山羊FGF5基因cDNA分子克隆及序列分析

采用逆转录-聚合酶链式反应(RT-PCR)技术,首次从山羊脑组织总RNA中扩增出成纤维细胞生长因子5(FGF 5)基因的cDNA编码序列.通过对序列的分析表明,克隆的山羊FGF 5cDNA编码序列与其他动物的序列具有高度的同源性.与G enbank中人和小鼠序列比较,山羊与人和小鼠的FGF 5核苷酸序列的同源性分别为91%和87%.编码的氨基酸序列比较,山羊和人的相似性为87%,山羊与小鼠的相似性为83%.如FGF s家族的其他成员一样,山羊的FGF 5蛋白也有一信号肽序列.山羊FGF 5基因外显子在编码氨基酸时,对同义密码子的使用表现有强烈的偏好性.不同物种对同义密码子使用的偏倚程度与偏好类型各不相同,也具有种属特异性.山羊FGF 5基因所编码的蛋白质中,20种氨基酸的含量差异很大,极性氨基酸含量高于非极性氨基酸含量.     

绵羊肺炎支原体新疆分离株黏附素与溶血素A基因的克隆及序列分析

根据Gen Bank报道的绵羊肺炎支原体(MO)基因组序列,设计特异性引物,对MO新疆分离株黏附素基因和溶血素A基因进行PCR扩增、克隆及测序;应用分子生物学软件对该基因及其编码蛋白进行分子特征分析,并与其他支原体相应序列进行同源性分析,构建该基因系统进化树。结果显示:黏附素基因长为3426 bp,溶血素A基因长为777 bp;黏附素在第1-23位氨基酸残基为信号肽、1个跨膜区,可能为分泌型蛋白;溶血素A无信号肽和跨膜区、有RNA结合位点和甲基转移酶结合位点。系统发生分析表明,黏附素基因与猪肺炎支原体P146株进化关系较近,与其他的支原体进化关系远;溶血素A基因与猪肺炎支原体232株、结膜支原体HRC581株位于同一分枝上,进化关系近。本研究克隆了MO黏附素基因和溶血素A基因,为了解MO毒力相关基因生物学功能及其致病中的作用奠定了前期基础。     

血吸虫膜相关抗原基因的克隆

目的:筛选日本血吸虫(Schistosoma japonicum,S.japonicum)新的疫苗候选抗原基因.方法:以S.japonicum成虫DNA为模板,PCR扩增相关基因,然后将其克隆XpBS-T载体,通过菌落PCR对产物进行检测,对阳性克隆子测序,与GeneBank中的已知相关序列进行比对分析.结果:菌落PCR获得一条与PCR产物一致的DNA片段,序列测定结果表明PCR产物与S.japonicum 22.6 kDa抗原分子核苷酸序列具有高度同源性.其目的基因大小636 bp,具有一个189 bp的ORF,能编码63个氨基酸.ORF不是全长的阅读框,只获得部分编码区.表达产物可含有一个包含9个氨基酸残基的潜在螺旋跨膜片段(25～33位)和一个酪氨酸激酶磷酸化位点(38～39位).讨论:成功克隆出一个与S.japonicum 22.6kDa抗原编码基因有高度同源性的基因.预测该基因为膜相关蛋白基因,可编码血吸虫体表膜相关蛋白.在生理机能上,该膜相关蛋白可能是信号传递分子.     

武汉地区丙型肝炎病毒包膜蛋白E1的生物信息学研究

使用生物信息学的方法,分析武汉地区不同基因型、亚型的丙肝病毒的包膜E1蛋白,预测其二级结构、核苷酸变异性、糖基化位点、亲水性、跨膜区、信号肽、蛋白修饰位点、B细胞抗原.结果显示各HCV的包膜E1蛋白二级结构差距不大;序列始末段有数个氨基酸残基的差距,序列上存在高变异位点,基因型2a的型内一致性最高;序列大量糖基化,有多个糖基化基化位点;序列分布着亲水性区域,基因型1b的分值很高;基因型1b、6a、3b、3a多数亚型有1个跨膜区,基因型2a多数亚型有两个跨膜区;基因型1b、6a、3b、3a无信号肽,基因型2a都有信号肽;蛋白序列上存在多个不同修饰位点和B细胞抗原,各基因型、亚型之间有明显差距,具有较大异质性.该研究为揭示病毒感染机制和研制地区性疫苗提供一定的科学依据.     

中华绒螯蟹内源性白斑综合症病毒基因EjsWSSV的克隆与鉴定(英文)

基于已获得的表达序列标签序列,应用RACE技术从中华绒螯蟹基因组中获得了一个白斑综合症病毒基因(EjsWSSV)的全长cDNA序列,全长为3 864 bp并包含一个3 732 bp的开放阅读框,编码一个138.25 kDa的含1 243个氨基酸的多肽,编码的氨基酸序列包含一个VWA结构域.通过序列比对、序列结构比较和一些生物信息学预测分析,表明EjsWSSV蛋白序列与白斑综合症病毒基因组的ORF16具有高同源性,它不含有信号肽和跨膜区,为非分泌型和α-螺旋型蛋白,可能定位于细胞质.通过PCR检测验证,EjsWSSV基因可能为内源性病毒基因.     

小反刍兽疫病毒融合蛋白基因的克隆、序列分析及表达

根据GenBank报道的小反刍兽疫病毒(PPRV)融合蛋白(F)基因序列,用特异性引物对PPRV疫苗株F蛋白基因进行了RT-PCR扩增,并将其克隆到pGEM-T载体中进行测序。结果表明:F基因ORF全长1641 bp,编码546个氨基酸;推导的氨基酸序列中第1~18位氨基酸构成信号肽序列,第488~510氨基酸为跨膜区。构建原核表达载体pETF1和pETF2,转化E.coliBL21(DE3),用IPTG诱导表达。SDS-PAGE和Western-blotting的分析结果表明,F1和F2基因在大肠杆菌中均获得了表达,且均具有良好的反应原性。用Ni-NTA试剂盒纯化F1和F2重组蛋白,为研发检测PPRV特异性抗体的诊断试剂奠定了基础。     

结核分枝杆菌FabG4的生物信息学分析及功能初探

目的预测结核分枝杆菌(H37Rv)的FabG4蛋白的结构、与其相互作用蛋白及T、B细胞抗原表位。方法从NCBI数据库获取FabG4基因序列和氨基酸序列,通过在线软件对FabG4的理化性质、二级和三级结构、T、B细胞抗原表位及其相互作用的蛋白进行预测分析。结果 H37Rv FabG4基因全长1365bp,编码454个氨基酸,预测到该蛋白性质稳定,无跨膜区,属于非分泌蛋白,二级结构显示α-螺旋(Th)、β-折叠(Ee)、β-转角(Tt)、无规则卷曲结构(Cc)分别为43. 61%,14. 98%,9. 03%,32. 38%;有多个T、B细胞抗原表位及多个与FabG4蛋白相互作用蛋白。mc2155 FabG4过表达株生长曲线显示,过表达菌株较野生型的生存能力明显增强,差异有统计学意义(P0. 01),形态粗、长。结论结核分枝杆菌FabG4蛋白稳定,所含保守结构域与其功能密切相关,筛选出6个B细胞优势表位,CTL和Th细胞优势表位各8个,为研究FabG4蛋白的结构、功能和开发靶向新药、抗结核疫苗提供理论依据。     

家蚕浓核病毒BmDNV-1(伊那株)结构蛋白基因VP4的克隆、表达及抗体制备

为研发新的日本血吸虫(Schistosoma japonicum)疫苗候选抗原基因,为血吸虫诊断和疫苗研制提供依据,提取和纯化S.japonicum成虫DNA,PCR扩增相关基因,然后将其克隆入pBST载体,对菌落PCR阳性克隆子测序和分析.菌落PCR获得一条与PCR产物一致的DNA片段,PCR产物与S.japonicum 22 600抗原分子DNA序列具有高度同源性,636 bp大小,oRF189 bp,ORF小是全长的蒯读框,只获得部分编码区,能编码63个氨基酸,表达产物含有一个包含9个氨基酸残基的潜在螺旋跨膜片段(25～33位)和一个酪氨酸激酶磷酸化位点(38～39位).本研究所获取的阳性克隆是与S.japonicum 22 600抗原编码基因有高度同源性的基因.通过Blastn和BLASTP分析,预测该基因为体膜相关蛋白基因,可编码血吸虫体壁相关蛋白,可能是信号传递分子.     

菘蓝色氨酸合成酶基因的克隆与生物信息学分析

对菘蓝色氨酸合成酶基因IiTSA进行克隆,并对其进行生物信息学分析.结果表明:菘蓝IiTSA基因全长2 111bp,包含8个外显子和7个内含子;cDNA全长1 035bp,编码311个氨基酸,编码的蛋白定位在叶绿体基质中,是一种无信号肽和跨膜结构域的疏水性蛋白;二级结构主要由α-螺旋、β-转角、延伸链和不规则卷曲组成;序列比对结果显示,菘蓝IiTSA核酸序列和氨基酸序列与其他多种植物的TSA核酸序列和氨基酸序列均具有较高的同源性.     

Bacillus sp.XJ1-05菌株GbsA基因的克隆及其生物信息学分析

根据地衣芽孢杆菌Bacillus licheniformis ATCC 14580的GbsA基因(GeneID:3027576)的核甘酸序列设计1对特异性引物.通过PCR的方法扩增由本实验室分离的中度嗜盐菌Bacillus sp.XJ1-05的GbsA基因,经T/A克隆,插入到pBS-T载体上进行序列测序,结果表明得到的GbsA基因的开放阅读框(ORF)全长为1473bp.在Genbank数据库中进行同源性检索,结果表明:其与Bacillus licheniformis ATCC 14580的GbsA基因的氨基酸序列同源性高达97%.生物信息学分析表明:该GbsA基因编码蛋白无信号肽,无跨膜区域,高亲水性,有2个醛脱氢酶作用位点.     

梅花鹿抗病毒蛋白的生物信息学分析

通过在线生物软件分析梅花鹿四种抗病毒蛋白A3Z2、BST-2A、BST-2B和SAMHD1蛋白的生物信息学特性。从转录组中获得四种蛋白的基因序列,应用Prot Param、Protscale、SOPMA、TMHMM、Target P、Signal P、Motif Scan、Interproscan以及BLAST等在线软件分析蛋白的理化性质、二级结构、穿膜域、结构域等等。首次获得了四种抗病毒因子的基因和蛋白序列,梅花鹿A3Z2基因编码393个氨基酸,相对分子质量为47.59 k Da,碱性,不稳定性亲水蛋白,无信号肽和跨膜结构域,胞内定位,具有糖基化和磷酸化位点,两个胞嘧啶脱氨酶结构域。梅花鹿BST-2A和2B基因编码159个氨基酸,相对分子质量为17.69 k Da和18.12 k Da,酸性,不稳定性亲水蛋白,无信号肽,BST-2A有两个跨膜结构域,BST-2B有一个跨膜结构域,膜定位,具有糖基化和磷酸化位点。梅花鹿SAMHD1基因编码613个氨基酸,相对分子质量为70.51 k Da,碱性,不稳定性亲水蛋白,无信号肽和跨膜结构域,胞内定位,具有磷酸化位点,d NTP磷酸水解酶活性结构域。梅花鹿A3Z2、BST-2A、BST-2B和SAMHD1蛋白具有潜在的抗病毒能力。     

金龟子绿僵菌酸性海藻糖酶基因的克隆及其序列特征分析

根据酸性海藻糖酶的蛋白质序列设计引物,PCR扩增出CQMa102 ATM1的cDNA和DNA序列,登录号分别为:DQ237957,EF190950.序列分析表明,ATM1 DNA序列含有3个内含子,其开放阅读框(ORF)编码1个含1 073个氨基酸的蛋白质,具有1个20个氨基酸的信号肽序列和含有30个可能的N-糖基化位点(Asn-Xaa-Ser/Thr).NCBI序列比对(Blastn)显示该蛋白与Aspergillus fumigatus的alpha、alpha-trehalose glucohydrolase,Aspergillus nidulans的酸性海藻糖酶前体和Talaromyces emrsonii的酸性海藻糖酶分别有62%、59%和57%的氨基酸相似性,与另外两个真菌Saccharomyces cerevisiae(Ath1p)和Candida albi-cans(Atc1p)的酸性海藻糖酶也具有约25%的氨基酸相似性.Southern杂交表明,ATM1基因在CQMa102基因组中为单拷贝.     

The protein-coding sequence of the bovine ACTH-beta-LPH precursor gene is split near the signal peptide region

The pituitary hormones corticotropin (ACTH) and beta-lipotropin (beta-LPH) are formed from a large common precursor. Recently, we have elucidated the whole primary structure of the bovine ACTH-beta-LPH precursor (designated alternatively as preproopiocortin) by determining the nucleotide sequence of cloned DNA complementary to the mRNA coding for the precursor protein. The amino acid sequence assigned has disclosed a characteristic repetitive structure of the ACTH-beta-LPH precursor. The repetitive units of the precursor protein each contain a melanotropin (MSH) sequence (alpha-, beta- or gamma-MSH) as well as other peptide components such as beta-endorphin and corticotropin-like intermediate lobe peptide (CLIP). The repetitive units as well as their peptide components are each bounded by paired basic amino acid residues, which apparently represent the sites of proteolytic processing. Several studies have confirmed the translational initiation site and protein structure assigned (see also ref. 11 and refs therein). In view of the recent knowledge about the organization of eukaryotic genes (see refs 12, 13 for reviews), it would be of particular interest to investigate the relationship between the repetitive structure of the ACTH-beta-LPH precursor containing different functional components and the arrangement of the protein-coding sequence in its gene. We have now isolated and characterized bovine genomic DNA fragments encoding this precursor protein and have demonstrated that the protein sequence is encoded by two non-consecutive DNA segments. An intron (intervening sequence) of approximately 2.2 kilobase pairs separates the smaller exon (mRNA-coding sequence), which contains the gene sequence encoding the signal peptide, from the larger exon, which contains the gene sequence for most of the protein structure, including the known biologically active component peptides.     

T细胞功能学与结构免疫学结合方法鉴定HLA-A2限制性的细胞毒性T淋巴细胞表位富集区

 T细胞表位在抗病毒的T细胞免疫中发挥核心作用,目前已在多种病原微生物的蛋白序列上发现存在T细胞表位的聚集现象。本文建立了一套功能学与结构学结合的策略鉴定病原体上细胞毒性T细胞(Cytotoxic T Lymphocyte,CTL)表位富集区的方法,并以严重急性呼吸道综合征(SARS)相关冠状病毒(SARS-CoV)的M蛋白为例,成功地鉴定了一个HLA-A2限制性的表位富集区。首先通过生物信息学的方法预测并合成M蛋白跨膜区的HLA-A2潜在结合多肽,通过体外复性实验和T2细胞结合实验验证多肽与HLA-A2的结合力;然后在HLA-A2.1/Kb转基因小鼠中检测这些多肽的免疫原性;最后通过X射线衍射技术,成功解析了其中一条多肽与HLA-A^*0201的复合物结构,其结构显示该多肽具有典型的HLA-A^*0201表位的结构特点,但却呈现出与以往鉴定多肽不同的构象和锚定残基。本文对于理解机体对SARS-CoV等病原体产生的T细胞免疫反应,以及为更广泛的人群设计T细胞疫苗具有重要意义。     

Sequence, structure, receptor-binding domains and internal repeats of human apolipoprotein B-100

Apolipoprotein (apo) B-100, the major protein component in low density lipoprotein (LDL), is the ligand that binds to the LDL receptor. It is important in the metabolism of LDL and elevated plasma levels of LDL-apo B are strongly associated with increased risk of coronary artery disease. Although apo B-100 is of great clinical and biological importance its primary structure has defied chemical elucidation, mainly because of its enormous size, insolubility, and tendency to aggregate. Less than 5% of the apo B-100 sequence has been reported, despite the efforts of many laboratories over the past twenty years. Here we report the complete amino acid sequence of human apo B-100 as deducted by sequence analysis of complementary DNA clones; 2,366 of the 4,536 residues were also confirmed by direct sequencing of apo B-100 tryptic peptides. The distribution of trypsin-accessible and -inaccessible peptides of the protein on LDL is non-random and they can be grouped into 5 hypothetical domains. Of 20 potential N-glycosylation sites identified in the sequence, 13 were found by direct peptide sequencing to be glycosylated, and 4 unglycosylated. Examination of the primary structure of apo B-100 reveals that it contains a large number of long (greater than 70 residues) internal repeats and an even larger number of shorter ones, suggesting that the apo B-100 sequence was derived largely from internal duplications. Finally, using synthetic peptides of a specific region of apo B-100, we have identified a potential LDL receptor-binding domain (residues 3,345-3,381) which can bind to the LDL receptor and suppress 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase activities in cultured human fibroblasts.     

Cloning and identification of measles virus receptor gene from marmoset cells

The measles virus (MV) strains with mutated hemagglutinin gene (ha) lost the capacity to infect its sensitive host cells (Vero cells), but it may infect the marmoset B-lymphoblastoid cell line B95a. From above, we can presume that there is a novel cellular receptor for those measles virus strains on B95a cell s. Using the yeast two-hybrid system, we screened and cloned a novel gene--bip (B-lympho- blastoid interaction protein of marmoset) from B95a cell cDNA library, which encoded a protein interacting with measles virus hemagglutinin protein (Ha). The bip cDNA was 1540 base pairs in length and contained a unique open rea ding frame (ORF) of 1011 base pairs encoding a transmembrane protein of 337 amino acid residues. The primary structure of amino acids residue is predicted that the Bip comprised a hydrophobic transmembrane domain and a hydrophobic leader region. The researches about the deletion mutants showed that the deletion of tran smembrane domain in Bip did not affect the interaction between Bip and Ha protei ns. Expression of bip in measles virus non-permissive cell line--CHO (Chinese hamster ovary) cells was performed to prove that CHO/Bip can be infected by meas les virus and then turned to the MV permissive cells. We concluded that the bip gene is a novel measles virus receptor gene in marmoset B-lymphoblastoid cells.     

基于全基因组序列的尖孢镰刀菌分泌蛋白预测及其特征分析

尖孢镰刀菌是一种以土壤习居为主要特征的植物病原菌,广泛分布于世界各地,且寄主广泛,能引起豆科、茄科、葫芦科等科100多种植物根腐、茎腐、茎基腐等病害,严重时造成植株萎蔫死亡,严重影响着植物的产量和品质.同时,目前生产上对于该菌的防治多使用甲霜灵·锰锌、多菌灵、百菌清等化学药剂,然而,防治效果不佳,急需开发针对该菌新的作用靶标的药剂.前人已经明确植物病原真菌分泌蛋白在侵染、操控植物等过程中发挥着重要功能,然而,学术界尚未见有关该菌中分泌蛋白的报道.本研究以尖孢镰刀菌菌株(Fusarium oxysporumf.sp.lycopersici 4287)的蛋白序列为基础数据,以分泌蛋白所具有的N-端含有信号肽、不含有跨膜结构域、没有GPI锚定位点、将蛋白分泌在胞外4大特征为依据,利用生物信息学在线分析程序明确该菌中含有778个分泌蛋白,并对上述蛋白开展特征分析,明确上述蛋白的氨基酸长度主要集中在101～400aa、氨基酸组成中以G,T,S,A较多,信号肽长度主要集中在16～21aa、信号肽氨基酸组成中以P,S,A,T,G较多,信号肽切割位点为T-X-T类型.为今后进一步开展尖孢镰刀菌的分泌蛋白功能解析和开发新型药剂靶标提供了重要的理论基础.     

Sequence similarity analysis of non-self CTL epitopes and mouse proteins using sequence alignment

This research analyzed amino acid sequence similarity between non-self T cell epitopes recognized by mouse antibodies and mouse proteins. Using sequence alignment,we found that only 8 of 1 108 epitopes are highly similar to mouse protein sequences. The result shows that non-self T cell epitopes are not similar or have little similarity to mouse protein sequences. Furthermore,reviewing the related literature,we also found that the eight epitopes would trigger immune responses in some particular environment,which are ignored by T cells in normal condition. The result suggests that no or low-similarity peptide vaccines can reduce the chance of collateral cross-reactions and enhance the antigen-specific immune response to vaccine.