[Generation and characterization of monoclonal antibody against human PIK3IP1].

AIM: To obtain monoclonal antibody against PIK3IP1 for further study of the structure and biological function of PIK3IP1 protein. METHODS: BALB/c mice were immunized with recombinant GST-PIK3IP1(62-168), Hybridoma cell lines secreting monoclonal antibodies against PIK3IP1 were screened by regular cell fusion and subcloning approach. The specificities of the monoclonal antibody was determined by ELISA, Western blot and Immunofluorescence assay. RESULTS: One hybridoma cell line (5C6) stable in secreting specific monoclonal antibody was successfully obtained. The subclass of IgG belonged to IgG1. The ascite titers of this monoclonal antibody reached 1:10(7). It could specifically bind to recombinant GST-PIK3IP1(62-168); protein and overexpressed PIK3IP1 and variant PIK3IP1-v1 proteins proved by Western blot. This antibody failed to react with E.coli lysates and glutathione S transferase (GST). At the same time, endogenous PIK3IP1 was not detected using 5C6 antibody. Immunofluorescence results revealed that overexpressed PIK3IP1 and variant PIK3IP1-v1 protein located in cytoplasm and distributed in fleck manner. CONCLUSION: Monoclonal antibody against PIK3IP1 with high titer and specificity has been successfully generated, which could be utilized as a useful reagent for the analysis of biochemical, structural, and functional properties of PIK3IP1.

[Expression and significance of severe acute respiratory syndrome associated coronavirus (SARS-CoV)-X4 protein in lungs of SARS patients].

OBJECTIVE: To investigate the significance of severe acute respiratory syndrome associated coronavirus (SARS-CoV)-X4 protein expression in lungs of patients with SARS. METHODS: Pathological features of the lungs from 4 SARS patients were examined and the expression of SARS-CoV-X4 protein in the lungs was evaluated with immunohistochemical staining using specific antibodies against protein X4. RESULTS: Microscopically, all lungs from 4 cases showed edema, erythrocyte and fibrin exudates in the alveoli, hyperplasia of alveolar epithelium, necrosis, hyaline membrane formation and fibroblast foci. Immunohistochemical stains showed a strong positivity of X4 protein in denudation cells, vascular endothelial cells and also erythrocytes and neutrophils in the alveoli of the lung tissues from the 4 cases. CONCLUSIONS: Expression of SARS-CoV-X4 protein in the lungs may be involved in the pathogenesis and progression of SARS.

The protein X4 of severe acute respiratory syndrome-associated coronavirus is expressed on both virus-infected cells and lung tissue of severe acute respiratory syndrome patients and inhibits growth of Balb/c 3T3 cell line.

BACKGROUND: The genome of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) includes sequences encoding the putative protein X4 (ORF8, ORF7a), consisting of 122 amino acids. The deduced sequence contains a probable cleaved signal peptide sequence and a C-terminal transmembrane helix, indicating that protein X4 is likely to be a type I membrane protein. This study was conducted to demonstrate whether the protein X4 was expressed and its essential function in the process of SARS-CoV infection. METHODS: The prokaryotic and eukaryotic protein X4-expressing plasmids were constructed. Recombinant soluble protein X4 was purified from E. coli using ion exchange chromatography, and the preparation was injected into chicken for rising specific polyclonal antibodies. The expression of protein X4 in SARS-CoV-infected Vero E6 cells and lung tissues from patients with SARS was performed using immunofluorescence assay and immunohistochemistry technique. The preliminary function of protein X4 was evaluated by treatment with and over-expression of protein X4 in cell lines. Western blot was employed to evaluate the expression of protein X4 in SARS-CoV particles. RESULTS: We expressed and purified soluble recombinant protein X4 from E.coli, and generated specific antibodies against protein X4. Western blot proved that the protein X4 was not assembled in the SARS-CoV particles. Indirect immunofluorescence assays revealed that the expression of protein X4 was detected at 8 hours after infection in SARS-CoV-infected Vero E6 cells. It was also detected in the lung tissues from patients with SARS. Treatment with and overexpression of protein X4 inhibited the growth of Balb/c 3T3 cells as determined by cell counting and MTT assays. CONCLUSION: The results provide the evidence of protein X4 expression following SARS-CoV infection, and may facilitate further investigation of the immunopathological mechanism of SARS.