Biomedical subjects

X F Wu

Publications and source records attributed to X F Wu.

At least 19 recordsLinked to original sources

PubMed2002

[Cloning and expression of gynecophoral canal protein gene of Schistosoma japonicum (Chinese strain)].

A 1949 bp cDNA fragment was amplified by RT-PCR from adult Schistosoma japonicum (Chinese strain) mRNA with 3 pair of primers that were designed according to published SmGCP gene encoding gynecophoral canal protein of Schistosoma mansoni and SjGCP1 gene encoding the conservative region of gynecophoral canal protein of Schistosoma japonicum. Sequence analysis indicated that this fragment, named SjGCP, with 85% identity to SmGCP, contained a complete open reading frame (ORF) of gynecophoral canal protein gene of Schistosoma japonicum (Chinese strain). The amino acid sequence shared 83.7% identity with gynecophoral canal protein of Schistosoma mansoni. This fragment was cloned into the expression vector pET28c(+) and subsequently expressed in Escherichia coli. SDS-PAGE revealed that the molecular weight of this expressed product was 80 kD. Western blotting showed that the recombinant protein reacted well with the rabbit serum immunized with Sj worm antigen, indicating that this expressed product had good antigenicity.

Amino Acid Sequence↗

PubMed2002

[The hemolytic site of the basic phospholipase A(2) from Agkistrodon halys pallas].

The gene of the basic phospholipase A(2) from Agkistrodon halys Pallas (BPLA(2) )was mutated site-directedly by polymerase chain reaction (PCR) and the residue Arg(34) of the encloding protein was substituted by Glu and Gln respectively. The mutant gene has been cloned into the expression vector pBLMVL2 and has been expressed in E.coli RR1 effectively. The protein was produced as insoluble inclusion bodies. After partial purification, the inclusion bodies were denatured and renatured into active form, and the renatured recombinant protein was purified by gel-filtration. The expression product has the same enzymatic activity as the denatured-refolded BPLA(2) and its hemolytic activity dropped distinctly, which suggest that the basic residue Arg(34) of BPLA(2) is a crucial amino acid residue during the process of hemolysis.

Agkistrodon↗

PubMed2001

[Gene chimeric fusion and expression of nucleocapsid NS3 regions and NS4 regions of hepatitis C virus genome].

Genes encoding HCV core and NS4 antigen epitopes and C33c antigen were cloned from HCV genome by PCR, respectively. Two fused genes were constructed. One contained these three genes, another contained genes encoding C33c antigen and NS4 antigen epitopes. These fused genes were cloned into expression plasmid pET-24(a)+ and pET-22(b)+ under T7 promoter and transformed into E. coli BL21 (DE3) respectively. SDS-PAGE analysis revealed that these fused antigens CCN, CN were highly expressed after the induction by 1 mmol/L IPTG. These Expression products were detected by western blotting with anti-HCV serum.

Escherichia coli↗

PubMed2001

[The new antiendotoxin strategies].

Lipopolysaccharide(LPS) is important in the pathogenesis of sepsis infected by gram-negative bacteria in humans, and part or all of this pathophysiology is mediated by a complex secondary inflammatory response. Administration of purified LPS or a variety of LPS-free recombinant mediators can reproduce much of the pathophysiology of gram-negative sepsis. There has been an explosion of new information over the last several years regarding the interactions of LPS with proteins and cells. Blocking sepsis at the bacterial toxin level is appealing because it is upstream, so that the secondary pathologic inflammatory cascade may be limited or prevented. A variety of antiendotoxin strategies have been proposed. Therapies under investigation include agents that bind and neutralize LPS, agents or systems that enhance LPS clearance, and agents that inhibit LPS interaction with serum elements or cellular receptors.

Acute-Phase Proteins↗

PubMed2001

[Cloning of secondary lymphoid-tissue chemokine (SLC) and its expression in prokaryotic system].

The total RNA from lymphoid tissue in Chinese was extracted, and the gene encoding the mature peptide of secondary lymphoid-tissue chemokine (SLC) was cloned by RT-PCR. Nucleotide sequence analysis showed that there is only one nucleotide different from that reported, but it doesn't alter the amino acid encoded. The SLC cDNA was inserted into an expression vector pET-28a(+) under T7 promoter and constructed recombinant plasmid pET28a-SLC. pET28a-SLC was transformed to E. coli BL21(DE3) and the expression strain was gotten. After inducing with IPTG for 3-5 hours the bacterium were sonicated. After centrifuging the supernatant was analysed by SDS-PAGE. An obvious expression band about 18 kD can be seen. The expressed product was purified by Ni2+ affinity chromatography column, and the purity is up to 90 percent.

Chemokine CCL21↗

PubMed2000

Purification, characterization, and cDNA sequence of halysetin, a disintegrin-like/cysteine-rich protein from the venom of Agkistrodon halys Pallas.

By means of DEAE-Sepharose CL-6B column chromatography, gel filtration on Sephadex G-75 and Superose 12 FPLC, halysetin, an antiplatelet protein, was purified from the venom of Agkistrodon halys Pallas with molecular mass of 29 kDa on SDS-PAGE and 23,168 Da by mass spectrometry. The p1 was about 5.0. Halysetin was devoid of phospholipase A2, fibrino-(geno)lytic, esterase, hemorrhagenic activities. Halysetin dose-dependently inhibited the aggregation of human platelet, which was stimulated by collagen with IC50 of 420 nM, but not that stimulated by ADP. The N-and C-terminal sequences of halysetin were characterized. Its full-length cDNA was cloned by RT-PCR from the total RNA extracted from the snake venom gland. It encoded a protein of 212-amino-acid residues with disintegrin-like/cysteine-rich domains and was highly homologous with SYMPs (snake venom metalloprotease).

Adenosine Diphosphate↗

PubMed2000

Preparation of recombinant human GDNF by baculovirus expression system and analysis of its biological activities.

Recombinant human glial cell line-derived neurotrophic factor (GDNF) has been expressed at high levels and produced in large quantities in baculovirus-infected Trichoplusia ni cells (Tn-5B1-4). The glycosylated protein was purified using immunoaffinity chromatography and gel filtration. Pure, recombinant human GDNF promoted the survival and morphological differentiation of embryonic dopaminergic neurons and enhanced axonal regeneration after sciatic nerve transection. Because recombinant bioactive human GDNF can be obtained in large quantities, and purified to near homogeneity, they are suitable for evaluation in animal models.

Animals↗

PubMed2000

Expression of isolated C-type carbohydrate recognition domains.

A galactose-binding lectin from the venom of the snake Trimeresurus stejnegeri consists of isolated carbohydrate recognition domains, belonging to group VII of the C-type animal lectins. As a first step toward determining the tertiary structure of the galactose-specific lectin, we produced the lectin in Escherichia coli. By in vitro refolding and affinity chromatography, modest amounts (8 mg/liter) of active recombinant proteins were obtained. The recombinant protein was homogeneous, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. Its amino acid sequence without the initiated methionine at the N-terminus was also characterized by mass spectrometry. The data of hemagglutination and enzyme-linked lectin binding assays demonstrated that the recombinant lectin showed similar sugar-binding activity as the native protein. In addition, fluorescence spectroscopy and circular dichroism also showed obviously their structural similarity.

Animals↗

PubMed2000

Human glial cell-line-derived neurotrophic factor: a structure-function analysis.

Glial cell-line-derived neurotrophic factor (GDNF) is a protein known to enhance the survival of dopaminergic and motor neurons. It has been shown to have therapeutic potential in the treatment of Parkinson's disease and other neurodegenerative diseases. GDNF gene was modified by deletion and insertion mutagenesis using PCR methods. The various mutants were all highly expressed in Escherichia coli. The recombinant proteins were purified and their survival-promoting activities were determined by motor neurons. The result showed that the C-terminus was critical for structure stability of GDNF, and the alpha-helix, finger1 and finger2 regions were involved in receptor binding, while the N-terminus was not essential for the biological functions of GDNF.

Animals↗

PubMed2000

High-dose and long-term therapy with interferon-alfa inhibits tumor growth and recurrence in nude mice bearing human hepatocellular carcinoma xenografts with high metastatic potential.

Postoperative recurrence of human hepatocellular carcinoma (HCC) is the major issue that must be addressed to further improve prognosis. This study was undertaken to investigate the effects of interferon-alfa-1b (IFN-alpha-1b) on recurrent tumor and metastasis after curative resection in nude mice bearing an HCC xenograft with high metastatic potential. Tumor tissues from LCI-D20, a metastatic model of HCC in nude mice, were orthotopically implanted in 105 nude mice. Eleven days later, 64 mice underwent curative resection of liver tumors. IFN-alpha at different doses was administered subcutaneously to mice with or without resection. In mice without resection, when comparison was made among control, IFN 7.5 x 10(6) U/kg/day, 1.5 x 10(7) U/kg/day for treated groups, and 3 x 10(7) U/kg/day; tumor volume was 8,475 mm(3) +/- 2,636 mm(3), 7,963 mm(3) +/- 3,214 mm(3), 769 mm(3) +/- 287 mm(3), and 13 mm(3) +/- 9 mm(3); incidence of lung metastasis being 100%, 80%, 40%, and 0%; life span was 45 +/- 4 days, 53 +/- 8 days, 81 +/- 6 days, and 105 +/- 24 days, respectively. In mice with curative resection, when comparison was made among control, IFN 5 x 10(5) U/kg/day, 1 x 10(6) U/kg/day, 4 x 10(6) U/kg/day, 7.5 x 10(6) U/kg/day, 1.5 x 10(7) U/kg/day, and 3 x 10(7) U/kg/day for treated groups; incidence of recurrent tumor was 100%, 100%, 87.5%, 100%, 87.5%, 62.5%, and 12.5%; lung metastasis being 100%, 75%, 87.5%, 50%, 62.5%, 0%, and 0%, respectively. IFN-alpha inhibited neovascularization induced by LCI-D20 tumor specimens implanted into the micropocket of nude mice corneas. In conclusion, high-dose and long-term therapy with IFN-alpha dose-dependently inhibits tumor growth and recurrence after resection of HCC. The effect of IFN-alpha may be attributed to antiangiogenesis in this experiment. These results provide potential clinical implication, particularly for the prevention of recurrence after curative resection of HCC.

Animals↗

PubMed2000

[High expression of human EPO gene in the larvae and pupae of the silkworm, Bombyx mori].

Erythropoietin (EPO) is a glycoprotein hormone produced primarily by the kidney, and is principal factor regulating red blood cell poduction. The synthesized EPO cDNA was inserted into the transfer vector pBlueBac III to generate the recombinant transfer plasmid pBlueBacEPO. Cotransfection of BmN cells with pBlueBacEPO DNA and Wild BmNPV DNA generated the recombinant virus rBmNPVEPO carrying EPO gene driven by the strong promoter of AcNPV polyhedrin gene. The results of Southern blot and PCR reaction confirmed that EPO gene had been correctly inserted in the target position in the BmNPV genome. ELISA assay showed that the EPO gene was expressed with high level in the larvae and pupae of the silkworm. The larvae and pupae produced as high as 62,800 u and 74,000 u in 1 mL hemolymph on the 4th day (larve) and the 5th day(pupae) after infection with the recombinant virus rBmNPVEPO, respectively. Western blot analysis showed the molecular weight of rhEPO produced in the lavae or pupae was about 26 kD. Biologic assay showed the rhEPO had high activity (about 63,000 u in per milliliter of hemolymph) in vitro.

Animals↗

PubMed2000

[Expression of Bacillus thuringiensis (Bt) crystal toxin gene in the chloroplast of tobacco].

The 3.5 kb wild-type Bt Cry I A(c) gene and its 3' truncated forms (2.1 kb, 1.8 kb) were placed under the control of plastid expression signals consisting of the strong light-induced psbA promoter and its 3' untranslated region with the aadA cassette (Prrn, aadA and psbA3') as a selectable marker. The resulting vectors pBT3, pBT8 and pBT22 also contain flanking tobacco plastid DNA homology regions to direct insertion of the Bt transgene into the tobacco plastid genome between psbA and trnK by homologous recombination. Transformed plastid genomes were selectively amplified by growing the cells on spectinomycin medium. Several independently transformed lines were obtained at last. The results of Southern and Western blot demonstrated that these three kinds of Bt genes had been introduced into tobacco plants, and their filial generations are resistant to spectinomycin. Insecticidal activity assay with transgenic tobacco leaves indicate that some plants have strong toxicity to cotton bollworm. This is the first report in China that Bt gene has been introduced and successfully expressed in the chloroplast of higher plants.

Bacillus thuringiensis Toxins↗

PubMed2000

[Expression of human GDNF in methyltrophic yeast Pichia pastoris and silkworm larvae].

The cDNA encoding glial cell derived neurotrophic factor (GDNF) was cloned into the Pichia expression vector pPIC9K and then transformed into his4 mutant yeast GS115 by electroporation. Multicopy transformants were screened by various G418 concentrations and induced by methanol. The human GDNF gene was cloned into the baculovirus transfer vector pBacPAK8. The recombinant transfer vector pBacPAK-GDNF was coinfected with linear Bm-BacPAK6 DNA into BmN cells. The recombinant virus was screened and plaque-purified. The silkworm larvae were infected with the recombinant virus and collected 5 days later. SDS-PAGE and Western blot confirmed that GDNF was expressed in Pichia culture medium and silkworm larvae hemolymph. The GDNF protein expressed in Pichia and silkworm larvae could significantly promote the survival and neurite outgrowth of dopaminergic neurons.

Animals↗

PubMed2000

[Fusion and expression of the gene encoding human Mn-SOD to anti-CEA single-chain antibody in Escherichia coli].

The gene encoding human manganese-superoxide dismutase (Mn-SOD) was fused to anti-carcinoembryonic antigen single-chain antibody gene to construct the fusion gene, then was ligated into prokaryotic expression vector pET-22b(+), The fusion gene was expressed in E. coli at high level, accounting for 24% of the total bacteria soluble protein; and was characterized by SDS-PAGE and Western-blot analysis; the expression product had the CEA-binding ability in RIA, and also had the SOD activity by pyrogallol autoxidation assay. So, the Mn-SOD moiety retains substantial enzymatic activity, where the ScFv moiety can deliver the fusion protein to tumor, Mn-SOD is a potential tumor-suppressor gene, maybe the fusion protein can provide a new pathway to tumor therapy.

Amino Acid Sequence↗

PubMed2000

[Cloning of the zot gene of Vibrio cholerae and its expression in Escherichia coli].

The zot gene encoding Zonula occludens toxin was amplified from classic Vibrio cholerae genomic DNA by PCR. The result of sequencing indicated that zot gene encodes 399 amino acid residues. The sequence of zot gene was a little bit different from that of reported including 14 nucleotides and four amino acid residues. The expression plasmid pET-ZOT was constructed by inserting zot gene into plasmid pET-28a(+) containing the T7 promoter. The expression plasmid was induced into E. coli BL21 (DE3) and expression strain BLZOT was selected. SDS-PAGE analysis revealed that the ZOT protein was expressed and accumulated up to above 15% of bacterial soluble protein after induced by IPTG. A protein of 47 kD was expressed as including body. Western blot analysis revealed that the expressed protein was ZOT.

Base Sequence↗

PubMed1999

Cloning of a galactose-binding lectin from the venom of Trimeresurus stejnegeri.

A galactose-binding lectin isolated from the venom of Trimeresurus stejnegeri is a homodimer C-type lectin. The cloned cDNA encoding the monomer of Trimeresurus stejnegeri lectin (TSL) was sequenced and found to contain a 5'-end non-coding region, a sequence which encodes 135 amino acids, including a typical 23 amino acid signal peptide followed by the mature protein sequence, a 3'-end non-coding region, a polyadenylation signal, and a poly(A) region. To completely characterize the deduced amino acid sequence, on-line HPLC-MS and tandem MS were used to analyse the intact monomer and its proteolytic peptides. A modified peptide fragment was also putatively identified by HPLC-MS analysis. The deduced amino acid sequence was found to contain a carbohydrate-recognition domain homologous with those of some known C-type animal lectins. Thus TSL belongs to group VII of the C-type animal lectins as classified by Drickamer [(1993) Prog. Nucleic Acid Res. Mol. Biol. 45, 207-232]. At present, a number of C-type lectins have been purified from snake venom, but most of them have been characterized only at the protein level. To our knowledge, this is the first known cDNA sequence of a true C-type lectin from snake venom.

Amino Acid Sequence↗

PubMed1999

The gene expression of coagulation factor VIII in mammalian cell lines.

Both the full-length and B domain-deleted cDNA of factor VIII were constructed in plasmid pcDNA3, respectively, and successfully expressed in Cos-7 cells. The yield of recombinant factor VIII-deltaB (0.4 U/mL/10(6) cells/day) was approximately four times higher than that of the recombinant factor VIII. In addition, it was indicated that the gene expression of factor VIII is specific for cells from different tissues. The highest expression level was found in the hepatocellular carcinoma line SMMC-7721, followed by kidney, ovary, and lung cell lines. To compare the efficiency of gene expression of recombinant factor VIII, the factor VIII-deltaB gene was further reconstructed in different forms in the expression plasmid pCMV-dhfr for transient gene expression in Chinese hamster ovary cells. The redundant 5'- and 3'-untranslated sequences of factor VIII-deltaB were deleted. The cDNA encoding the heavy and light chains of factor VIII were constructed, respectively. Among them the high yield of the recombinant factor VIII was found in the coexpression of the heavy and light chain cDNA fragments of factor VIII. The deletion of the redundant 5'-untranslated sequence of factor VIII-deltaB was also beneficial for gene expression. As expected, the gene coexpression of factor VIII-deltaB and von Willibrand Factor cloned by the long-polymerase chain reaction method was also helpful for enhancing the expression level of recombinant factor VIII. A monoclonal antibody raised against factor VIII was prepared and used for the specific assay of recombinant factor VIII by the competitive ELISA method, the assay results were consistent with those determined by the one-stage bioassay.

Animals↗

PubMed1999

Cloning of a cDNA encoding a nerve growth factor precursor from the Agkistrodon halys Pallas.

Based on the high conservation in the 5' and 3' untranslated regions of NGF cDNAs, oligonucleotides complementary to all these known sequences were synthesized. By RT-PCR, we successfully isolated the complementary DNA encoding NGF precursor from the Agkistrodon halys Pallas (a Chinese snake strain). The nucleotide sequence which presents 90.5%, 88.6% and 63.4% homology to that of Krait Bungarus multicinctus, cobra and human NGF respectively, encoded a prepro-NGF molecule with 241 amino acids and a mature NGF molecule with 119 amino acids. The NGF cDNA inserts were subcloned into pCDNA3 expression vector and then transfected into COS-7 cells. The supernatant of the transfected cells turned out NGF biological activity as assayed by the survival rate of PC12 pheochromocytoma cells.

Agkistrodon↗