Biomedical subjects

Xiang-Fu Wu

Publications and source records attributed to Xiang-Fu Wu.

At least 55 records · Page 3Linked to original sources

PubMed2000

Fusion and Expression of the Genes Encoding Murine-Human Chimeric Heavy Chain to Carcinoembryonic Antigen with Core-streptavidin Gene.

In order to reduce the human anti-murine antibody (HAMA) in radioimmunotherapy, the gene encoding the heavy chain variable region (V(H)) of the murine monoclonal antibody with high specificity and affinity to carcinoembryonic antigen was fused to the human C(gamma3) gene to construct the murine-human chimeric heavy chain antibody gene, then was linked to core-streptavidin which can specifically bind to biotin, facilitating its purification and radioisotope labeling. The fusion gene was expressed in E.coli at high level, accounting for 24% of the total bacteria protein, and was characterized by SDS-PAGE and Western-blots. When using RIA the content of inclusion bodies was denatured and followed by renaturation, it was shown by using RIA to possess ability to bind to its specific antigen of CEA. Using horseradish peroxidase (HRP) labeled biotin as antibody in Western-blots, one band of 70 kD only was detected, demonstrating the fusion protein not only had the ability to bind CEA, but also could bind biotin specifically.

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PubMed2000

High Expression and Purification of Recombinant Human Serum Albumin from Pichia pastoris.

Recombinant human serum albumin (rHSA) was produced in methylotrophic yeast Pichia pastoris. By optimization of expression, about 150 mg/L of rHSA was obtained from broth of Pichia pastoris GS115/HAS (his+Mut(S)) supernatant. The rHSA was isolated and purified by hollow-fiber ultrafiltration, Phenyl-Sepharose hydrophobic chromatography and antibody-immunoadsorbent chromatography. Finally, rHSA was purified to electrophoretic purity.

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PubMed2000

Cloning and Functional Expression of Neurotoxin cDNA from Naja naja atra.

Three new cDNAs named NL1, NL2 and NL3 were cloned from the total RNA of Naja naja atra by RT-PCR. The protein sequences encoded by them showed 77%, 72% and 98% structure identity to cobrotoxin which is a postsynaptic neurotoxin from Taiwan cobra (Naja naja atra), respectively. The five conservative residues, Tyr(25), Lys(27), Trp(29), Arg(33) and Lys(47), essential for the function of cobrotoxin were also found in the three cDNAs. The NL3, the most homologous to cobrotoxin, was expressed in E.coli BL21(DE3) by cloning into pET28b+. The expressed product was insoluble inclusion bodies and could be purified up to 90% purity in the range of 6 mg per liter culture cells by a single affinity step. The purified protein was refolded in vitro and the toxicity was assessed to be less than the native cobrotoxin in mice.

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PubMed1999

Expression of a Gene for Single-chain Antibody to Carcinoembryonic Antigen in Bombyx mori cells and Its Larvae.

Recombinant Bm-BacScFv virus which contains a cDNA encoding anti-CEA single-chain antibody was generated by cotransfection into Bm N cells with the transfer plasmid pBacPAK-(His)(6)-ScFv and the modified Bombyx mori nuclear polyhedrosis virus Bm-BacPAK genomic DNA. Bombyx mori cells and larvae were infected by the recombinant virus, respectively. The anti-CEA single-chain antibody were expressed both in Bombyx mori cells and larvae, the former accounted for 6% of the total cell protein, the later 0.3 mg per larvae. The histidine-tagged ScFvs were respectively purified with nickel-iminodiacetic acid affinity chromatography. The purity of the former reached over 90% and that of the latter was lower. Purified protein products was able to bind to CEA with an affinity constant of 5.4x10(8)/mol.L(-1) and 2.3x10(8)/mol.L(-1), slightly lower than its parental monoclonal antibody E(7)B(10) which had 2.7x10(9)/mol.L(-1).

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PubMed1999

Cloning and Expression of a Novel Mutated Osteoprogerin/Osteoclastogenesis Inhibitory Factor Gene.

Total RNA was isolated from normal Chinese human liver cell line L02. A mutated osteoprotegerin/osteoclastogenesis Inhibitory Factor(OPG/OCIF) cDNA was amplified by RT-PCR using the total RNA as template and was inserted into pBS-sk plasmid. Sequence analysis showed that the OPG/OCIF cDNA from L02 cells was a mutated OPG/OCIF gene which had a nonsense mutation(Ochre) at the codon of Gln(394). The OPG/OCIF isoform was 8 amino acid residues less at the C terminal than the OPG/OCIF reported. The 3'fragment of OPG/OCIF gene from genomic DNA of cell line 293(ATCC CRL 1573) was also cloned. Sequence analysis indicated that the sequence of genomic DNA was the same as that of cDNA. The mutated OPG/OCIF gene was inserted into yeast expression plasmid pPIC3.5K, and the recombinant plasmid was used to transform Pichia pastoris GS115. SDS-PAGE analysis revealed that the human OPG/OCIF isoform was highly expressed and accumulated up to over 30% of soluble protein of yeast after the induction by methanol for 3 to 5 days. The longer the transformant was induced, the higher the ratio of glycosylated protein was.

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PubMed1999

Cloning and Expression of Human Manganese-superoxide Dismutase cDNA.

The cDNA encoding human manganese-superoxide dismutase was amplified from the human liver cell (L02) total RNA by RT-PCR and sequenced. The human Mn-SOD cDNA was ligated into expression vector pET-24a(+) under T7 promoter. After 5 h induction with 1 mmol/L IPTG 800 mol/L Mn(2+) human Mn-SOD was highly expressed in E.coli BL21(DE3). The protein product was up to 50% of the bacteria total protein in soluble form and had specific SOD activity.

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PubMed1999

Expression and Biochemical Characterization of Acidic Phospholipase A(2)I from Agkistrodon acutus.

A cDNA encoding acidic phospholipase A(2)I(A.aAPLA(2)I)from Agkistrodon acutus was inserted into a bacterial expression vector and effectively expressed in E.coli RR1. The protein was produced as insoluble inclusion bodies. After partial purification by washing the inclusion bodies with Triton X-100, denaturing and refolding, the renatured recombinant protein was purified by FPLC column Superose(TM)12. The enzymatic acti-vity and platelet aggregation inhibiting effect of the expressed A.aAPLA(2)I is close to those of denatured-refolded native acidic PLA(2) from Agkistrodon halys Pallas, and has the same hemolytic activity as denatured-refolded basic phospholipase A(2) from Agkistrodon halys Pallas. The roles of various amino acid residues in the enzymatic activity and pharmacological activities of phospholipase A2 are discussed.

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PubMed1999

Synthesis and Biological Activity of Human Calcitonin Analogue.

The potency of salmon calcitonin (sCT) is higher but the structural homology between sCT and human calcition (hCT) is only 50%. Based on the comparison of the structure between sCT and hCT, we have designed and synthesized a hCT analogue (mhCT-2) by solid phase method, using air oxidation in diluted solution to obtain a peptide with intramolecular disulfide bond. Through HPLC purification, we obtained a capillary electrophoresis-homogeneous mhCT, results of analysis of its mass spectrum and N-terminal sequencing were in accordance with the theoretical values. The results of calcitonin bioassay by estimating the blood calcium levels in rats showed that the potency of mhCT-2 was around 2 000 IU/mg, one order of magnitude higher than that of hCT. In RIA assay, we have found that the immunoactivity of mhCT-2 and hCT was much different because of their different binding abilities to anti-hCT antibody. This indicated that the conformation of mhCT-2 was changed as compared with hCT. In rat osteoporosis model, the results showed that pharmacologic effects of mhCT-2 was the same as that of sCT. The synthetic mhCT-2 seems promising to be a clinically useful peptide with high potential in osteoporosis therapy, because it is similar in biological properties to, but less immunogenic than sCT.

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PubMed1999

Sequence Analysis of SOD Gene of Bombyx mori Nuclear Polyhedrosis Virus and Its Expression in E.coli.

Superoxide dismutases scavenge superoxide radicals and protect cells from oxidative stress. SOD gene of Bombyx mori nuclear polyhedrosis virus has been cloned by PCR, and is expressed in E.coli with the activity of SOD being 576.13 u/mL. DNA sequence analysis shows that SOD gene of BmNPV encodes 151 amino acids. The homology between BmNPV and AcNPV of the nucleotide sequences of SOD gene is 97.2%, and 56% between BmNPV and human SOD1.

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PubMed1999

Analysis of N-Terminal Nucleotide Sequence of NDV Fusion Protein.

Fusion protein of Newcastle disease virus is a glycoprotein with antigenicity. The cDNA of fusion gene of NDV strain F48E8 was cloned by RT-PCR, and the sequence of F48E8 was compared with other two virulent strains, Australia-victoria and Italien. Results show that the sequences of N-terminus are very similar, and cysteine residues and the potential glycosylation sites are relatively conserved. However, there are a few differences in the region of signal peptides.

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PubMed1999

Cloning of Human Glial Neurotrophic Factor and Its High Expression in Insect Cells.

The human glial cell derived neurotrophic factor (GDNF) was expressed in Tn-5B1-4 insect cells using the Bac to Bac baculovirus expression system, accounting for 30% of total cellular soluble proteins. The expressed product was purified by affinity chromatography and the activity assays showed that it could significantly promote the survival of dopaminergic neurons. The study can be the basis for further structural-functional analysis of GDNF.

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PubMed1999

Helicobacter pylori HspA Heat-shock Protein Gene Cloning, Expression and Immunogenicity.

Helicobacter pylori is the causative agent of gastritis and peptic ulcer in human. The heat-shock protein A (HspA)may stimulate the immunoresponse protecting human body against challenge of H.pylori. The gene encoding the structural A subunit of H.pylori heat-shock protein was amplified from H.pylori chromosomal DNA by PCR, and was inserted in the prokaryotic expression vector pET-22b(+), and then was transformed into the BL-21(DE3)E.coli strain to express the HspA recombinant protein. HspA gene was measured to be 354 base pairs, and the recombinant protein gene encoded polypeptides of 118 amino acid residues, corres-ponding to calculated molecular weight of 15 kD. Western blot analysis of HspA recombinant protein was confirmed that it could be specifically recognized by the serum of H.pylori-infected patients, and could also be re-cognized by the serum of immunized Balb/c mice with HspA itself. This result suggests that HspA may be an effective protein vaccine for prevention and treatment of the infection of H.pylori.

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PubMed1999

Cloning and Expression of Annexin V cDNA in E.coli.

The cDNA encoding the mature annexin V was isolated by using RT-PCR method from total RNAs of fresh human placenta. The result of sequencing indicated that the sequence of isolated annexin V cDNA was the same as the reported nucleotide sequence of annexin V. The annexin V cDNA was cloned into expression plasmid pET24a(+) under T7 promoter and then transformed into E.coli BL21(DE3). SDS-PAGE analysis revealed that the human annexin V was highly expressed and accumulated up to 38% of total bacterial proteins in soluble form after the induction by 1 mmol/L IPTG. The purified human annexin V can significantly prolong activated partial thromboplastin time (APTT).

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PubMed1999

Expression of Nerve Growth Factor from Agkistrodon halys Pallas in Silkworm Larvae.

The cDNA encoding nerve growth factor (NGF) precursor from Agkistrodon halys Pallas (a Chinese snake species) was cloned into pBacPAK8 a baculovirus shuttle vector. The recombinant shuttle vector pBacPAK-NGF 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. The SDS-PAGE and the NGF activity by bioassay of PC12 cells have shown a high expression level of NGF of good biological activity in the larvae.

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PubMed1999

The Human Recombinant GDNF and Study of Its Biological Activity.

The cDNA encoding the mature human glial cell derived neurotrophic factor(GDNF) was isolated by using RT-PCR method from total RNA extracted of fetal human brain. The expression plasmid pET-GDNF was constructed by inserting GDNF cDNA into plasmid pET-28a(+) containing T7 promoter and transformed into E.coli BL21(DE3). An expression strain BLGDNF was selected. SDS-PAGE analysis revealed that the human GDNF protein was highly expressed and accumulated up to above 30% of the total bacterial proteins in the form of inclusion body after the induction. The antibody to GDNF was prepared by immunization of rat using purified GDNF protein. Purified and refolded GDNF protein could significantly promote the survival of dopaminergic neurons.

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PubMed1999

Cloning, Sequencing and Expression of Human Copper, Zinc-superoxide Dismutase cDNA.

The cDNA encoding the human copper, zinc-superoxide dismutase(Cu, Zn-SOD) was amplified from the human liver by RT-PCR and sequenced. The cloned human Cu, Zn-SOD cDNA was ligated into expression vector pET-22b(+) under T7 promotor. After 3 h induction with 1 mmol/L IPTG, human Cu, Zn-SOD was highly expressed in E.coli BL21(DE3). The expression product was up to 30% of the total protein of the bacteria in soluble form, which had specific SOD activity.

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PubMed1999

Cloning and Sequencing of Genes Encoding Phospholipase A(2) from Agkistrodon acutus.

Synthetic oligonucleotides were used to amplify phospholipase A(2) (PLA(2)) gene by RT-PCR from total RNA of snake Agkistrodon acutus venom gland. The PCR products were subcloned and positive clones were screened with acidic PLA(2) gene from Agkistrodon halys Pallas. Finally, four cDNAs of PLA(2) isoenzymes were isolated. Their complete sequences were determined by bidirectional sequencing and their amino acid sequences were deduced. They were designated as A.aAPLA(2)I A.aAPLA(2)II A.aBPLA(2) and A.aLys(49)-PLA(2) according to their isoelectric points calculated by computer and special structure characteristics respectively. The amino acid sequence of 1 10 residues of A.aAPLA(2)I deduced from the cDNA is identical to that of acidic PLA(2) which had been isolated from Agkistrodon acutus. A.aLys(49)-PLA(2) is unique because of the usual Asp(49) is replaced by Lys(49), which may lower its enzymatic activity. Their similarity scores were calculated and compared by computer. The successful cloning of these isoenzymes genes may provide more information for the study on structure-function relationship of PLA(2) family.

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PubMed1999

Modeling and Analysis of Structures of Phospholipase A(2)'s from Venom of Agkistrodon haly Pallas.

We have modeled three-dimensional structures of basic-acidic hybrid phospholipase A(2)-II and neutral phospholipase A(2) from venom of snake Agkistrodon halys Pallas, based on the known structures of basic and acidic phospholipase A(2)'s from the same source. We have compared these structures of phospholipase A(2)'s, explained the results of fluorescent spectrum study on the phospholipase A(2)'s and calculated the electrostatic potential maps on the catalytic active site. We suggest that the electrostatic potential around the catalytic active site of PLA(2) containing a calcium ion favors the binding of the PLA(2) to its substrate with negative charge.

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