Biomedical subjects

Sheng-Qi Wang

Publications and source records attributed to Sheng-Qi Wang.

45 records · Page 3Linked to original sources

PubMed2003

[Preliminary study on HLA-B genotyping by oligonucleotide chips].

HLA genes constitute a highly polymorphic multigene system. In the present study, HLA-B oligonucleotide chips were manufactured by using a set of sequence-specific oligonucleotide probes derived from polymorphic regions in exon 2 and exon 3 of HLA-B gene spotted by microarrayer onto the aldehyde modified glass slides. In addition, the sequenced HLA-B gene clones used as standard samples were amplified from exon 2 and exon 3 by PCR. Together with the correct hybridization and wash conditions, the PCR products were bound with the array probes on the chip, and the hybridization patterns were transformed to HLA-B genotypes. The results showed that the genotypes of standard samples by the HLA-B oligonucleotide chips were completely identical with the sequenced clones. In conclusion, the oligonucleotide chip method presented here for HLA-B genotyping is a rapid, accurate, sensitive and attractive high throughput biochemical way.

Genotype↗

PubMed2003

Fabrication and optimization of HLA-DRB1-12 oligonucleotide microarray.

Oligonucleotide microarray is developed on the basis of hybridization on the solid substrate. The pre-activated glass substrates and the terminal modification of the oligonucleotides are the two important factors in the process of fabrication for microarray. In order to compare the hybridization signal intensity of the different terminal modified oligonucleotide probes, the eight kinds of oligonucleotides were designed according to the sequence of HLA-DRB1-12, including the amino modified oligonucleotides with PEG spacer and the one without spacer, the phosphorothioate modified oligonucleotides with PEG spacer and the one without spacer. They were modified on 5' terminal and 3' terminal, respectively. In addition, the oligonucleotides probes with the internal spacer of different number of PEG were designed to observe the relationship between the spacer of PEG and the hybridization efficiency. These probes were respectively fixed on the bromoacetylation activated and glutaraldehyde activated slides to manufacture the two kinds of microarray which hybridized with the fluorescence labeled PCR product of HLA-DRB1-12 gene. The results from the study demonstrated that the signal intensity of 3' amino-modified probes with the internal spacer of different number of PEG on the bromoacetylation activated slides was stronger than the others. It is concluded that the 3' amino-modified oligonucleotide with an internal PEG spacer and the bromoacetylation activated slide enhanced the hybridization efficiency and were worthy to be proposed for the fabrication of HLA microarray or other kinds of microarrays for detecting fluorescence labeled PCR product in the future study.

HLA-DR Antigens↗

PubMed2003

[Over-expression in Escherichia coli and purification of nucleocaspid and membrane protein of SARS coronavirus].

Genes encoding nucleocaspid (N) and membrane (M) protein of SARS coronavirus were obtained by RT-PCR and were cloned into expression vector pET22b and pBV222. DNA sequencing showed that the genes cloned from a patient in Beijing were identical to the gene sequences from reported Toronto strain. The genes were over-expressed in E. coli either as inclusion body or as soluble form. The recombinant proteins were purified by ion-exchange, or ion-exchange followed by metal chelate affinity chromatography. The recombinant N protein was demonstrated highly antigenic and could be employed as antigen to detect SARS antibodies in ELISA system for SARS diagnosis.

Chromatography, Affinity↗

PubMed2002

[Detection of P53 and K-ras gene mutations in lung cancer with oligonucleotide chip].

Different factors including hybridization solution components, hybridization temperature, and the concentration and proportion of the labelled primer, which affected the sensitivity and specificity of single mutation identification, were exploited. Asymmetric PCR increased the hybridization sensitivity, and the asymmetric multi-PCR did not affect the specificity, while the sensitivity was improved a little. Among 30 lung cancer samples detected with the oligonucleotide microarray, 12 was found P53 gene mutations and 5 had K-ras gene mutations. The P53 gene mutations identified by the oligonucleotide microarray was proved 80% same as the sequencing results. The obvious statistical relations of K-ras and P53 gene mutations with tumor type, tumor stage and smoking were not obtained because of less samples and mutation sites.

Genes, ras↗

PubMed2002

[Preparation and analysis of oligonucleotide microarray for expression detection of mouse cytokine-associated gene].

A new method for the preparation of oligonucleotide microarray for gene expression detection was found. The key techniques and standards of quality controlling for preparation of oligonucleotide microarray was explored using gene of human 23 kD highly protein and Luciferase and mouse cytokine-associated genes. By the using of a software system MProbe, oligonucleotide probes were designed and BLAST. All the probes have a very high specificity, i.e. except target sequence, the similarity between the probe and non-target sequences is less than 70% and the hairpin structure are not exist in all probes. All the probes have the same length 40. GC contents in all probes are in a narrow scope (from 45% to 55%). All the probes are modified with amino at 5' or 3' terminal. The satisfied images with good sensitivity and very high specificity were obtained by the using of the methods above and also using of positive and negative controls and some internal controls(house keeping gene) to quantitate and balance expression of genes. High specificity, good sensitivity and stability have been verified by three continuous experiments using the oligonucleotide microarray to study gene expression profile of normal mouse breast grand tissue. The oligonucleotide microarray for expression detection prepared using our method have high specificity, good sensitivity and stability et al. It may be a more advanced method for analysis of gene expression profile.

Animals↗

PubMed1997

Sequencing Analysis of Oligodeoxynucleotide by Matrix-assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry.

A mixed matrix alpha-cyano 4-hydroxycinnamic acid (alpha-Cyano)/3-hydroxypicolinic acid (3HPA) was found to be a good matrix for the analysis of oligodeoxynucleotides by matrix-assisted laser desorption ionization time-of-flight mass spectrometry ( MALDI-TOF-MS). Using the mixed matrix, a similar sensitivity was obtained for the different oligodeoxynucleotides: d(T)(10), d(A)(10) and d(C)(10). This methodology can be used in combination with the partial digestion of oligodeoxynucleotides by 5'- and 3'-exonucleases as a powerful tool for sequencing analysis of oligonucleotides.

Journal Article↗

PubMedDate not supplied

Synthesis of antisense oligonucleotide-peptide conjugate targeting to GLUT-1 in HepG-2 and MCF-7 Cells.

A simple procedure for the preparation of oligonucleotide-peptide conjugate was developed. p-Hydroxy-benzoic acid was used as a linker for the connection of the fragments of peptide and oligonucleotide. It was found that such formed linkage was stable under the conditions of conjugate synthesis. The designed conjugate targeting to GLUT-1 showed up to 50% inhibition of cell proliferation in HepG-2 and MCF-7 cells. Comparing to the results from the expressed antisense RNA in cancer cells, it was proposed that the conjugate of signal peptide mimic and antisense oligonucleotide could improve the permeability of antisense oligonucleotide through cell membrane.

Blotting, Northern↗

PubMedDate not supplied

A concise method for the preparation of peptide and arginine-rich peptide-conjugated antisense oligonucleotide.

Peptide-oligonucleotide conjugates were synthesized using two strategies: a mimetic signal peptide-conjugated oligonucleotide was assembled stepwise on CPG by using 2,2-dimethyl-3-hydroxypropionic acid as a linker. To solve the precipitation problem in the coupling reaction caused by the electrostatic interaction of arginine-rich peptides and oligonucleotide, oligonucleotides were absorbed on an anion-exchange resin, and then the on-resin fragment was applied for the conjugation with arginine-rich peptide. The peptide-antisense oligonucleotides showed permeability to the cell membrane of HepG-2 cells.

Arginine↗

PubMedDate not supplied

Microarray analysis of altered gene expression in diallyl trisulfide-treated HepG2 cells.

Diallyl trisulfide (DT) is a natural compound derived from garlic. Despite its reported lipid-lowering effects, the mechanisms of its actions are not clear yet. To further understand the molecular mechanisms of actions of this compound, microarray technology was used in the present study to investigate the lipid-lowering effects of DT on HepG2 cells. To optimize the concentration of DT treatment on HepG2 cells, a series of concentration of DT were incubated with HepG2 cells for 24 h. The data indicted that the concentrations of DT in the range from 20 to 50 microM were effective in lowering cellular total triglyceride and cholesterol, with no significant cytotoxicity. Using oligonucleotide microarrays and RT-PCR technology, the genes that were differentially regulated by DT were identified. The results showed that peroxisome proliferator activated receptor alpha (PPAR-alpha) and hepatocyte nuclear factor 4alpha (HNF-4alpha) mRNA were up-regulated, and CYP7A1 mRNA was down-regulated following DT treatment, suggesting that the lipid-lowering effects of DT may be at least in part mediated through the regulation of PPAR-alpha dependent pathways.

Allyl Compounds↗