加氧酶基因(tfd C)的克隆及其在大肠杆菌中表达

根据已报道的邻苯二酚１,２－双加氧酶基因（ｔｆｄ Ｃ）序列,设计ＰＣＲ引物,从一株邻单胞菌（Ｐｌｅｓｉｏｍｉｎａｓ）的ｐＬ１质粒上扩增到ｔｆｄ Ｃ基因片段,连接到ｐＧＥＭ－Ｔ载体上,并转化大肠杆菌ｌＭ１０９菌株,筛选到阳性克隆。序列分析结果表明,ＰＣＲ产物全长８０１ｂｐ,有一阅读框,编码２５５个氨基酸,与增氧产碱菌（Ａｌｃａｌｉｇｅｎｅｓ ｅｕｔｒｏｐｌｕｓ）的ｔｆｄ Ｃ基因相比,在６９３位相差一     

鹅新城疫病毒P基因的RNA编辑及其相关蛋白的分子特征

应用RT-PCR方法对鹅新城疫病毒安徽分离株WF00GP基因进行了RNA编辑分析,发现w‰GP基因在保守的编辑位点上插入一个G,使得P基因增加编码V蛋白,而且它的最大ORF的长度为1188bp,编码395个氨基酸的P蛋白。wkG株和参考毒株的P基因的同源性和系统发育分析表明,WF00G株与水禽源毒株NA．1、ZJ1、FPI／02、PX2／03、Duck／1／05和SF02等亲缘关系较近,与传统疫苗株或弱毒株HB92、LaSota和Clone30以及F48E9等的亲缘关系相对较远。进一步对其V蛋白羧基端序列分析发现,虽然编辑位点氨基酸序列（132KKG134）和羧基端的5个Cys残基位点是高度保守的,但是V蛋白c末端氨基酸存在较大变异,APMV-1毒株V蛋白羧基端氨基酸的突变呈现“基因型一致性”。     

Molecular cloning and nucleotide sequence of a gene for alkaline cellulase from Bacillus sp. KSM-635

A gene for alkaline cellulase from the alkalophilic Bacillus sp. KSM-635 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. Although the recombinant plasmid contained two HindIII inserts of 2.6 kb and 4.0 kb, the inserts were found to be contiguous in the Bacillus genome by hybridization analysis. Nucleotide sequences of a 2.4 kb region which was indispensable for the production of cellulase, and the flanking, 1.1 kb region, were determined. There was an open reading frame (ORF) of 2823 bp in the 3498 bp sequence determined, which encoded 941 amino acid residues. Two putative ribosome-binding sites and a sigma 43-type, promoter-like sequence were found upstream from an initiation codon in the ORF. The deduced amino-terminal sequence resembles the signal peptide of extracellular proteins. A region of amino acids, 249 to 568, of the deduced amino acid sequence of the cellulase from this organism is homologous with those of alkaline and neutral enzymes of other micro-organisms, but nine amino acid residues were found to be conserved only in the alkaline enzymes.     

白鱀豚BDNF基因的扩增和序列分析

利用聚合酶链式反应,首次从白鱀豚基因组DNA 中扩增和克隆到脑源神经营养因子的编码区。在该段序列中含有一个长为747 bp 的开放阅读框,无内含子,编码一个由248 个氨基酸组成的蛋白质,预计分子量为27 953.7道尔顿。其中包括由18 个氨基酸残基组成的信号肽区,111 个氨基酸残基组成的前肽区及119 个氨基酸残基组成的成熟区。序列分析表明,白鱀豚脑源神经营养因子基因编码区的核苷酸序列与其它哺乳动物相似性超过90%,而与猪牛相似性相对较高（分别为95% 和94.7%）。氨基酸序列比较发现,白鱀豚BDNF 前体蛋白的氨基酸序列与其它哺乳动物具有94.5% ～99.5%的相似性,显示了极高的保守性。通过邻接法进行的系统发生分析中,鲸目和食肉目的物种分别聚为单系;与其它哺乳动物相比,鲸类与有蹄类的牛和猪的亲缘关系相对较近,这与鲸类和有蹄类之间具有相对较近的亲缘关系相符。
     

Molecular cloning of the nahG gene encoding salicylate hydroxylase from Pseudomonas fluorescens

A gene encoding the salicylate hydroxylase was cloned from the genomic DNA of Pseudomonas fluorescens SME11. The DNA fragment containing the nahG gene for the salicylate hydroxylase was mapped with restriction endonucleases and sequenced. The DNA fragment contained an ORF of 1,305 bp encoding a polypeptide of 434 amino acid residues. The nucleotide and amino acid sequences of the salicylate hydroxylase revealed several conserved regions with those of the enzyme encoded in P. putida PpG7: The homology of the nucleotide sequence is 83% and that of amino acid sequence is 72%. We found large conserved regions of the amino acid sequence at FAD and NADH binding regions. The FAD binding site is located at the amino terminal region and a lysine residue functions as a NADH-binding site.     

Synthethic peptide used to develop antibodies for detection of polyhedrin from monodon baculovirus (MBV)

The use of previously published primers to amplify the monodon baculovirus (MBV) polyhedrin gene sequence by polymerase chain reaction (PCR) from post larvae (PL) of Thai Penaeus monodon resulted in failure. As a result, the putative polyhedrin protein of MBV was isolated from infected PL by homogenization, differential centrifugation and density gradient centrifugation with verification by transmission electron microscopy (TEM). By SDS-PAGE, a single major protein band at 58 kDa was obtained from the putative polyhedrin fraction and this corresponded to a previous report of the molecular weight of polyhedrin from MBV. When used for N-terminal sequence analysis, the putative polyhedrin protein yielded a sequence of 25 amino acids (M F D D S M M M E N M D D L S G D Q K M V L T L A) that did not correspond to the deduced amino acid sequence derived from a previous report of a putative MBV polyhedrin gene amplicon. Despite this, a synthetic peptide of our 25 amino acid sequence (25Pmbv) was conjugated with bovine serum albumin and used as an antigen for antiserum production in mice. Using immunohistochemistry with tissue sections of PL infected with MBV or other viruses, the mouse anti-25Pmbv antiserum showed strong immunoreactivity to occlusion bodies of MBV only. It also showed strong reactivity to the 58 kDa putative polyhedrin protein in Western blots. Altogether, the results suggest that the 58 kDa protein is Thai MBV polyhedrin and that a previously reported MBV polyhedrin gene sequence may represent another protein or polyhedrin from a different variety of MBV.     

Structural characterization of protein secretion genes of the bacterial phytopathogen Xanthomonas campestris pathovar campestris: relatedness to secretion systems of other gram-negative bacteria

Summary The nucleotide sequence was determined of a 5.3 kb region of the Xanthomonas campestris pathovar campestris genome carrying a gene cluster encoding protein secretion and pathogenicity functions. A putative promoter sequence and five open reading frames (ORF) which may be part of an operon were revealed. The five predicted primary translation products comprise 531, 390, 147, 169 and 138 amino acids with M_r values of 58854, 42299, 15548, 18214 and 15108 respectively. A sixth, partial ORF is also present. Between ORF1 and ORF2 is a sequence of unknown function showing 7 by duplications. The deduced amino acid sequence of ORF1 is related to the Klebsiella pneumoniae PulE protein, to the Bacillus subtilis ComG ORF1 and to the Agrobacterium tumefaciens VirB ORF11 products. In addition, the deduced amino acid sequence of ORF2 showed homology to the Pu1F and to the ComG ORF2 products. The proteins encoded by ORF3, 4 and 5 showed amino acid homology to PulG, H and I products respectively. The proteins encoded by ORF2, 3, 4 and 5 showed significant hydrophobic domains which may represent membrane-spanning regions. By contrast the protein encoded by ORF1 was largely hydrophilic and had two putative nucleoside triphosphate binding sites.The nucleotide sequence data in this paper have been deposited in the EMBL, Genbank and DDBJ nucleotide sequence databases under the accession number X59079     

水稻矮缩病毒第一号组份基因和编码蛋白的序列分析

水稻矮缩病毒（RiceDwarfVirus,简称RDV）是我国南方水稻病毒病的重要病原,属植物呼肠孤病毒。从中国福建分离物中克隆了基因组第一号片段（S1）的全长cDNA并对其进行全序列分析,结果表明RDV福建分离物S1克隆片段全长4422bp,含有一个长4332bp的开放阅读框架,编码一个由1444个氨基酸组成的多肽（P1）,分子量为164kD．根据基因序列,对推测的P1氨基酸序列分析表明,序列中含有依赖于RNA的RNA聚合酶（RNA-dependentpolymerase-RDRP）保守序列：motifＩ（DXXXXD）、motifⅡ（SGXXXTXXXN）和motifⅢ（GDD）,除此之外,在模式Ⅲ后还存在一个很保守的区域EXXKXY。由此说明RDVS1编码的蛋白P1可能是病毒的一种RDRP。将RDV福建分离物引核苷酸和编码蛋白氨基酸序列与日本流行株系相比,同源性分别为95％和97％。RDV福建分离物S1序列已被DenBank接受,号码为U73201。     

绵羊CAST基因2型和4型转录本的克隆及特性分析

钙蛋白酶抑制蛋白(Calpastatin, CAST)是一种内源性的需要Ca²⁺激活的钙蛋白酶抑制剂, 在肌肉组织的蛋白质降解过程中起重要的调节作用。文章利用牛^CAST基因的mRNA序列, 通过逆转录RT-PCR首次克隆获得绵羊CAST基因2型转录本和4型转录本的部分cDNA序列, 并对序列进行生物信息学分析。CAST基因2型转录本的扩增片段为4 385 bp, 完整的开放阅读框为2 361 bp, 编码786个氨基酸; CAST基因4型转录本的扩增片段为1 467 bp, 完整的开放阅读框为1 317 bp, 编码438个氨基酸。CASTⅡ型蛋白序列存在4个保守结构域, CASTⅣ型蛋白序列存在3个保守结构域; 两者的二级结构均以螺旋为主, 富含疏水区域, 其氨基酸序列存在多个磷酸化位点以及蛋白激酶C(Protein kinase C, PKC)的磷酸化位点。通过RT-PCR分析CAST基因2型转录本和4型转录本的组织表达谱, 结果表明CAST基因2型转录本在所检测的10个组织中均表达, CAST基因4型转录本仅在睾丸组织中表达。