The nucleotide sequence of Shigella flexneri 1A: A common Indian isolate

We report complete sequences of 2 genes of S.flexneri 1a an Indian isolate for the first time. Shigella is causative agent of shigellosis or bacillary dysentery. Genomic library was constructed by shotgun approach. Sequencing was carried out using Big Dye terminator chemistry using ABI 3730 48 capillary DNA analyzer. 170 recombinants were subjected to nucleotide sequencing. Sequence data was analyzed, of these 2 clones showed presence of complete genes out of the total clones sequenced. Annotations were done using various bioinformatics tools. Gene Sfo676 on contig SF21B11, 513 bp long codes for a protein 170 aa long with molecular weight of 18836.5 daltons. The protein is 99 % identical to S. flexneri 2a 301 and not with any other strain of Shigella. It has 7 different sites for phosphorylation, myristoylation and glycosylation. Predicted cellular localization is cytoplasmic membrane. SF0368 is another full-length gene SF0368 on contig SF69C1 is a 312 nucleotide long. It is 103 aa long with molecular weight 11394.0 daltons. Protein is 100% identical to S. flexneri 2a 301 and 99% with S. sonnei strain 046. The gene shows difference when compared with S.sonnei in mono and dinucleotide frequency as well as amino acid composition.     

Identification of a LDL-receptor in the lymphatic filarial parasiteWuchereria bancrofti

Little is known about host-parasite inter-relationship in the lymphatic filarial parasites. There is no information available about the ability of these parasites to acquire cholesterol, though it is known that in general, nematodes lack the ability to synthesise cholesterolde novo. In this study, we have shown that the filarial parasites also lack the ability to incorporate labelled acetate into cholesterol, indicating the absence of the machinery for cholesterol biosynthesis. We have further shown that they elaborate a 43 kDa surface receptor for acquiring LDL-bound cholesterol. We have shown by polymerase chain reaction the presence of a 860 bp fragment indicating the presence of the gene for LDL-related protein (LRP) in the human filarial parasiteWuchereria bancrofti in the genomic DNA. We have also shown that it is expressed as seen in the cDNA clones identified from an expression library.     

人CD34＋干/祖细胞异常表达基因hCLP46功能的初步探讨

hCLP46是从MDS-AML的CD34＋细胞cDNA文库中筛选出的一个新的基因，为了进一步对该基因进行功能、性质研究，本实验利用RT-PCR技术对该基因进行了组织特异性表达检测，并将该基因构建到真核表达载体pcDNA3.1(+)中，转染U937细胞，发现过量表达该基因的U937细胞能够抑制TGF-对p15基因的上调。此外，利用生物信息学手段对该基因进行序列分析，得到其启动子区存在一个典型的“CpG island”。提示该基因有可能参与甲基化调节途径。通过本文的研究，为进一步研究该基因的作用机制打下基础。     

Biochemical characterization of an immunodominant allergen/antigen ofAspergillus fumigatus

An 18kDa protein was identified as a major immunodominant allergen/antigen secreted by a wild type isolate and various clinical isolates ofA. fumigatus. The protein was purified to homogeneity and the N-terminal amino acid was found to be alanine. The N-terminal 20 amino acid sequence of 18kDa was found to be similar to restrictocin, a cytotoxin secreted byAspergillus restrictus. Mass spectroscopic analysis of the purified allergen revealed a molecular size of 17.01 kDa. Immunoreactivity of the purified allergen with monoclonal antibodies and specific IgG and IgE antibodies of the patients of aspergillosis confirmed that this protein is Asp fl.     

Production of recombinant enveloped structural proteins from the Chinese WSSV isolate

The white spot syndrome virus (WSSV) is one of the deadly pathogens of penaeid shrimps and other crustaceans. The WSSV virion consists of an enveloped rod-shaped nucleocapsid enclosing a large circular double stranded DNA genome of 305 Kb with 181 open reading frames. The two major structural genes, VP19 and VP28 were amplified from the genomic DNA of Chinese isolate of WSSV and cloned in pUCm-T vector and sub cloned in pET-30a (+) vector. The expressions of genes inE. coli (BL21) were confirmed by SDS-PAGE analysis. The clones were sequenced, submitted to the gene bank and the Xiang Shan strain of WSSV were compared with the previous reported sequence of WSSV of various regions which revealed that VP19 and VP28 gene sequences had certain differences from the sequences of similar genes of the isolate already reported. The recombinant proteins expressed, purified and characterized.     

溶藻弧菌铁调蛋白基因的克隆、表达及其特征分析

铁为一切有机体所必需。对铁摄取与利用的调控在细菌生长和铁毒性中起重要作用。在革兰氏阴性细菌中,铁摄取调节蛋白(Fur)主管铁的代谢。溶藻弧菌是人和海洋动物共感染的一种主要病原弧菌。本研究克隆表达了溶藻弧菌的fur基因,并分析了其相关特征。该基因序列全长994 bp,其中包含450 bp的开放读码框,编码150个氨基酸残基的蛋白,推导的相对分子量为16.78 kD.在编码序列的上下游,RBS序列-、10序列-、35序列和二个潜在的转录终止子分别得到确定,但没有发现存在于大肠杆菌fur基因中的"铁盒子"。氨基酸序列的中段从G46位至D104位,为高度保守区域,其中包括含组氨酸丰富铁结合模型(H3-D-H-L-V-C-L-D-C-G)。SDS-PAGE分析表明,fur基因可在大肠杆菌中大量表达,带His-Tag的重组融和蛋白经镍亲和层析得到纯化,Western-blot分析结果显示,Fur蛋白具有免疫反应性。为进一步研究Fur蛋白的结构与功能打下基础。     

Zn/Cd超富集植物天蓝遏蓝菜(Thlaspi caerulescens)中TcCaM2基因的克隆及在酵母中的重金属耐受性分析

从重金属超富集植物天蓝遏蓝菜(T. caerulescens)Cd胁迫的cDNA文库中，通过基因表达的差异筛选得到的阳性克隆之一，核苷酸序列分析表明与拟南芥CaM2的相似性高达92%，故命名为TcCaM2(T. caerulescens CaM2, GenBank No. EF053035)。其核苷酸序列与迄今已知的钙调蛋白基因同源率在83%以上，其氨基酸序列同源性高达95%以上，与大多数高等植物的CaM，如印度芥菜、拟南芥、水稻等同源性则更高。将TcCaM2基因置于酵母表达启动子之下，构建酵母表达载体，并将其转入重金属敏感型酵母突变菌株，酵母重金属耐受实验表明，TcCaM2在酵母中的过量表达提高了酵母对Co2+或Ni2+的耐受性，增加了对Cd2+的敏感性，对Zn2+胁迫抗性稍有增加但差异不显著；说明TcCaM2通过其对重金属离子的结合作用及在信号转导系统中的功能，在植物细胞对重金属的富集、耐性中发挥了重要的调节作用。     

Molecular characterization of matrix metalloproteinase-1 (MMP-1) in Lucilia sericata larvae for potential therapeutic applications

BackgroundThe salivary glands of Lucilia sericata are the first organs to express specific endopeptidase enzymes. These enzymes play a central role in wound healing, and they have potential to be used therapeutically.MethodsRapid amplification of cDNA ends and rapid amplification of genomic ends were used to identify the coding sequence of MMP-1 from L. sericata. Different segments of MMP1 gene, namely the middle part, 3′ end, and 5′ end, were cloned, sequenced, and analyzed using bioinformatics tools to determine the distinct features of MMP-1 protein.ResultsAssembling the different segments revealed that the complete mRNA sequence of MMP-1 is 1932 bp long. CDS is 1212 bp long and is responsible for the production of MMP-1 of 404 amino acid residues with a predicted molecular weight of 45.1 kDa. The middle part, 3′ end, and 5′ end sequences were 933, 503, and 496 bp. In addition, it was revealed that the MMP-1 genomic sequence includes three exons and two introns. Furthermore, the three-dimensional structure of L. sericata MMP-1 protein was evaluated, and its alignment defined that it has high similarity to chain A of human MMP-2 with 100% confidence, 72% coverage, and 38% identity according to the SWISS-MODEL modeling analysis.ConclusionsMMP-1 of L. sericata has a close relationship with its homologs in invertebrates and other insects. The present study significantly contributes to understanding the function, classification, and evolution of the characterized MMP-1 from L. sericata and provides basic required information for the development of an effective medical bioproduct.     

鸡白细胞介素18全长基因的克隆、表达及分子进化分析

根据鸡白细胞介素18（IL-18）cDNA基因序列设计了1对特异性引物,应用RT-PCR技术,从脂多糖（lipopolysaccharide,LPS）刺激的我国地方品种萧山鸡原代鸡脾细胞中扩增并克隆鸡IL-18全长基因（Genbank accession,AY628648）。扩增片段全长591bp,共编码197个氨基酸的前体蛋白,其中含有表达完整功能蛋白所必需的起始密码子和终止密码子。该序列与国外报道的鸡IL-18全长基因核苷酸序列及推导的氨基酸序列的同源性为98．99％。只在引导序列中出现两个氨基酸残基的缺失,分子进化分析表明萧山鸡IL-18基因与火鸡以及家鸭基因形成一个独立的分支。将萧山鸡IL-18成熟蛋白序列插入pGEX-4T-2载体并在Ecoli中得到表达,获得45kD的GST-IL-18融合蛋白。经纯化后用于制备多克隆抗体。本研究对萧山鸡白细胞介素18的全长基因进行了克隆及表达,并对其分子进化进行了分析。为深入研究其功能奠定了基础。     

商陆扩展蛋白基因PaEXP1在逆境胁迫下的表达

利用同源克隆的方法,从商陆中克隆得到扩展蛋白基因的全长cDNA序列,命名为PaEXP1(GenBank 登录号为GQ851947).PaEXP1长1128bp,含有759bp的完整开放阅读框,编码252个氨基酸,分子量27.1kD,等电点8.55.PaEXP1氨基酸序列N端含有8个半胱氨酸残基,1个组氨酸(His-Phe-Asp,HFD)域,C末端存在4个保守的色氨酸残基,具有扩展蛋白的典型结构.系统进化树分析表明,PaEXP1属于扩展蛋白的α-扩展蛋白家族.Real time-PCR结果表明,PaEXP1主要在根中表达,低温、盐、重金属和渗透胁迫均强烈地抑制其基因的表达.酵母转化实验表明,转PaEXP1可以提高酵母的平均直径,说明PaEXP1参与细胞壁的松弛扩展和细胞的增大过程.     

Immune response to n-terminal and c-terminal deletion mutants of Aspergillus fumigatus major allergen ASP F 3

The ubiquitous fungus Aspergillus fumigatus causes allergic rhinitis, asthma, sinusitis and allergic bronchopulmonary aspergillosis. A number of major allergens from A. fumigatus are purified, but their structure-function role in the pathogenesis of disease is not known. Such information is essential for devising alternative therapy of fungal allergic diseases. In the present study, N-terminal and C-terminal deletion mutants ofAsp f 3 were constructed and their immunopathological responses studied in a mice model of allergy. Three mutants viz,Asp f 3 (aa 33–168), (aa 1–142), and (aa 23–142) were made by deleting certain amino acids from epitopic regions of full lengthAsp f 3, a major allergen of A. furnigatus. TheAsp f 3 and three mutated proteins were expressed in pET vector. The C-terminal deletion mutantAsp f 3 (aa 1–142) induced elevated IFN-γ but low levels of IL-4 by spleen cells. This mutant also showed significant downregulation of peripheral blood eosinophils and lung inflammation in immunized mice. The N-terminal deletion mutantAsp f 3 (aa 33–168) also exhibited an immuno-suppressive effect in terms of IgE production and induction of Th2 cytokine. The results indicate thatrAsp f 3 and its deletion mutants induced distinct immune-inflammatory responses in mice on challenge with these proteins. The non-IgE binding deletion mutants ofAsp f 3 (aa 1–142 and aa 33–168) could deviate Th2 immune response with a concomitant reduction in airway inflammation and infiltration of inflammatory cells.     

家蚕丝胶蛋白基因1(ser-1)启动子的克隆及其活性分析

家蚕ser-1基因是中部丝腺中特异表达组织特异性基因。为研究家蚕ser-1基因在时空上的调控机制,用PCR的方法克隆了家蚕ser-1基因启动子并进行序列分析,进一步构建了由ser-1基因启动子驱动报告基因DsRed的新型表达质粒pSK-ser-DsRed-PolyA,并通过蚕体和家蚕BmN细胞进行了瞬时表达。结果显示,家蚕ser-1基因启动子的TATA框的保守序列为TATAAAA,位于-24～-30处,CAAT框位于-112～-115处;ser-1基因启动子可以驱动红色荧光基因DsRed在家蚕幼虫的中部丝腺组织和培养的BmN细胞中瞬时表达。     

Characterization of OAZ1 and its potential functions in goose follicular development

BackgroundOrnithine decarboxylase antizyme 1 (OAZ1) is an important regulator of polyamine synthesis and uptake. Our previous studies indicated that high OAZ1 expression in the ovaries of laying geese is responsible for poor egg production. In the present study, the molecular characterization of goose OAZ1 gene was analyzed, as well as the expression profile in various follicular tissues.ResultsAn 873-bp cDNA sequence of the OAZ1 gene (Accession No. KC845302) with a + 1 frameshift site (+ 175T) was obtained. The sequence consisted of a 652-bp two overlapping open reading frames (a putative protein with 216 amino acids). The OAZ domain, OAZ signature and OAZ super family domain were prominent conserved regions among species. As the follicle size increased, OAZ1 abundance showed an increasing trend during follicular development, while it decreased during follicular regression. The level of OAZ1 mRNA expression was the lowest in the fifth largest preovulatory follicle, and was 0.65-fold compared to the small white follicle (P < 0.05). OAZ1 mRNA expression in the largest preovulatory and postovulatory follicle was 2.11- and 2.49-fold compared to the small white follicle, respectively (P < 0.05).ConclusionsThe goose OAZ1 structure confirms that OAZ1 plays an important role in ornithine decarboxylase-mediated regulation of polyamine homeostasis. Our findings provide an evidence for a potential function of OAZ1 in follicular development, ovulation and regression.     

人Dcp2基因克隆表达、多克隆抗体的制备及其应用

mRNA在细胞质内的稳定性是调控基因表达的重要方式。细胞以mRNA降解的方式对过时的、突变有害的mRNA进行及时清除以保证基因的正确表达和细胞正常的生命活动。其中脱帽酶Dcp1/Dcp2在mRNA降解中起到主要作用，但对其降解mRNA的详细作用机制了解甚少。到目前为止，尚没有该基因市售的抗体出现，阻碍了对该基因机理的研究。本文目的是利用PCR方法，从人的胎肝文库中克隆到Dcp2基因，制备其多克隆抗体血清。实验结果显示，通过溴化氰偶联柱子对该多克隆抗体血清经纯化得到的多克隆抗体，用于Western Blotting 试验，可以检测到该蛋白内源性的表达；用于激光共聚焦定位实验显示该内源性蛋白定位在细胞核内；此抗体同时可用于免疫沉淀实验。因此Dcp2基因和多克隆抗体的获得，为进一步研究该基因的功能创造了条件。     

Transient expression of a green fluorescent protein in tobacco and maize chloroplast

BackgroundMaize is one of the most important crops worldwide and has been a target of nuclear-based transformation biotechnology to improve it and satisfy the food demand of the ever-growing global population. However, the maize plastid transformation has not been accomplished due to the recalcitrant condition of the crop.ResultsIn this study, we constructed two different vectors with homologous recombination sequences from maize (Zea mays var. LPC13) and grass (Bouteloua gracilis var. ex Steud) (pZmcpGFP and pBgcpGFP, respectively). Both vectors were designed to integrate into rrn23S/rrn16S from an inverted repeat region in the chloroplast genome. Moreover, the vector had the mgfp5 gene driven by Prrn, a leader sequence of the atpB gene and a terminator sequence from the rbcL gene. Also, constructs have an hph gene as a selection marker gene driven by Prrn, a leader sequence from rbcL gene and a terminator sequence from the rbcL gene. Explants of maize, tobacco and Escherichia coli cells were transformed with both vectors to evaluate the transitory expression–an exhibition of green and red fluorescent light under epifluorescence microscopy. These results showed that both vectors were expressed; the reporter gene in all three organisms confirmed the capacity of the vectors to express genes in the cell compartments.ConclusionsThis paper is the first report of transient expression of GFP in maize embryos and offers new information for genetically improving recalcitrant crops; it also opens new possibilities for the improvement in maize chloroplast transformation with these vectors.How to cite: Arévalo-Gallegos S, Varela-Rodríguez H, Lugo-Aguilar H, et al. Transient expression of a green fluorescent protein in tobacco and maize chloroplast. Electron J Biotechnol 2020;44. https://doi.org/10.1016/j.ejbt.2020.01.008     

Molecular mechanism of pathogenesis of dengue virus: Entry and fusion with target cell

Dengue fever is one of the major health problems in India. Interaction with specific receptor(s) at the cell surface is one of the first events in the pathogenesis of Dengue virus. However, relatively little is known about these receptors. Cellular receptors in human monocytes and mouse neural cells are main target for the viral infection. The envelope protein of the virus (E-protein) plays important role in attachment of virus to target cells and their interaction with cellular receptors. The modulation of receptor gene(s) and/or protein(s) can be used as a method for interfering with virus entry and can thus become a new method for disease prevention. The receptors can be purified by affinity chromatography using E-protein as ligand. It has been reported that addition of highly sulfated heparan sulfate prevents E-protein binding to target cells suggesting that heparan sulfate is utilized by dengue envelope protein to bind to target cells.     

氧化型低密度脂蛋白诱导LOX-1 在基因和蛋白水平表达

目的研究氧化型低密度脂蛋白(oxidized LDL, ox-LDL)是否能在基因和蛋白两个水平诱导内皮细胞表达凝集素样氧化低密度脂蛋白受体(lectin-like oxidized LDL receptor, LOX-1),以探讨LOX-1在动脉粥样硬化形成和发展中的作用.方法将不同浓度ox-LDL(20,40,60,80 mg/L)与内皮细胞共孵育24 h及浓度40 mg/L的ox-LDL作用内皮细胞不同时间(0、3、6、12、24 h),反转录聚合酶链反应检测LOX-1 mRNA水平表达,细胞酶联免疫法测定LOX-1蛋白水平表达.结果加入Ox-LDL 20 mg/L使LOX-1 mRNA和蛋白表达量增加(P＜0.01),40 mg/L使其表达量达最高峰,随后逐渐下降.而同一浓度下从0 h～24 h的趋势是LOX-1 mRNA和蛋白逐渐增加(P＜0.01),在12 h增加最明显.结论氧化型低密度脂蛋白呈剂量依赖性和时间依赖性地上调LOX-1 mRNA和蛋白表达,LOX-1可能在动脉粥样硬化形成和发展中起重要作用.     

串联亲和纯化技术筛选hCLP46的相互作用蛋白

hCLP46(human CAP10-like protein46)是从MDS-AML患者的CD34+干细胞cDNA文库中筛选出的基因.我们利用串联亲和纯化技术来筛选与hCLP46有相互作用的蛋白.通过体内交联-甘氨酸洗脱策略,检测到7条有差异的蛋白带,经液相色谱-质谱联用鉴定,得到了CNX和PDI等一系列内质网伴侣蛋白.所以hCLP46可能是一个糖蛋白,其成熟过程利用了BiP/Grp94和CNX/CRT 2套伴侣蛋白系统.