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Biomedical subjects

Yi Gong

Publications and source records attributed to Yi Gong.

At least 37 records · Page 2Linked to original sources
PubMed2005

Application of phage-displayed single chain antibodies in western blot.

A phage display single chain fragment variable library constructed on pIII protein of M13 filamentous phage was screened using B-lymphocyte stimulator and FP248 as selective molecules. After four rounds of panning, there was a remarkable enrichment in the titer of bound phages. Twenty phage clones were selected from the last round and screened by means of phage-ELISA. With the antibody phages as primary antibodies in Western blot, we developed a method for detecting the specific antigen. The dilutions of antibody phages depend on the affinity between antibody-displayed phage particles and antigens.

Animals↗
PubMed2005

[Construction, Expression, Purification and Antiviral Activity of Fused Interferon-BLA (IFN-BLA).].

A fusion protein, Interferon-BLA (IFN-BLA), was constructed with IFN-beta-1b and IFN-alpha-2b separating by a linker -GGGS-. The laboratory-scale expression conditions in E.coli BL21 CodonPlus (DE3)-RIL had been optimized and IFN-BLA was expressed higher than 35% of total protein in the cells mainly as inclusion body. The inclusion body of IFN-BLA was denatured and refolded by dialysis and purified by ion-exchange chromatography. The overall yield of IFN-BLA was about 45 mg/L with purity higher than 90%. Antiviral activity assay suggested that this newly fused protein may have synergetic or additive antiviral activities.

Antiviral Agents↗
PubMed2004

Large-scale cDNA transfection screening for genes related to cancer development and progression.

A large-scale assay was performed by transfecting 29,910 individual cDNA clones derived from human placenta, fetus, and normal liver tissues into human hepatoma cells and 22,926 cDNA clones into mouse NIH 3T3 cells. Based on the results of colony formation in hepatoma cells and foci formation in NIH 3T3 cells, 3,806 cDNA species (8,237 clones) were found to possess the ability of either stimulating or inhibiting cell growth. Among them, 2,836 (6,958 clones) were known genes, 372 (384 clones) were previously unrecognized genes, and 598 (895 clones) were unigenes of uncharacterized structure and function. A comprehensive analysis of the genes and the potential mechanisms for their involvement in the regulation of cell growth is provided. The genes were classified into four categories: I, genes related to the basic cellular mechanism for growth and survival; II, genes related to the cellular microenvironment; III, genes related to host-cell systemic regulation; and IV, genes of miscellaneous function. The extensive growth-regulatory activity of genes with such highly diversified functions suggests that cancer may be related to multiple levels of cellular and systemic controls. The present assay provides a direct genomewide functional screening method. It offers a better understanding of the basic machinery of oncogenesis, including previously undescribed systemic regulatory mechanisms, and also provides a tool for gene discovery with potential clinical applications.

Animals↗
PubMed2004

The toxic effects of nonylphenol on the reproductive system of male rats.

Male Sprague-Dawley (SD) rats were exposed to 4-t-nonylphenol (NP) by gavage at dosages of 0, 125 and 250 mg/kg/day for 50 days. Organ weights of liver, kidney, testis and epididymis were measured. Sperm number in the head of epididymis was counted. Several hormones including testosterone, luteinizing hormone and follicle stimulating hormone were measured. Testicular sections were observed by light and electron microscopy. Terminal dideoxynucleotidyl transferase dUTP nick end labeling assay was performed to probe the apoptotic cells in seminiferous tubules. When rats were treated with nonylphenol at 250 mg/kg/day, the absolute and relative weight of epididymis decreased dramatically, while the relative weights of kidney and liver increased by 14 and 22%, respectively. In addition, the sperm density of the head of epididymis and the testosterone level descended at 250 mg/kg/day. The levels of luteinizing hormone and follicle stimulating hormone increased in both nonylphenol treated groups. Pathological changes were detected by microscopy and the transferase dUTP nick end labeling (TUNEL) assay showed that the number of apoptotic cells in testes increased with nonylphenol in a dose-dependent manner.

Administration, Oral↗
PubMed2004

Fusion protein of interleukin 4 and diphtherial toxin with high cytotoxicity to cancer cells.

Receptor of human interleukin 4 (hIL4R) has been found to be present on many types of cancer, so it may be a good target for cancer therapy. Here, fusion toxin gene DT4H has been constructed by fusing DNA sequence encoding the first 389 amino acids of diphtherial toxin (DT), which can not bind its own receptor, to human interleukin 4 (hIL4) gene. In order to improve the affinity of fusion toxin for hIL4R, a circularly permuted form of hIL4 (cpIL4) was used. The fusion gene was expressed in Escherichia coli where the fusion toxin DT4H was highly expressed. Purified DT4H was very cytotoxic to cancer cell line U251 cells, and moderate cytotoxic to HepG2 and MCF-7 cells. SGC-7901 cells were insensitive to it. The cytotoxic action of DT4H was specific because it was blocked by excess hIL4. These results suggest that DT4H may be a useful agent in the treatment of certain malignancies.

Antineoplastic Agents↗
PubMed2004

Recombinant scFv antibodies against E protein and N protein of severe acute respiratory syndrome virus.

Three single chain antibodies (scFv) against the proteins of severe acute respiratory syndrome coronavirus (SARS-CoV) were isolated by phage display from an scFv antibody library. Bio-panning was carried out against immobilized purified envelope (E) and nucleocapsid (N) proteins of SARS-CoV. Their binding activity and specificity to E or N protein of SARS-CoV were characterized by phage-ELISA. Two of them, B10 and C20, could recognize non-overlapping epitopes of the E protein according to the two-site binding test result. Clone A17 could recognize N protein. The sequence of the epitope or overlapping epitope of scFv antibody A17 was PTDSTDNNQNGGRNGARPKQRRPQ. The affinity (equilibrium dissociation constant, K(d)) of SARS-CoV E protein was 5.7 x 10(-8) M for B10 and 8.9 x 10(-8) M for C20. The affinity of A17 for N protein was 2.1 x 10(-6) M. All three scFv antibodies were purified with affinity chromatography and determined by Western blot.

Amino Acid Sequence↗
PubMed2003

Construction of an EGF receptor-mediated histone H1(0)-based gene delivery system.

PURPOSE: To construct an EGF receptor (EGF-R)-mediated histone H1(0)-based gene delivery system for gene therapy. METHODS: A recombinant DNA containing histone H1(0), EGF-R ligand, and endosomalytic domains was constructed in a prokaryotic vector and expressed in E. coli. Expression of the beta-galactosidase (beta-gal) gene in the tumor cells and tissues was observed after transduction of the beta-gal gene packaged by purified fusion proteins in vitro and in vivo. RESULTS: As an extension of the research on previously reported chemically synthetic composite polypeptide gene delivery systems, this genetically engineered polypeptide has proved to be capable of targeting the beta-galactosidase (beta-gal) gene into EGF-R-positive cancer cells both in vitro and in vivo. We also studied the time course of beta-gal gene expression in tumor tissues delivered in vivo by this polypeptide vector. At 24 h after administration, expression of the beta-galactosidase gene in tumor reached peak levels. The dosage optimization of administered polyplex was also investigated. The optimal dose of polyplex per mouse was 1 microg DNA packaged by 3 microg of composite polypeptide. CONCLUSIONS: The genetically engineered polypeptide based on histone H1(0) is a promising gene delivery system targeting EGF-R.

Animals↗
PubMed2003

Molecular cloning, characterisation and tissue-specific expression of human LAG3, a member of the novel Lag1 protein family.

We identified and characterised the cDNA coding for human Lag3, a member of the novel eukaryotic Lag1 protein family which was related to the Saccharomyces cerevisiae longevity-assurance gene LAG1. The composite nucleotide sequence revealed a coding region of 1140 bp, a 5'-UTR of 210 bp and a 3'-UTR of 900 bp. The deduced sequence of 380 amino acids had six transmembrane regions and the conserved Lag1 motif. Northern blot analysis revealed that the approximately 2.2 kb LAG3Hs transcript was expressed in all 12 human tissues, high in kidney and liver, moderate in brain, heart, skeletal muscle, placenta and lung and low in colon, thymus, spleen, small intestine and peripheral blood leukocyte. Because of its structural similarity to LAG1, it was postulated that LAG3Hs may mediate sphingolipids metabolism.

5' Untranslated Regions↗
PubMed2003

[Severe acute respiratory syndrome epidemiology in Sichuan province].

OBJECTIVE: To explore the epidemiological characters of severe acute respiratory syndrome (SARS) in Sichuan province in order to provide evidence for prevention and control. METHODS: To generate data on SARS in Sichuan province through descriptive and analytical studies on time, geographic distribution, population, source of infection, the way of case finding, symptom, diagnosis and treatment of the cases. RESULTS: The peak of the epidemic last from April 16 to May 7. The number of cases in Luzhou and Guangyuan cities took up 60% of the total. Mobile population occupied 68% of the cases. Most of the patients were above the age of 20 with a sex ratio of 1.5:1 (m/f). 80% of the cases had a history of working in Guangdong province and recently returned to their hometowns. The main symptoms and signs of the SARS patients would include fever, cough and chest X-ray changes. CONCLUSION: All cases were imported. Fluctuation of the epidemics was mainly affected by the mobility of working population who recently returned to their hometowns. Measures concerning the prevention and control of the epidemics would mainly target on the isolation of confirmed and suspected patients who might serve as the sources of infection through setting up quarantine station, assigned hospitals and special 'fever-clinics'.

Adult↗
PubMed2003

Interaction of C17orf25 with ADP-ribose pyrophosphatase NUDT9 detected via yeast two-hybrid method.

The gene C17orf25 was isolated from the liver by RACE PCR. nudt9 gene was screened by yeast two-hybrid method in MatchMaker human HeLa cDNA library. NUDT9 is an enzyme that has pyrophosphatase activity with ADP-ribose as its substrate. Fusion expression of C17orf25 and GFP and computer analysis showed that C17orf25 was probably located in mitochondria. Furthermore, C17orf25 may suppress the cell growth by interaction with NUDT9.

Amino Acid Sequence↗
PubMed2003

Expression of C-peptide multiple gene copies in Escherichia coli and stabilities of C-peptide in aqueous solution.

A gene fragment encoding three copies of proinsulin C-peptide was synthesized and expressed in E. coli and the recombinant proinsulin C-peptide was produced through site-specific cleavage of the resulting gene products. The fusion protein was expressed at high level, about 80 mg/L, as a soluble product in the cytoplasm. Ni-NTA affinity chromatography efficiently separated the expressed fusion protein from the supernatant, to obtain about 37.5 mg/L of the fusion protein with 70% purity. Enzymatic digestion by trypsin and carboxypeptidase B of the fusion protein efficiently released native C-peptide, the overall yield of recombinant C-peptide at a purity over 95% was 1.5 mg/L. The good agreement of amino acids composition, together with shown similarities of the recombinant C-peptide to C-peptide standard in the comparative RP-HPLC analysis and IMMULITE C-Peptide quantitative assay, suggested that the recombinant C-peptide obtained in this report was the native human C-peptide. The investigation of the chemical stability of recombinant human C-peptide in aqueous solutions by RP-HPLC was also reported. The degradation of the recombinant C-peptide showed a marked dependence on pH and temperature. The degradation reaction of C-peptide occurred immediately in pH 3 or pH 9 buffered solution. The degradation reaction of C-peptide followed first-order kinetics in pH 3 buffered solution at 37 degrees C or 70 degrees C, only 40.3% of C-peptide was remained after 10 h at 70 degrees C. The maximum stability was achieved at pH 7.4, more than 90% of C-peptide were detected at pH 7.4 and 37 degrees C after 10 h and at pH 7.4 and 70 degrees C after 5 h. 99% and 96% of C-peptide was remained at pH 7.4 and 37 degrees C after 10 h with and without 10 g/L BSA respectively.

Amino Acid Sequence↗
PubMed2003

Cloning and expression of adiponectin and its globular domain, and measurement of the biological activity in vivo.

3T3-L1-adipocytes produce the adipocyte complement related protein of 30 kD (ACRP30), which is exclusively expressed in differentiated adipocytes. Decreased expression of ACRP30 correlates with insulin resistance in mouse models of altered insulin sensitivity. Adiponectin, the human homologue of ACRP30, circulates in human plasma at high levels. Plasma adiponectin levels have been reported to be decreased in some insulin-resistant states, such as obesity and type II diabetes mellitus. Here, full-length adiponectin and its C-terminal globular head domain (gAdiponectin) were expressed in Escherichia coli and gAdiponectin was used to immunize a rabbit to obtain polyclonal antiserum with titer of 10,000. Adiponectin was detected in human plasma with the use of gAdiponectin anti-serum by Western blot analysis, which was also detected by gACRP30 anti-serum. Injection in alloxan-treated rats with purified recombinant fusion adiponectin or gAdiponectin transiently abolished hyperglycemia. So adiponectin and gAdiponectin might have activity as a glucose lowering agent and potentially as a therapeutic for metabolic disease. All these results suggested that the recombinant protein had biological activity, and provided a useful tool in further studies.

3T3-L1 Cells↗
PubMed2003

[Purification and identification of human recombinant IFN-beta expressed in yeast Pichia pastoris].

To find an effective and quick way of purifying and identifying recombinant human IFN-beta (rhIFN-beta) expressed in yeast Pichia pastoris, Blue Sepharose 6 fast flow (Blue S6FF) and immunological affinity chromatography (IAC) were compared in this report. rhIFN-beta was produced in 15 liter bioreactor and purified using the two methods mentioned above. The protein concentrations of rhIFN-beta and residual mouse IgG in purified rhIFN-beta were determined with ELISA. The molecular weight and specificity were demonstrated by PAGE and Western blot. The density of the specific precipitation bands was determined by gel scanning. The relative bioactivities were determined by cyto pathogenic effect inhibition (CPEI). The results showed that 2.65 and 3.03 mg of rhIFN-beta were obtained, respectively, by purifying with Blue S6FF or IAC from 2 liter of fermentation supernatant. The molecular weight was 22 kD. The concentrations of the special precipitation of rhIFN-beta were 95.1% and 96.2% respectively. The relative bioactivity of rhIFN-beta purified by Blue S6FF and IAC were 1.63x10(7) IU/mg and 1.43x10(7) IU/mg, respectively. The residual mouse IgG in purified rhIFN-beta by IAC was less than 50 microg/L. The results indicated that rhIFN-beta could be purified effectively and quickly from fermentation supernatant of yeast Pichia pastoris by IAC. The rhIFN-beta products purified by Blue S6FF and IAC had almost the same purity and bioactivity. The data accumulated from the experiment are useful to the preparation of rhIFN-beta on a larger scale.

Animals↗
PubMed2003

[The immunopotentiation of human B lymphocyte stimulator C-terminal peptide].

The cDNA of human B lymphocyte stimulator C-terminal peptide (C-BLyS) was amplified by nested PCR from cDNA library of human fetal brain. The expression plasmid pT7450-C-BLyS was constructed and transformed into E. coli BL21 CodonPlus (DE3) RIL which can recognize many rare codons. The C-BLyS protein was expressed as inclusion body in E. coli BL21 CodonPlus (DE3) RIL and the inclusion body of C-BLyS was denatured and then refolded by dialysis and purified by ion exchange chromatography. The refolded and purified C-BLyS can specifically bind with BCMA-Fc, a fusion protein of BLyS receptor and human IgG1 Fc. Furthermore, C-BLyS is proved to be an effective stimulator in mouse splenocytes proliferation in vitro and effective immunostimulant in vivo.

Animals↗
PubMed2003

[Use of PCR related methods in detection of gene mutation].

Many inherited diseases and drug resistance have been attributed to mutations in corresponding genes. In this paper, several techniques based on PCR used in diagnosis were concluded. The development and research progress of Mismatch PCR were discussed in details. Some information about an assay that we developed for detection of antimicrobial resistance to quinolones was also described.

English Abstract↗
PubMed2003

[HRCA and application in detection of genetically modified plant].

In this article primary studies of the application of hyperbranched rolling cycle amplification (HRCA) in exogenous genes detection of transgenic plants were done. Four padlock probes were designed according to the sequences of four genes/DNA fragments that are used widely in transgenic plants; part of the sequence of pKK233 was chosen as the linking part of padlock probes and a pair of HRCA primers was designed according to the sequence of linking part. Study of the specificity of ligation in HRCA with isotope labeled padlock probes indicated padlock probes could be ringed effectively only when corresponding target DNA exited in the same reaction system and could not be ringed when there was no corresponding target DNA exited. Ligation time is very different according to the characteristic of target DNA being used. 5 min to 10 min is enough if the target DNA is plasmid; 30 min to 60 min is needed if the target is genome DNA of plant because it's sequence is more complex than that of plasmid's. HRCA time was analyzed which indicated longer reaction time can obviously increase the amount of products. Quantity of enzyme in HRCA was also analyzed. Different amount of enzyme (from 0.5 unit to 4 units) can give similar result when other conditions are not changed. On the basis of the research, transgenic tobacco was detected with these four padlock probes and the results were just as prospective. In order to increase the efficiency of detection, multiplex HRCA (MHRCA)was used. In MHRCA more than one padlock probes are used at the same time in the same reaction system to detect more than one targets. Because the amplification products of MHRCA will be complex and it is almost impossible to analyze with electrophoresis, so reverse-blot is used. Detection results of transgenic tobacco with this method are the same with anticipation. Compare to MPCR method we established before MHRCA is more convenient to operate and more effective in detecting exogenous genes in transgenic plants.

Nucleic Acid Amplification Techniques↗
PubMed2003

[Study of the expression membrane protein LASS2].

A human membrane protein LASS2 (Homo sapiens longevity assurance homologue 2 of yeast LAG1), which has important physiologic functions, was expressed in three different expression systems. Only the LASS2 protein carboxyl terminal hydrophilic fragment could be expressed in the prokaryote expression system and its polyclonal antibody was produced. The full length of LASS2 protein could be expressed successfully in both eukaryotic in vitro translation system and Bacuvirus expression system: Bac-to-Bac system. SDS-PAGE analysis revealed that the molecular weight of expressed product of LASS2 was about 28 kD. The product was also proved by Western blot. This recombinant LASS2 protein was purified by metal affinity resin and the purity is above 90%.

Animals↗