Cloning, Expression, Purification of a Fusion Protein of SARS-CoV Nucleocapsid and Spike Protein Fragments and Its Immunological Characteristics / 中国生物化学与分子生物学报

The spike (S) and nucleocapsid (N) proteins, which are responsible for viral binding to cell surface receptors and the formation of ribonucleoprotein complexes during virion assembling, are major structure proteins of severe acute respiratory syndrome coronavirus (SARS-CoV). The expression of recombinant protein may give more accurate result for detecting SARS-CoV infection. A novel fusion protein,comprising of two fragments of N and S proteins from SARS-CoV, was prepared. Our computer-assisted analysis suggested that the immunodominant domains were located in the amino acid residues 1-227 of N protein and 450-650 of S protein, further the fusion of the two fragments did not change the immunochemical characteristics. The complementary DNA(cDNA) encoding N1-227 fused with S450-650 was obtained by sequence overlapping extension (SOE), and named NLS. It was cloned into pET-28a ( + ), an expression vector for His-tag fusion protein. This new constructed fusion protein was prokaryotic expressed in E. coli,and purified by metal chelate affinity chromatography with the purity over 95 %. The purified fusion protein was identified by anti-His monoclonal antibody and convalescence SARS patients serum. The NLS protein based ELISA showed that NLS maintained appropriate antigenicity and specificity to react with the sera of convalescent SARS patients. The functional NLS protein were successfully expressed and purified. And the fusion protein based ELISA can be used for detection of antibodies (Abs) against the S and N proteins of SARS-CoV. It may provided a novel diagnostic tool and have the potential application in developing of anti-SARS vaccine.

Cloning, expression and protein structure prediction of a novel SCF-TPO fusion gene / 中国病理生理杂志

AIM: To obtain the high expression of recombinant human stem cell factor - thrombopoitin (SCF-TPO) fusion gene and predict its structure property. METHODS: Tour primers were designed according to known sequence of TPO and SCF to amplify the functional amino acid domain of TPO and SCF by RT- PCR, respectively from fetus hepatocytes. The expression plasmid pET32a/SCF- TPO was constructed by VOE gene fusion technique and expressed in BL21(DE3)plysS. The fusion protein property, such as second structure, flexibility, and hydrophilicity were predicted by DS Gene and Protscale software. RESULTS: The expression vector, pET32a/SCF - TPO was constructed and the high expression of the SCF/TPO fusion protein was obtained, with the expression amount of up to 40% of the total cellular protein. DS Genel .5 and Protscale predict no new antigenicity in fusion protein, and the second structure and ioelectric point have no changes except four amino acids change in first structure. There are high flexibility and low hydrophilieity in the linker peptide. CONCLUSION: High expression of SCF- TPO fusion protein has been obtained and protein prediction shows that the fusion protein design is reasonable, which lay foundation for further study of biological fundation of SCF - TPO fusion protein.

Analysis of biochemical and physical properties for a new recombinant B7-Pseudomonas exotoxin fusion protein B7-2-PE40KDEL / 中国免疫学杂志

Objective:Identification of some biochemical and physical properties for a new recombinant B7-2-PE40KDEL exotoxin fusion protein.Methods:12%SDS-PAGE separating and gel imaging analyzing,peptide mass fingerprinting,Western blot and MTT assasying were used respectively for identification of the protein.Results:Molecular weight of the recombinant B7-2-PE40KDEL was 72 628,5% of the difference to its theoretical value 69 561.The result of Western blot indicated that the purified recombinant B7-2-PE40KDEL could specifically bind with mAb anti-human B7-2 and the antibody against PEA,while the negative control did not.The recombinant B7-2-PE40KDEL digested with trypsin and then detected by MOLTI-TOF-MS.It was shown that the detected 15 peptides lied in the extracellular part of B7-2 and the truncated Pseudomonas extoxin PE40KDEL.Searching in the peptident data bank of Expasy website,we did not find any known proteins which was accordant with the above terms.The cytotoxic activity of the recombinant toxin with MTT method showed that the B7-2-PE40KDEL selectively killed Jurkat cell line which expressesed CD28 receptor well and had no killing effect on the Raji cell line unexpressing CD28 receptor.Conclusion:Recombinant B7-2-PE40KDEL exotoxin fusion protein we construct proves to be a new one with targeted killing bioactivity to B7:CD28 system.