Cloning and sequence analysis of the 10 kDa antigen gene of Mycobacterium tuberculosis

The gene encoding a major protein antigen of Mycobacterium tuberculosis has been cloned and sequenced using oligonucleotide probes derived from the N-terminal sequence of the analogous protein from Mycobacterium bovis BCG. The gene analysis revealed a sequence encoding a protein of 99 amino acid residues, with a molecular mass of 10.7 kDa. Computer prediction suggests that the protein contains three T-cell-determined epitopes (of which one has been demonstrated experimentally) and three B-cell-determined epitopes. The protein sequence was homologous to two prokaryote heat-shock proteins and the gene possessed heat-shock-like promoter sequences upstream of the initiation codon. A hairpin loop identified in the upstream sequence may also be important in regulation of the gene.     

长角血蜱卵泡抑素相关蛋白的定性

参照GenBank中长角血蜱致病性Okayama株卵泡抑素基因的核苷酸序列(GenBank Accession No.DQ248886)设计合成一对引物,从本实验室保藏的单克隆洁净长角血蜱饥饿成蜱中快速提取总RNA,通过RT-PCR扩增出814bp的卵泡抑素基因,序列比对结果显示:与长角血蜱致病性Okayama株的核苷酸序列及氨基酸序列一致性分别为97.8%和99%,将其亚克隆到表达载体pGEX-4T-1中进行表达,GST融合重组蛋白预期分子量为57kD。表达重组蛋白经MagneGSTTM蛋白纯化系统纯化后作为抗原分别与抗不同发育阶段长角血蜱(卵、幼蜱、若蜱、成蜱)多克隆抗体作为一抗进行免疫印迹,结果表明:与长角血蜱卵制备的多克隆抗体有很强的免疫反应,而与其他发育阶段(幼蜱、若蜱、成蜱)饥饿长角血蜱制备的多克隆抗体反应性很弱。以上结果表明:长角血蜱卵泡抑素蛋白在长角血蜱产卵及卵成熟发育时期的表达水平较其他发育阶段(幼蜱、若蜱、成蜱)的蛋白表达水平高。     

A cDNA encoding a putative lipid transfer protein expressed in sunflower seeds

Based on the N-terminal sequence of a sunflower antifungal protein, a full length cDNA (Ha-LTP5) encoding a putative lipid transfer protein from sunflower seeds was cloned using a RT-PCR based strategy. However, the sequence of the deduced protein is not identical to that of the antifungal protein previously isolated. The nucleotide sequence presents an ORF of 116 amino acids with a putative signal peptide, thus encoding a mature protein of 90 amino acids that is basic and hydrophobic. In contrast to the pattern of expression described for most LTP-like genes from dicots, Northern blot analyses detected constitutive expression of Ha-LTP5 in seeds, but not in aerial parts of sunflower plants.     

Synthesis and characterization of chimeric silkworm silk

A synthetic gene encoding a chimeric silklike protein was constructed that combined a polyalanine encoding region (Ala)(18), a sequence slightly longer than the (Ala)(12-13) found in the silk fibroin from the wild silkworm Samia cynthia ricini, and a sequence encoding GVGAGYGAGAGYGVGAGYGAGVGYGAGAGY, found in the silk fibroin from the silkworm Bombyx mori. A tetramer of the chimeric repeat sequence encoding a approximately 29 kDa protein was expressed as a fusion protein in Escherichia coli. In comparison to S. c. ricini silk, the chimeric protein demonstrated improved solubility because it could be dissolved in 8 M urea. The purified protein assumed an alpha-helical structure based on solid-state (13)C CP/MAS NMR and was less prone to conformational transition to a beta-sheet, unlike native silk proteins from S. c. ricini. Model peptides representing the crystalline region of S. c. ricini silk fibroin, (Ala)(12) and (Ala)(18), formed beta-sheet structures. Therefore, the solubility and structural transitions of the chimeric protein were significantly altered through the formation of this chimeric silk. This experimental strategy to the study of silk structure and function can be used to develop an improved understanding of the contributions of protein domains in repetitive silkworm and spider silk sequences to structure development and structural transitions.     

Identification and cloning of an aspartyl proteinase from Coccidioides immitis

A 45 kDa protein was isolated from a soluble vaccine prepared from formaldehyde-killed spherules of Coccidioides immitis. From the N-terminal amino acid sequence, the protein yielded a 17-amino-acid peptide that was homologous to sequences of other fungal aspartyl proteinases. The coccidioidal cDNA encoding the proteinase was amplified using oligonucleotide primers designed from the 45 kDa N-terminal amino acid sequence and a fungal aspartyl proteinase consensus amino acid sequence. The PCR product was cloned and sequenced, and the remaining 5' upstream and 3' downstream cDNA was amplified, cloned, and sequenced. The cDNA encoding the coccidioidal aspartyl proteinase open reading frame was cloned and the fusion protein containing a C-terminal His-tag expressed in E. coli. The recombinant aspartyl proteinase was purified by immobilized metal affinity chromatography. This recombinant protein will be used for further studies to evaluate its antigenicity, including protective immunogenicity.     

Identification of a gene encoding an immuno-reactive membrane protein from Campylobacter jejuni

A gene encoding a putative membrane protein has been identified from Campylobacter jejuni NCTC 11168 following an immuno-screen of a lambda ZAP II genomic DNA library with antiserum raised against glycine-extractable proteins. The nucleotide sequence of the entire genomic insert revealed six open reading frames, all but one of which have sequence homologues in the complete genome sequence of Helicobacter pylori. The gene encoding the immuno-reactive protein was further identified by independent expression of these reading frames in Escherichia coli. The gene encodes an integral membrane protein, expression of which in E. coli results in a profound filamentous phenotype.     

A trypanosomal CCHC-type zinc finger protein which binds the conserved universal sequence of kinetoplast DNA minicircles: isolation and analysis of the complete cDNA from Crithidia fasciculata.

Replication of the kinetoplast DNA minicircle light strand initiates at a highly conserved 12-nucleotide sequence, termed the universal minicircle sequence. A Crithidia fasciculata single-stranded DNA-binding protein interacts specifically with the guanine-rich heavy strand of this origin-associated sequence (Y. Tzfati, H. Abeliovich, I. Kapeller, and J. Shlomai, Proc. Natl. Acad. Sci. USA 89:6891-6895, 1992). Using the universal minicircle sequence heavy-strand probe to screen a C. fasciculata cDNA expression library, we have isolated two overlapping cDNA clones encoding the trypanosomatid universal minicircle sequence-binding protein. The complete cDNA sequence defines an open reading frame encoding a 116-amino-acid polypeptide chain consisting of five repetitions of a CCHC zinc finger motif. A significant similarity is found between this universal minicircle sequence-binding protein and two other single-stranded DNA-binding proteins identified in humans and in Leishmania major. All three proteins bind specifically to single-stranded guanine-rich DNA ligands. Partial amino acid sequence of the endogenous protein, purified to homogeneity from C. fasciculata, was identical to that deduced from the cDNA nucleotide sequence. DNA-binding characteristics of the cDNA-encoded fusion protein expressed in bacteria were identical to those of the endogenous C. fasciculata protein. Hybridization analyses reveal that the gene encoding the minicircle origin-binding protein is nuclear and may occur in the C. fasciculata chromosome as a cluster of several structural genes.     

cDNA sequence analysis of a novel member of the three loop protein family from the Chinese continental banded krait.

The cDNA encoding a novel three loop protein was cloned from cellular RNA isolated from the venom gland of Bungarus multicinctus multicinctus by RT-PCR. The mature protein has 82 amino acid residues. It shared only 25-38% similarity with some cardiotoxins and did not have sequence similarity with neurotoxins, while its cDNA was about 70% similar to both the cDNAs encoding neurotoxins and the cDNAs encoding cardiotoxins.     

Analysis of the elements of catabolite repression in Clostridium acetobutylicum ATCC 824

The PTSH gene, encoding the phosphotransferase protein HPr, from Clostridium acetobutylicum ATCC 824 was identified from the genome sequence, cloned and shown to complement a PTSH mutant of Escherichia coli. The deduced protein sequence shares significant homology with HPr proteins from other low-GC gram-positive bacteria, although the highly conserved sequence surrounding the Ser-46 phosphorylation site is not well preserved in the clostridial protein. Nevertheless, the HPr was phosphorylated in an ATP-dependent manner in cell-free extracts of C. Acetobutylicum. Furthermore, purified His-tagged HPr from Bacillus Subtilis was also a substrate for the clostridial HPr kinase/phosphorylase. This phosphorylation reaction is a key step in the mechanism of carbon catabolite repression proposed to operate in B. Subtilis and other low-GC gram-positive bacteria. Putative genes encoding the HPr kinase/phosphorylase and the other element of this model, namely the catabolite control protein CcpA, were identified from the C. Acetobutylicum genome sequence, suggesting that a similar mechanism of carbon catabolite repression may operate in this industrially important organism.     

禽巴氏杆菌C48-3株成熟黏附蛋白基因cpm39的克隆及序列分析

目的：对禽巴氏杆菌C48-3躺株编码成熟黏附蛋白的基因cpm39进行克隆和序列分析。方法：通过PCR从禽巴氏杆菌C448-3。基因组DNA中扩增出cpm39基因,克隆到pMD18-T载体中,转化大肠杆菌DH5d,并对目的基因进行核苷酸序列测定;用Clustal X和Mega 2．1软件将测定的序列与GenBank中已登录的16种血清型巴氏杆菌株核苷酸序列进行同源性分析。结果：测序结果表明cpm39基因大小为1002bp,与已知的16个血清型巴氏杆菌cpm39基因核苷酸序列的同源性为81．5％～100％。结论：克隆得到禽巴氏杆菌C。躺株编码成熟黏附蛋白的cpm39基因,该基因在不同血清型巴氏杆菌中具有很高的同源性,该蛋白可以作为研制预防巴氏杆菌病亚单位疫苗的候选抗原。     

Sequence of a Euplotes crassus macronuclear DNA molecule encoding a protein with homology to a rat form-I phosphoinositide-specific phospholipase C.

A 604-base pair macronuclear DNA molecule from the hypotrichous ciliate Euplotes crassus was cloned and its DNA sequence determined. The DNA sequence contains an open reading frame capable of encoding a protein 141 amino acids in length. The putative protein contains significant sequence similarity to other eukaryotic proteins, including the rat form-I phosphoinositide-specific phospholipase-C.     

Cloning and expression of cDNA coding for bouganin.

Bouganin is a ribosome-inactivating protein that recently was isolated from Bougainvillea spectabilis Willd. In this work, the cloning and expression of the cDNA encoding for bouganin is described. From the cDNA, the amino-acid sequence was deduced, which correlated with the primary sequence data obtained by amino-acid sequencing on the native protein. Bouganin is synthesized as a pro-peptide consisting of 305 amino acids, the first 26 of which act as a leader signal while the 29 C-terminal amino acids are cleaved during processing of the molecule. The mature protein consists of 250 amino acids. Using the cDNA sequence encoding the mature protein of 250 amino acids, a recombinant protein was expressed, purified and characterized. The recombinant molecule had similar activity in a cell-free protein synthesis assay and had comparable toxicity on living cells as compared to the isolated native bouganin.     

The nucleotide sequence of the yeast MEL1 gene.

The complete nucleotide sequence of the MEL1 gene of the yeast, Saccharomyces cerevisiae, encoding alpha-galactosidase was determined. The nucleotide sequence contains an open reading frame of 1413 bp encoding a protein of 471 amino acids. Comparison with the known N-terminal amino acid sequence of the mature secreted protein indicated that alpha-galactosidase is synthesized as a precursor with an N-terminal signal sequence of 18 amino acids. The general features of this signal peptide resemble those of other yeast signal peptides. Molecular weight of the mature alpha-galactosidase polypeptide deduced from the nucleotide sequence is 50.049 kd. The 5' regulatory region has sequences in common with other yeast genes regulated by the GAL4-protein.     

The primary structures of helices A to G of three new bacteriorhodopsin-like retinal proteins.

The primary structures of helices A to G of all bacteriorhodopsin (BR)-like retinal proteins identified in newly isolated halobacteria have been determined from the nucleotide sequence of the BR-like protein genes. Using PCR methods, gene fragments encoding the A- to G-helix region of BR-like proteins were directly amplified from the total genomic DNA of the seven new halobacterial strains. Oligonucleotide primers corresponding to highly conserved regions in the helices A to G were designed from the nucleotide sequences of bacterioopsin (bop) and archaeopsin-I (aop-I), and some primers were effective for the amplification of the gene encoding C- to G-helix region of all new BR-like proteins. The primer corresponding to A-helix region was designed either from the nucleotide sequence of bop and aop-I or from the N-terminus amino acid sequence of a BR-like protein. Three new BR-like proteins were identified from the amino acid sequence, which was deduced from the nucleotide sequence of the genes encoding A- to G-helix region of the BR-like proteins. It was found that not only the amino acid sequence, but also the nucleotide sequence of the gene encoding the C- and G-helix region, in which a number of important residues for proton translocation are located, is highly conserved in three new BR-like proteins. Analysis of the primary structures of the A- to G-helix region of new BR-like proteins revealed that one has about 85% homology with aR-I and aR-II, and the rest have about 55% homology with halobium BR, aR-I and aR-II.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular characterization of the S-layer gene,sbpA, of Bacillus sphaericus CCM 2177 and production of a functional S-layer fusion protein with the ability to recrystallize in a defined orientation while presenting the fused allergen

The nucleotide sequence encoding the crystalline bacterial cell surface (S-layer) protein SbpA of Bacillus sphaericus CCM 2177 was determined by a PCR-based technique using four overlapping fragments. The entire sbpA sequence indicated one open reading frame of 3,804 bp encoding a protein of 1,268 amino acids with a theoretical molecular mass of 132,062 Da and a calculated isoelectric point of 4.69. The N-terminal part of SbpA, which is involved in anchoring the S-layer subunits via a distinct type of secondary cell wall polymer to the rigid cell wall layer, comprises three S-layer-homologous motifs. For screening of amino acid positions located on the outer surface of the square S-layer lattice, the sequence encoding Strep-tag I, showing affinity to streptavidin, was linked to the 5' end of the sequence encoding the recombinant S-layer protein (rSbpA) or a C-terminally truncated form (rSbpA(31-1068)). The deletion of 200 C-terminal amino acids did not interfere with the self-assembly properties of the S-layer protein but significantly increased the accessibility of Strep-tag I. Thus, the sequence encoding the major birch pollen allergen (Bet v1) was fused via a short linker to the sequence encoding the C-terminally truncated form rSpbA(31-1068). Labeling of the square S-layer lattice formed by recrystallization of rSbpA(31-1068)/Bet v1 on peptidoglycan-containing sacculi with a Bet v1-specific monoclonal mouse antibody demonstrated the functionality of the fused protein sequence and its location on the outer surface of the S-layer lattice. The specific interactions between the N-terminal part of SbpA and the secondary cell wall polymer will be exploited for an oriented binding of the S-layer fusion protein on solid supports to generate regularly structured functional protein lattices.     

Sequence of a Euplotes crassus Macronuclear DNA Molecule Encoding a Protein with Homology to a Rat Form-I Phosphoinositide-specific Phospholipase C

A 604-base pair macronuclear DNA molecule from the hypotrichous ciliate Euplotes crassus was cloned and its DNA sequence determined. The DNA sequence contains an open reading frame capable of encoding a protein 141 amino acids in length. The putative protein contains significant sequence similarity to other eukaryotic proteins, including the rat form-I phosphoinositide-specific phospholipase C.     

球毛壳菌（Chaetomium globosum）过氧化物膜蛋白过敏原基因克隆、序列分析及原核表达

以球毛壳菌cDNA文库中获得过氧化物膜蛋白（pero）基因片段（GenBank Accn:BP099709）为基础,用RACE 技术获得该基因的全长cDNA序列。序列长747bp,由412bp的3′RACE产物和508bp的5′RACE产物拼接而成。开放阅读框501bp,编码166个氨基酸,蛋白分子量为17.5kD,理论等电点为5.75。利用cDNA两侧非编码区序列作引物克隆出该基因的DNA序列,序列分析表明该基因由2个内含子和3个外显子组成。ClustalX多序列比对表明：该基因与粗糙脉孢菌（Neurospora crassa）的过氧化物膜蛋白过敏原同源性最高（83%）。将pero基因编码区克隆到原核表达载体pET28a中,构建成表达质粒pET28a-pero并转化大肠杆菌BL21,IPTG诱导后SDS-PAGE检测表达情况,结果发现在21kD处有一特异性融合蛋白带,大小与预期相符,说明该基因已经在大肠杆菌中表达。克隆的cDNA序列、DNA序列及推测的氨基酸序列在GenBank登录(登录号分别为AY555771,AY584753,AAS66898)。     

Nucleotide sequence of a genomic and a cDNA clone encoding an extracellular alkaline protease of Aspergillus fumigatus

An Aspergillus fumigatus extracellular alkaline protease (ALP) which is an enzyme of the subtilisin family is a potential virulent factor of the fungus. The gene encoding ALP was isolated from a genomic library made from DNA of an A. fumigatus isolate. The nucleotide sequence of this gene was compared to that of a cDNA encoding A. oryzae ALP and to that of a cDNA from A. fumigatus encoding the mature ALP protein. Mature A. fumigatus ALP contains 282 amino acids and is encoded by three exons. The pre-proenzyme has a leader sequence of 121 amino acids.