梅花鹿S100A4 基因的克隆及融合蛋白的表达

为了研究梅花鹿S100A4 (S100 calcium binding protein A4)基因在鹿茸生长过程中的作用。用RT-PCR 法
从生茸骨膜细胞总RNA 中克隆了梅花鹿S100A4 基因,在NCBI 中对基因序列进行比对;将完整的基因序列与逆
转录病毒表达载体pLEGFP-C1 重组,获得了重组质粒pLEGFP-S100;用脂质体法将pLEGFP-S100 与pVSV-G (被
膜载体)共转染包装细胞GP2 - 293,获得重组病毒上清液,感染角柄骨膜细胞后逆转录病毒携带的基因进入宿
主细胞。结果显示:S100A4 基因是一个相对保守的基因,与多个物种的匹配度达到90% ;重组逆转录病毒载体
pLEGFP-S100 可以形成重组逆转录病毒粒子,将S100A4 基因导入靶细胞,并表达S100A4 与GFP (Green fluorescent
protein)的融合蛋白。     

Cloning and expression of beta1,4-galactosyltransferase gene from Helicobacter pylori

Helicobacter pylori, which is a human pathogen associated with gastric and duodenal ulcer, has been shown to express human oncofetal antigens Lewis X and Lewis Y. Although the mammalian glycosyltransferases that synthesize these structures are well characterized, little is known about the corresponding bacterial enzymes. We report that a novel beta1,4-galactosyltransferase gene (HpgalT) involved in the biosynthesis of lipopolysaccharides in H. pylori has been cloned and expressed in Escherichia coli. The deduced amino acid sequence of the protein (HpGal-T) encoded by HpgalT consists of 274 residues with the calculated molecular mass of 31,731 Da, which does not show significant similarity to those of beta1,4-galactosyltransferases from mammalian sources and Neisseria It was confirmed that HpGal-T catalyzed the introduction of galactose from UDP-Gal in a beta1,4 linkage to accepting N-acetylglucosamine (GlcNAc) residues by means of high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). When the E.coli cells which overexpressed HpgalT was coupled with the UDP-Gal production system, which consisted of recombinant E.coli cells overexpressing its UDP-Gal biosynthetic genes and Corynebacterium ammoniagenes, N-acetyllactosamine, a core structure of lipopolysaccharide of H.pylori, was efficiently produced from orotic acid, galactose, and GlcNAc.     

Cloning and expression of a beta tubulin gene of Physarum polycephalum

A beta tubulin gene of Physarum polycephalum has been isolated from a genomic library in the phage EMBL4. Southern-blot hybridization to genomic DNA indicates that the cloned DNA is derived from the betB1 locus of the beta tubulin gene family. A tubulin-specific subfragment of the phage DNA was used as a hybridization probe to construct a restriction map of the betB1 locus. The probe consisted of the almost complete coding region of the 5' half of the tubulin gene, interrupted by one intron. The derived amino acid sequence of this subclone deviates from the protein sequence for Physarum amoebal beta tubulin (amino acids 4-207) in two of 207 amino acids. We used both recA and recBC sbcB bacterial host strains, which have been recommended for cloning of instability-conferring sequences of the Physarum genome, but were unable to subclone the 3' part of the gene from the phage DNA. Primer-extension analysis indicates that the betB gene is expressed in the vegetatively proliferating amoebal and plasmodial stages of the life cycle as well as in differentiating (sporulating) plasmodia.     

Immunohistochemical localization of S100 beta in human nervous system tumors by using monoclonal antibodies with specificity for the S100 beta polypeptide

The immunohistochemical localization of the calcium-binding protein, S100 beta, in human nervous system tumors has been examined by using a monoclonal antibody with specificity for the S100 beta polypeptide. S100 beta-specific immunoreactivity is detected in astrocytoma, glioblastoma, Schwannoma, ependymoma, and craniopharyngioma, whereas no reactivity is seen in oligodendroglioma, meningioma, neuroblastoma, or medulloblastoma. These data suggest that analysis of S100 beta localization with these monoclonal antibodies may be useful for research or diagnostic purposes.     

Cloning and expression pattern of the human NDRG3 gene

We report the cloning and expression pattern of a novel N-myc downstream-regulated gene 3 (NDRG3), located on human chromosome 20q11.21-11.23. The NDRG3 cDNA is 2588 base pair in length, encoding a 363 amino acid polypeptide highly related to mouse Ndr3 protein. Northern blot reveals that NDRG3 is highly expressed in testis, prostate and ovary. By in situ hybridization, the NDRG3 mRNA was localized to the outer layers of seminiferous epithelium, indicating that it may play a role in spermatogenesis.     

Cloning and expression of human core 1 beta1,3-galactosyltransferase.

The common core 1 O-glycan structure Galbeta1--> 3GalNAc-R is the precursor for many extended mucin-type O-glycan structures in animal cell surface and secreted glycoproteins. Core 1 is synthesized by the transfer of Gal from UDP-Gal to GalNAcalpha1-R by core 1 beta3-galactosyltransferase (core 1 beta3-Gal-T). Amino acid sequences from purified rat core 1 beta3-Gal-T (Ju, T., Cummings, R. D., and Canfield, W. M. (2002) J. Biol. Chem. 277, 169-177) were used to identify the core 1 beta3-Gal-T sequences in the human expressed sequence tag data bases. A 1794-bp human core 1 beta3-Gal-T cDNA sequence was determined by sequencing the expressed sequence tag and performing 5'-rapid amplification of cDNA ends. The core 1 beta3-Gal-T predicts a 363-amino acid type II transmembrane protein. Expression of both the full-length and epitope-tagged soluble forms of the putative enzyme in human 293T cells generated core 1 beta3-Gal-T activity that transferred galactose from UDP-Gal to GalNAcalpha1-O-phenyl, and a synthetic glycopeptide with Thr-linked GalNAc and the product was shown to have the core 1 structure. Northern analysis demonstrated widespread expression of core 1 beta3-Gal-T in tissues with a predominance in kidney, heart, placenta, and liver. Highly homologous cDNAs were identified and cloned from rat, mouse, Drosophila melanogaster, and Caenorhabditis elegans, suggesting that the enzyme is widely distributed in metazoans. The core 1 beta3-Gal-T sequence has minimal homology with conserved sequences found in previously described beta3-galactosyltransferases, suggesting this enzyme is only distantly related to the known beta3-galactosyltransferase family.     

Synthesis and expression of a gene coding for the calcium-modulated protein S100 beta and designed for cassette-based, site-directed mutagenesis

As an initial step in studies aimed at addressing the question of what common and unique features of the S100 family of proteins are related to their specific functions and localizations, a gene coding for one of the S100 proteins, S100 beta, has been prepared by ligation of 12 overlapping, synthetic oligonucleotides. Automated DNA sequence analysis demonstrated that the final construct has the expected structure. The gene was inserted into a plasmid vector that contains a tac promoter and ampicillin-resistance gene, thus allowing both amplification and direct expression cloning in Escherichia coli. The gene was designed to allow rapid, efficient changes of single or multiple amino acids by using cassette-based mutagenesis while the gene is resident in the vector. The expressed protein (VUSB-1) is indistinguishable from bovine brain S100 beta in terms of electrophoretic mobility, reactivity with antibodies to S100 beta, amino acid composition, and partial amino acid sequence analysis. Preparations of expressed protein are also functionally similar to bovine brain S100 beta as determined by aldolase activator activity and neurite extension factor activity, supporting the concept that these activities are a property of the S100 beta polypeptide.     

Molecular cloning and characterization of the human S100A14 gene encoding a novel member of the S100 family

S100 proteins form a growing subfamily of proteins related by Ca2+-binding motifs to the Efhand Ca2+-binding protein superfamily. By analyzing a human lung cancer cell line subtraction cDNA library, we have identified and characterized a new member of the human S100 family that we named S100A14 (GenBank acc. no. NM_020672). It encodes a mRNA present in several normal human tissues of epithelial origin, with the highest level of expression in colon. The full-length cDNA is 1067 nt in length, with a coding region predicting a protein of 104 amino acids that is 68% homologous to the S100A13 protein. The deduced amino acid sequence of the human S100A14 and its mouse homolog (identified as GenBank entry) contains two EF-hand Ca2+-binding domains, a myristoylation motif, a glycosylation site, and several potential protein kinase phosphorylation sites. We have mapped this gene to human chromosome 1q21, within a region where at least 15 other S100 genes are tightly clustered. A 3.2-kb genomic fragment containing the entire S100A14 was cloned and sequenced. The gene is split into four exons and three introns spanning a total of 2165 bp of genomic sequence. We examined the intracellular distribution of the epitope-tagged S100A14 protein in two human lung carcinoma cell lines and one immortalized monkey cell line. Pronounced staining was observed in the cytoplasm, suggesting an association with the plasma membrane and in the perinuclear area. We also provide evidence for heterogenic expression of S100A14 in tumors, demonstrating its overexpression in ovary, breast, and uterus tumors and underexpression in kidney, rectum, and colon tumors, a pattern suggesting distinct regulation with potentially important functions in malignant transformation.     

酿酒酵母CICC1747醛糖还原酶基因的克隆及表达

采用PCR技术以酿酒酵母CICC1747基因组DNA为模板扩增得到醛糖还原酶基因GRE3,插入到pET-15b载体的NdeⅠ和BamHⅠ酶切位点之间,构建了酿酒酵母醛糖还原酶原核表达载体pET-15b-GRE3。将该载体转化到大肠杆菌菌株Rosetta(DE3)中,重组菌株用IPTG诱导表达,采用紫外分光光度法测定醛糖还原酶活力,并对其表达条件进行初步优化。SDS-PAGE电泳结果显示在分子量约37 kD处有明显的特异性蛋白质条带。发酵液的比酶活最高为54.94 mU/mg,与酿酒酵母野生菌株相比提高了近10倍。     

Cloning and functional expression of a human heparanase gene.

We have cloned a gene (HSE1) from a human placental cDNA library that encodes a novel protein exhibiting heparanase activity. The cDNA was identified through peptide sequences derived from purified heparanase isolated from human SK-HEP-1 hepatoma cells. HSE1 contains an open reading frame encoding a predicted polypeptide of 543 amino acids and possesses a putative signal sequence at its amino terminus. Northern blot analysis suggested strong expression of HSE1 in placenta and spleen. Transient transfection of HSE1 in COS7 cells resulted in the expression of a protein with an apparent molecular mass of 67-72 kDa. HSE1 protein was detectable in conditioned media but was also associated with the membrane fraction following cell lysis. The HSE1 gene product was shown to exhibit heparanase activity by specifically cleaving a labeled heparan sulfate substrate in a similar manner as purified native protein.     

Cloning of human lysozyme gene and expression in the yeast Saccharomyces cerevisiae

cDNA clones encoding human lysozyme were isolated from a human histiocytic cell line (U-937) and a human placenta cDNA library. The clones, ranging in size from 0.5 to 0.75 kb, were identified by direct hybridization with synthetic oligodeoxynucleotides. The nucleotide sequence coding for the entire protein was determined. The derived amino acid sequence has 100% homology with the published amino acid (aa) sequence; the leader sequence codes for 18 aa. Expression and secretion of human lysozyme in Saccharomyces cerevisiae was achieved by placing the cloned cDNA under the control of a yeast gene promoter (ADH1) and the alpha-factor peptide leader sequence.     

DNA sequences regulating human beta globin gene expression.

Human delta globin is expressed at approximately 1-2% of the level of human beta globin in erythroid cells despite the marked homology between these two globins. To determine the DNA sequences responsible for this effect, delta and beta globin genes and fusion products of these genes constructed in vitro were transfected and expressed in HeLa cells. The results indicate that when the small intervening sequence of the beta gene (beta IVS 1) is replaced by delta IVS 1, expression of the chimeric gene is the same as that of the normal beta globin gene. By contrast, when the large intervening sequence of the beta gene (beta IVS 2) is replaced by delta IVS 2, expression of the chimeric gene is markedly reduced. These results suggest that there are signals within IVS 2 of the delta and beta genes which affect their relative expression.