A novel approach for identifying the heme-binding proteins from mouse tissues / 基因组蛋白质组与生物信息学报·英文版

Heme is a key cofactor in aerobic life, both in eukaryotes and prokaryotes. Because of the high reactivity of ferrous protoporphyrin IX, the reactions of heme in cells are often carried out through heme-protein complexes. Traditionally studies of heme-binding proteins have been approached on a case by case basis, thus there is a limited global view of the distribution of heme-binding proteins in different cells or tissues. The procedure described here is aimed at profiling heme-binding proteins in mouse tissues sequentially by 1) purification of heme-binding proteins by heme-agarose, an affinity chromatographic resin; 2) isolation of heme-binding proteins by SDS-PAGE or two-dimensional electrophoresis; 3) identification of heme-binding proteins by mass spectrometry. In five mouse tissues, over 600 protein spots were visualized on 2-DE gel stained by Commassie blue and 154 proteins were identified by MALDI-TOF, in which most proteins belong to heme related. This methodology makes it possible to globally characterize the heme-binding proteins in a biological system.

The C-terminal portion of the nucleocapsid protein demonstrates SARS-CoV antigenicity / 基因组蛋白质组与生物信息学报·英文版

In order to develop clinical diagnostic tools for rapid detection of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) and to identify candidate proteins for vaccine development, the C-terminal portion of the nucleocapsid (NC) gene was amplified using RT-PCR from the SARS-CoV genome, cloned into a yeast expression vector (pEGH), and expressed as a glutathione S-transferase (GST) and Hisx6 double-tagged fusion protein under the control of an inducible promoter. Western analysis on the purified protein confirmed the expression and purification of the NC fusion proteins from yeast. To determine its antigenicity, the fusion protein was challenged with serum samples from SARS patients and normal controls. The NC fusion protein demonstrated high antigenicity with high specificity, and therefore, it should have great potential in designing clinical diagnostic tools and provide useful information for vaccine development.

The epitope study on the SARS-CoV nucleocapsid protein / 基因组蛋白质组与生物信息学报·英文版

The nucleocapsid protein (N protein) has been found to be an antigenic protein in a number of coronaviruses. Whether the N protein in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is antigenic remains to be elucidated. Using Western blot and Enzyme-linked Immunosorbent Assay (ELISA), the recombinant N proteins and the synthesized peptides derived from the N protein were screened in sera from SARS patients. All patient sera in this study displayed strong positive immunoreactivities against the recombinant N proteins, whereas normal sera gave negative immunoresponses to these proteins, indicating that the N protein of SARS-CoV is an antigenic protein. Furthermore, the epitope sites in the N protein were determined by competition experiments, in which the recombinant proteins or the synthesized peptides competed against the SARS-CoV proteins to bind to the antibodies raised in SARS sera. One epitope site located at the C-terminus was confirmed as the most antigenic region in this protein. A detailed screening of peptide with ELISA demonstrated that the amino sequence from Codons 371 to 407 was the epitope site at the C-terminus of the N protein. Understanding of the epitope sites could be very significant for developing an effective diagnostic approach to SARS.

A strategy for searching antigenic regions in the SARS-CoV spike protein / 基因组蛋白质组与生物信息学报·英文版

In the face of the worldwide threat of severe acute respiratory syndrome (SARS) to human life, some of the most urgent challenges are to develop fast and accurate analytical methods for early diagnosis of this disease as well as to create a safe anti-viral vaccine for prevention. To these ends, we investigated the antigenicity of the spike protein (S protein), a major structural protein in the SARS-coronavirus (SARS-CoV). Based upon the theoretical analysis for hydrophobicity of the S protein, 18 peptides were synthesized. Using Enzyme-Linked Immunosorbent Assay (ELISA), these peptides were screened in the sera from SARS patients. According to these results, two fragments of the S gene were amplified by PCR and cloned into pET-32a. Both S fragments were expressed in the BL-21 strain and further purified with an affinity chromatography. These recombinant S fragments were confirmed to have positive cross-reactions with SARS sera, either by Western blot or by ELISA. Our results demonstrated that the potential epitope regions were located at Codons 469-882 in the S protein, and one epitope site was located at Codons 599-620. Identification of antigenic regions in the SARS-CoV S protein may be important for the functional studies of this virus or the development of clinical diagnosis.

Identification of genes associated with human osteosarcoma metastasis suppression using suppression subtractive hybridization / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify genes associated with metastasis suppression and to investigate the molecular mechanism of osteosarcoma metastasis.</p><p><b>METHODS</b>The subtracted cDNA library of low metastatic human osteosarcoma cell line SOSP-9607 was constructed using suppression subtractive hybridization. Partial clones were sequenced. The acquired sequence data were aligned against the GenBank nucleotide database using Blastn to search for sequence matches. The interested clone was used to perform Northern blot and reverse transcriptase-PCR (RT-PCR) analysis on mRNA isolated from low metastatic cell line SOSP-9607 and OS-9901, high metastatic cell line SOSP-M and three pulmonic metastatic nodules of nude mice.</p><p><b>RESULTS</b>A cDNA clone from low metastatic cell line SOSP-9607 subtracted cDNA library was identified as telomeric repeat binding factor 2 (TERF2) by sequence analysis and Blastn search. Northern blot and RT-PCR analysis demonstrated that TERF2 expressed highly in low metastatic cell line SOSP-9607 and OS-9901, but not in high metastatic cell line SOSP-M and three pulmonic metastatic nodules.</p><p><b>CONCLUSION</b>TERF2 may be important for suppressing metastasis of osteosarcoma.</p>

Apoptosis in Human Osteosarcoma Cell Line SOSP-9607 Induced by Caspase-6 / 中国肿瘤生物治疗杂志

Objective:To explore caspase-6's effect on the apoptosis of osteosarcoma cell line SOSP 607. Methods: The expression level of caspase-6 in the osteosarcoma cell line SOSP-9067 was examined by RT-PCR method. The adeno-virus adv5 vector with caspase-6 gene was constructed and transferred into the osteosarcoma cell line SOSP-9607 by lipo-fection. The cell survival rate after transfection was assayed by MTT method. The cell morphological changes were observed by microscope and electron microscope, the apoptosis of transferred cells were examined by gel electrophoresis. Results:No expression of caspase-6 was examined in the osteosarcoma cell line SOSP-9607. A high expression of caspase-6 was identified by RT-PCR after the transfection. The cell growth curve declined after transferring caspase-6. Electrophoresis of DNA displayed the apoptosis ladder. Conclusion: Transferring caspase-6 into the osteosarcoma line SOSP-9607 may inhibit the growth of the osteosarcoma cell line SOSP-9607 and this effect might be achieved by inducing apoptosis.