Objective To clone, express Tp0319 gene from Treponemapallidum (T. pallidum), and to assess the immunocompetence of recombinant protein. Methods The immuno-dominant region of Tp0319gene was chosen by computer analysis, amplified from T. pallidum complete genome by PCR, subcloned into the expression vector pQE32 to construct a recombinant plasmid, pQE32/Tp0319, which was then expressed in E. coli M15. The recombinant protein was purified with Ni-NTA affinity chromatography, and identified by using sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western blot. New Zealand rabbits were immunized with the recombinant protein, and the titer of anti-Tp0319 antibodies in sera from immunized rabbits were measured with indirect ELISA. Also, indirect ELISA with the recombinant Tp0319 as coating antigen was performed to detect the anti-Tp0319 antibody in sera from 200 normal human controls and 200 patients with syphilis. Results The prokaryotic expression vector pQE32/Tp0319 was constructed successfully, and the recombinant protein Tp0319 with a molecular weight of about 30 000 was attained. Specific humoral response was elicited by the recombinant protein in New Zealand rabbits and the specific antibody titer was more than 1: 10 240 after immunization for 3 times. Western blot proved that the recombinant protein could specifically react with anti-T. pallidum IgG antibody-positive sera. Indirect ELISA was successfully developed with the recombinant Tp0319, and detected antibodies to T. pallidum in control sera with a sensitivity and specificity of 100% (40/40), respectively. Compared with T. pallidum particle agglutination (TPPA) assay, the sensitivity and specificity of the indirect ELISA were 92.6% and 100%, respectively, in the detection of T. pallidum in sera from patients and controls, and the concordance between the indirect ELISA and TPPA was 96%. Conclusions The prepared recombinant protein shows a satisfactory immunocompetence, which may lay a foundation for its further application in the serodiagnosis of syphilis.

AIM: To determine the expression level of Fas molecules on the T cells isolated from systemic lupus erythematosus (SLE) patients. METHODS: The expression of Fas on T cells and the apoptotic rate of 36 patients with SLE in active phase and 18 normal people were determined with flow cytometry. The patients were treated with impact therapy of prednisone, methylprednisolone or cyclophosphamide, respectively, according to their conditions. Within 3 days after admission, immediately before discharging (the average days in hospital is 22.6 d), and 4 weeks after discharging, the amount of Fas molecules expressed on T cell surface was determined respectively with flow cytometry in each patient. RESULTS: There was a positive correlation between SLEDAI, apoptotic rate of T cells and Fas molecules on T cells in SLE patients of active phase (P

Objective To establish an efficient baculovirus-insect cell expression system for the production of human immunodeficiency virus-1 ( HIV-1 ) envelope glycoprotein gp120 and to evaluate the physiochemical properties, antigenicity and immunogenicity of the recombinant protein. Methods The gene encoding HIV-1 NL4-3 gp120 was cloned into the downstream of pH promoter of the baculovirus transfer vec-tor pAcgp67B to construct the recombinant transfer vector pAc-gp120. Expression of the protein of interest was induced in baculovirus-infected High FiveTM insect cells. ELISA, analytical ultracentrifugation and size-exclusion chromatography were carried out to characterize physicochemical properties of the expressed gp120 protein. Immunogenicity of the recombinant gp120 protein was analyzed by HIV neutralization assay after im-munizing BALB/c mice with it. Results The recombinant HIV-1 gp120 protein was successfully obtained from the established insect cell expression system with a purity of more than 90％ and a mean yield of 13 mg/L in four batches. That recombinant HIV-1 gp120 protein was characterized by homogeneity in solution and possessed a good immunoreactivity to neutralizing antibodies and antisera against HIV. Immunogenicity analysis in BALB/c mice demonstrated that the recombinant gp120 protein could induce effective immune re-sponses against HIV-1 NL4-3. Conclusion A simple and scalable approach to obtain homogeneous and im-munogenic HIV-1 gp120 antigen is successfully established, which will promote further investigation of HIV vaccine candidates.

OBJECTIVETo investigate the expressions of Engrailed-2 (EN2) and β-catenin in bladder urothelial carcinoma and explore their significance.

METHODSSixty bladder urothelial carcinoma samples of different grades and stages and 10 normal bladder mucosal tissues were examined for expressions of EN2 and β-catenin proteins and mRNA using immunochemistry, Western blotting and RT-PCR. RESULTS Compared to normal bladder mucosa, bladder urothelial carcinoma tissues showed significantly increased expressions of EN2 and β-catenin proteins (P<0.05), and the high-grade carcinoma tissues exhibited significantly stronger expressions than the low-grade ones (P<0.05); the expressions of the proteins increased also significantly with advanced pathological stages of bladder urothelial carcinoma (P<0.05). The expressions of EN2 and β-catenin mRNAs showed a consistent pattern of changes with their protein expressions.

CONCLUSIONThe expressions of EN2 and β-catenin are significantly increased in bladder urothelial carcinoma. EN2 may contribute to the development and progression of bladder urothelial carcinoma by activating Wnt/β-catenin signal pathway.

Adult ; Aged ; Aged, 80 and over ; Carcinoma ; metabolism ; pathology ; Female ; Homeodomain Proteins ; metabolism ; Humans ; Male ; Middle Aged ; Nerve Tissue Proteins ; metabolism ; Urinary Bladder Neoplasms ; metabolism ; pathology ; beta Catenin ; metabolism

Lentiviral vectors were powerful gene delivery tools for gene therapy. We developed a new lentiviral vector pBobi-MIL that constitutively expressed O6-methylguanine-DNAmethyltransferase (MGMT) and Luciferase, linked by the internal ribosomal entry site (IRES), to realize drug tolerance and real time monitoring in vivo. All results from RT-PCR, drug treating clones forming, immunofluorometric assay and chemiluminescence detection showed that cells infected by recombinant lentivirus L-MIL simultaneously expressed these two genes. This lays the foundation for the further research in gene therapy and can also help identify lentivirus titer.
DNA Modification Methylases ; biosynthesis ; genetics ; DNA Repair Enzymes ; biosynthesis ; genetics ; Drug Resistance ; genetics ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; Humans ; Lentivirus ; genetics ; metabolism ; Luciferases ; biosynthesis ; genetics ; Tumor Suppressor Proteins ; biosynthesis ; genetics

We constructed the CAP2NC prokaryotic expression vector of HIV-1 NL4-3 strain and obtained relatively pure CAP2NC protein by optimizing its purification conditions to explore its in vitro self-assembly conditions. Primers were designed according to the CAP2NC DNA sequence of HIV-1 NL4-3 strain. The target gene was amplified by PCR and cloned into prokaryotic expression vector pTO-T7. Then the recombinant strain was transformed into Escherichia coli BL21 (DE3). IPTG induced protein expression, then the protein was purified by hydrophobic chromatography. SDS-PAGE and Western blotting were performed to analyze the target protein, and the biological activity of the antigen was identified through ELISA. The self-assembly of CAP2NC protein was analyzed by transmission electron microscopy and gel filtration chromatography. The protein had good reaction with the specific antibodies of p24 and formed different structures in various conditions. When 10% yeast RNA was added to the protein complex, the recombinant protein only formed into a tubular structure, which was similar to the self-assembled structure of the HIV-1 virus capsid. The results showed that the HIV-1 CAP2NC protein had in vitro self-assembly activity, and the RNA affected the structure of CAP2NC protein assembly. The protein can be used as a simple and effective molecular model to study its structure, and then it can provide a reference for the study of HIV immature virus particles.