Biomedical subjects

Shisong Fang

Publications and source records attributed to Shisong Fang.

12 recordsLinked to original sources

PubMed2005

Topology characterization of a benzodiazepine-binding beta-rich domain of the GABAA receptor alpha1 subunit.

Structural investigation of GABAA receptors has been limited by difficulties imposed by its trans-membrane-complex nature. In the present study, the topology of a membrane-proximal beta-rich (MPB) domain in the C139-L269 segment of the receptor alpha1 subunit was probed by mapping the benzodiazepine (BZ)-binding and epitopic sites, as well as fluorescence resonance energy transfer (FRET) analysis. Ala-scanning and semiconservative substitutions within this segment revealed the contribution of the phenyl rings of Y160 and Y210, the hydroxy group of S186 and the positive charge on R187 to BZ-binding. FRET with the bound BZ ligand indicated the proximity of Y160, S186, R187, and S206 to the BZ-binding site. On the other hand, epitope-mapping using the monoclonal antibodies (mAbs) against the MPB domain established a clustering of T172, R173, E174, Q196, and T197. Based on the lack of FRET between Trp substitutionally placed at R173 or V198 and bound BZ, this epitope-mapped cluster is located on a separate end of the folded protein from the BZ-binding site. Mutations of the five conserved Cys and Trp residues in the MPB domain gave rise to synergistic and rescuing effects on protein secondary structures and unfolding stability that point to a CCWCW-pentad, reminiscent to the CWC-triad "pin" of immunoglobulin (Ig)-like domains, important for the structural maintenance. These findings, together with secondary structure and fold predictions suggest an anti-parallel beta-strand topology with resemblance to Ig-like fold, having the BZ-binding and the epitopic residues being clustered at two different ends of the fold.

Amino Acids↗

PubMed2005

[Modified molecular beacon-based dual real-time PCR for detection of SARS virus and its application].

OBJECTIVE: To develop the modified molecular beacon-based dual fluorescent PCR assays for detection of SARS virus. The assay was applied to the early clinic diagnosis & animal tracking. METHODS: On the basis of the obtained core sequence of open reading frame 1b of the coronavirus polymerase gene sequences, which was published in GenBank, using modified molecular beacon probe, artifical virus techinique and two different fragments amplification with different fluoresce, one set of primers and probe were designed. Then fluorescent PCR assays for specific and sensitive detection of the SARS virus was established, while the ELISA & the traditional method were used as control. 368 clinical specimens such as the throat swab, serum, feces, and urine from different cases, 52 cell cultures and 50 animal specimens were detected by the molecular beacon-based PCR. RESULTS: The sensitivity of real -time PCR was 10 - 100 copies/ml, there was no cross reaction to other respiratory viruses such as influenza virus etc. Of 368 specimens, 20 were positive by using molecular beacon-based fluorescence PCR. The positive rate of SARS case (10/47) were 21.27%, the positive rate of the throat swab of SARS cases (10/23) were 43.87% . Among 52 SARS cell cultures, 29 were positive. The positive rate of SARS cell cultures was 55.77% . Of 50 animal specimens, 23 were positive. The positive rate was 46%. Furthermore, SARS virus RNA was detected in feces and in serum during the acute phase. CONCLUSION: The molecular beacon-based PCR is sensitive and specific, it could be applied to the early diagnosis and animal tracking. This molecular beacon-based PCR kit is useful for the different units.

Animals↗

PubMed2004

[Expression and identification of cecropin D gene cloned in Pichia pastoris yeast cells].

OBJECTIVE: Cecropin D gene was cloned into methanol nutritional vector-pPICZ alpha-A, linked with alpha-factor signal peptide. METHODS: Positive recombinant was gotten through PCR and EcoR I restriction enzyme. The positive recombinant vector was transformed into Pichia pastoris yeast strain GS115. RESULTS: The positive recombinant yeast velum was fermented and its expressive product was assayed by the method of anti-germ. CONCLUSION: Cecropin D gene was cloned into yeast cell and its expressive products has high activity of killing bacteria, Which could be developed into a new type of anti-bacterial medicine.

Anti-Bacterial Agents↗

PubMed2004

[Establishment of two-dimensional gel electrophoresis technical platform for proteome research].

OBJECTIVE: To establish and optimize two-dimensional gel electrophoresis technique for proteomics. METHODS: Immobiline pH gradients isoelectric focusing was used as 1D, vertical SDS-PAGE as 2D, some factors such as method and volume of loading sample, choice of gels, concentration of SDS gels, preset of electric program and parameters, protocol for staining the gels were improved. RESULTS AND CONCLUSION: Using the proper method described above, satisfactory 2DE maps of human embryonic lung fibroblast(HLF) and silkworm egg were obtained. 2DE technical platform for proteome research was set up.

Animals↗

PubMed2004

[Mechanism and prevention measures of gene silencing].

With the widely application of transgene technology in the fields of high-level biologies, more and more attention was payed to gene silence. Gene silence can take place at the phase of transcription and post-transcription. With the results of methylation, repeated copies and homologous sequence of inner genes, trans-gene are silenced. There are many kinds of mechanisms of gene silencing and here, the mechanisms explaining the gene silence and some prevention measures were summarized in this paper.

DNA Methylation↗

PubMed2004

[Safety assessment of GM yeast feed additive with cecropin CAD gene].

OBJECTIVE: To evaluate the safety of GM yeast feed additive with cecropin CAD and to study and set up a model of Safety assessment for GM feed and detecting method. METHODS: To ensure the safety of the GM products, it has been done that to detect and value the safety of receptor organisms and expression products of extrinsic gene, the genetic stability of biologic properties of genomic modified yeast feed and condition of transfer and cumulation of anti-bacterial peptide and its products in circumstance and the feeded animals. RESULT AND CONCLUSION: The receptor animals and expression products of extrinic gene are safe, and the genomic modified products have steady genetic characters. The cectopin CAD neither cumulates in feeded animal nor releases into environment. The genomic modified feed additive is safe.

Animal Feed↗

PubMed2004

[Study of preparation methods of polyclonal antibody of chymopapain].

OBJECTIVE: To study the preparation methods of polyclonal antibody of chymopapain with higher titre. METHODS: Three types of antigens-native chymopapain, chymopapain inactivated by H2O2 and inactivated by iodoacetic acid were injected into different male rabbits by hypodermic injections and by intravenous injection. The blood samples were obtained from veins in ear to test antisera after the 3rd injection, the used methods were Western blotting and ELISA. The data was analyzed by the SAS system. RESULTS: The antisera titre of two inactivated antigens were obviously higher than that of nature antigen (P < 0.05), while there was no significant difference between the two inactivated antigens. Intravenous injection can obviously increase the ability of immunoresponse and raise antisera titre rapidly. CONCLUSION: The inactivated antigens have better immunogenicity than native chymopapain. As compared with iodoacetic acid, using H2O2 was an economical and effective method. The combination method of hypodermic injections and intravenous injection can raise antisera titre effectively and shorten the period of antibody preparation.

Animals↗

PubMed2004

[Progress on phage antibody library technology].

The exterior protein gene and the specific protein gene coded on the surface of phage can be fusedly expressed on the surface of phage with the recently developed technology-phage display technique. Through the PCR technique, the whole light chain genes and heavy chain genes can be amplified. Cloning these genes into lambda phage expression vectors and constructing phage antibody library through antigen adsorption-elution-proliferation, many targeting clones could be selected and the responding mono-clone antibody could be produced. Detailed progress in this regard was reviewed in the paper.

Antibodies, Monoclonal↗

PubMed2003

[Studies on protein-based identification method of genetically modified capsicum].

The detection system based on protein is a method to evaluate the safety of genetically modified foods (GMF). Using cecropin BD gene in capsicum, a detecting method was set up. It is a system of evaluating the real expressive condition and safety of the foreign target protein of GMF. In this studies, with the preformative technic method, a satisfactory results by making use of hemolymph of immunized pupae of Antheraea pernyi as standard experimental material was achieved, comparing with the realities of the goal protein expressive condition of cecropin D gene in capsicum. The detecting steps were as following: the goal protein from material was extracted roughly, then with CM-Sepharose-FF ion-exchange chromatography twice, the goal protein was purified moderately. The purified product was identified by detecting the anti-bacterial activity, electrophoresis, biological auto-photography of the goal protein and MADDI-TOF mass spectrum. The results showed that the expressive foreign target protein in transgenic capsicum was in accordance with standard protein in the physical and chemical property, anti-bacterial activity and molecular weight. It indicated that expression of the target gene in capsicum is real, it corresponded to expected value. The separation, purification and identification methods of cecropin D were established in the study. By means of the comparative experiments about anti-bacterial activity and molecular weight of anti-bacterial peptide(ABP) from GM-capsicum and hemolymph of immunized pupae of Antheraea pernyi, the identification method of target protein from GM-capsicum was set up. The method is easy to be operated, fast and feasible.

Capsicum↗

PubMed2003

[Gene chip technology and it's application to genetic toxicology].

Gene chip, also called as DNA array, Oligonucleotide Microchip or Oligonucletide array, is a new technology developed in recent years. It can be applied prospectively in life science research, such as gene expression, gene diagnose and gene polymorphism etc. In this paper, the principle, types, procedures and application of gene chip to genetic toxicological research were reviewed.

Humans↗

PubMed2003

[Application of mass spectrometry in the proteome research].

Electrospray ionization(ESI) and matrix-assisted laser desorption ionization(MALDI) are two kinds of newly-developed mass spectrometry techniques. The application of mass spectrometry is described in the proteome research such as MS charting, determination of molecular weight, peptide mass fingerprinting(PMF) and peptide sequence validation. The advantages of mass spectrometry are sensitive, accurate, rapid, high-throughput and automated. They have been proved a powerful technique and can be used as an important method for proteome research.

Mass Spectrometry↗

PubMed2002

[Study on the identification of genomic modified foods].

Nucleotide-based amplification method is an important system for the identification of genomic modified foods (GMF). Roundup Ready Soybeans (Monsanto company), Bt 176 GM maize (Novartis/Ciba-Geigy company) and Cecropin D capsicum was used as material to search for the feasibility of investigating the safety of GMF by PCR method. Primers specific for inserted genes and crop endogenous genes in Roundup Ready Soybeans, Bt 176 maize and Cecropin D capsicum were applied. The discrimination system for GM soybeans, GM maize and GM capsicum from the counterpart of non-GM products and the detection system for correlating marker genes and transgenes are established. The method was easy and fast, and the corresponding results fixed the standard or declared data.

Capsicum↗