Objective:Choosing efficient expression vector express anti HBsAg Fd and L chain in E.coli to produce anti HBsAg human Fab.Purificating inclusion bodies to denaturating and refolding the protein.Methods:Expression vector of PQE32 Fd、PQE32 L was constructed and transformed into E.coli strain M15,efficient expression clone was screened by SDS PAGE.Results:After induced by IPTG,M15 PQE32 Fd、M15 PQE32 L expressed insoluble recombinant protein in inclusion bodies.The Fd、L chain gene sequence conform with that reported in NCBI BLAST.Conclusion:The M15 PQE32 expression system is stable and efficient for expressing anti HBs human Fd、L chain.The denaturation and protein refolding of the anti HBsAg Fab expressed in inclusion bodies need to be studied further.

Objective:To study the evolution of B lymphocyte and NK cell in Severe Acute Respiratory Syndrome (SARS) patients.Methods:B lymphocyte and NK cell in the peripheral blood was detected by flow cytometry in 53 patients with SARS.Results:During the disease progression,B lymphocyte decreased then increased or increased progressively in 92% cured SARS patients, in others have no change or decrease.The NK cell showed individual difference: typical immune response was showed in 73.7% patients, but in others have no change or increase.

Objective:To study the evalution of T lymphocyte in Severe Acute Respiratory Syndrome (SARS) patients.Methods:T lymphocyte in the peripheral blood was determined by Flow Cytometry in 53 patients with SARS. Results:The CD4 +T cell and CD8 +T cell decreased reversiblly in the cured SARS patients, but decreased progressively in the dead cases. The level and duration of the decrease are correlated with the patient's condition. The lowest CD4 +T cell count is 305?150 cells/?l(P

Objective:Express a human anti-HBsAg single chain antibody fragment(scFv)-consensus interferon (cIFN) fusion protein by bacterial expression system and analyse the function of the fusion protein.Methods:Human anti-HBsAg single chain monoclonal antibody cDNA encoding the variable regions of immunoglobulin from PBMC of Hepatitis B patient. Consensus interferon gene was produced by overlap PCR.A plasmid for production of cIFN-scFV fusion protein was constructed, then the expression vector pRA cIFN-scFV transformed with the E.coli strain BL21(DE3). The gene product was analysed SDS-PAGE and Western blotting, then was solubilized by guanidine hydyochloride, refolded by conventional dilution method, and purified using metal-chelating chromatography. The immune and functional analysis of the resulting fusion protein have been studied by ELISA,FACS(Flow cytometry),MTS assay and hemaglutination inhibition test.Results:The authors isolated and characterized the human anti-HBsAg single chain antibody fragment(scFv)-consensus interferon (cIFN) fusion protein. The resulting human anti-HBsAg scFv-cIFN fusion protein was bound to react with HBsAg and cIFN, this react show that highly specific and bioactivity.Conclusion:A human anti-HBsAg single chain antibody fragment(scFv)-consensus interferon (cIFN) fusion protein was produced by bacterial expression system in this study. This genetically engineered human anti-HBsAg scFv-cIFN fusion protein promises to be an important reagent for hepatitis B immunotherapy.

Objective Based on Total Quality Management (TQM) theory,our study aims to analyze the clinical Bio-Bank overall quality management implications,basic characteristics,principles,and management mechanisms,and provide theoretical basis for the clinical Bio-Bank quality construction.Methods Using theoretical and literature research methods,Bio-Bank overall quality management qualitative analysis was conducted,putting forward a framework of Bio-Bank comprehensive quality management.Results Biological sample overall quality management was defined theoretically including its connotation,concepts and basic characteristics.We also put forward an application principle and basic operation method at the application level.Conclusions Total Quality Management (TQM) is applied to the clinical Bio-Bank construction,from where,the scientific and unique management content can effectively optimize the Bio-Bank management regulation and standardization of the sample operation process in the PDCA cycle,which is critical to improve the quality of clinical Bio-Bank.

【Objective】 To investigate the regulation of HCBP6 mimic phosphorylation on triglyceride synthesis in hepatocytes so as to provide a molecular target for the treatment of metabolism-associated fatty liver disease. 【Methods】 We used site-directed mutagenesis to mimic constitutive phosphorylation and dephosphorylation of HCBP6 Ser-10 and Ser-151. Oil red O staining and triglyceride content determination were used to detect triglyceride levels in hepatocytes. The expressions of SREBP1c, ACC1 and FASN were detected by qRT-PCR and Western blotting. The Dual-Luciferase Report Gene System was used to detect SREBP1c promoter activity. 【Results】 HCBP6 Ser-10 phosphorylation promoted triglyceride synthesis. HCBP6 Ser-10 phosphorylation upregulated the expressions of SREBP1c, ACC1and FASN genes; HCBP6 Ser-10 phosphorylation enhanced the SREBP1c promoter activity. 【Conclusion】 HCBP6 Ser-10 phosphorylation can significantly enhance the activity of the SREBP1c promoter, upregulate the SREBP1c-FASN signal pathway transduction, and promote the synthesis of triglycerides.

Objective: To investigate the efficacy of 200IU hepatitis B immunoglobulin (HBIG) injection at 1 month after birth to interrupt the mother-to-children transmission (MTCT) of hepatitis B virus (HBV). Methods: Infants born to mothers who were hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) positive, with HBV DNA load ≥1.0×10⁶ IU/ml and who did not receive antiviral drug treatment during pregnancy, were randomly divided into 2 groups. Infants in the control group were treated with standard immunoprophylaxis: 200 IU HBIG and 10 μg recombinant hepatitis B vaccine injection within 2 h after birth and a vaccine booster at 1 and 6 months after birth. For infants in the HBIG group the standard immunoprophylaxis and an additional 200 IU HBIG were administered at 1 month. HBsAg, the antibody to HBsAg (anti-HBs), and HBV DNA load were measured at birth and after 7 months. later.Immunoprophylaxis failure was defined as the presence of HBV DNA and HBsAg positivity or the presence of HBV DNA and HBsAg negativity at 7 months. Results: In this prospective cohort study, of the 280 infants enrolled, 14 infants (HBIG/control: 6/8) were lost to follow-up and 266 subjects (HBIG/control: 134/132) completed the 7-month study. The log₁₀HBV DNA load of mothers in the HBIG group and control group were (7.31±0.66) log₁₀IU/ml and (7.32±0.74) log₁₀IU/ml, respectively (P=0.92). The MTCT rate of the two groups was similar (5.97% vs. 7.58%, P=0.63). At 7 months, the HBsAg positive rate and the level of anti-HBs in the two groups were 94.03%(126/134)vs. 91.67% (121/132) and 623.60±412.93 mIU/mL vs. 620.38±399.10 mIU/ml, respectively with no significant difference (P=0.48 and P=0.95, respectively). The log₁₀ HBV DNA load of mothers in immunoprophylaxis failure group and success group was similar (P=0.09). The number of infants who were serum HBsAg positive and HBV DNA positive at birth in the immunoprophylaxis failure group were higher than those in the success group (100% and 100% vs. 35.89% and 31.85%, P<0.01, respectively). The serum HBsAg levels in infants at birth was the only independent relevant factor for HBV MTCT, with risk rates of 11.18 (95% Confidence interval (CI), 1.23-101.88), 352.00 (95%CI, 15.82-7833.20), and 968.00 (95%CI 81.35-11519.19) for HBsAg levels of 0.05-< 1, 1-< 10, and ≥ 10 IU/ml, respectively, compared to infants with HBsAg levels < 0.05 IU/ml. Conclusions Administering 200IU HBIG injection at 1 month did not reduce the risk of HBV MTCT.

Objective: To investigate the differences in frequency and function of natural killer cells (NK) between chronic hepatitis B (CHB) and acute hepatitis B (AHB). Methods: Patients with AHB and those with CHB in immune active (IA) phase were enrolled. The frequencies of NK, CD56^dimNK, CD56^brightNK and the expression of functional molecules IFNAR2 and NKp46 on the surface of NK cells were detected respectively among patients with CHB in IA phase, patients with AHB, and those recovered from AHB. At the same time, their correlations with ALT, HBV DNA and HBV markers were analyzed. Results: Between IA and AHB, the frequencies of NK cells and NKp46^dim NK cells in AHB cases were significantly lower than those in IA cases, but the frequency of NKp46^high NK cells in AHB was higher than that in IA. For patients who recovered from AHB, the frequency of NK cells and NKp46^dim NK cells increased; the varied ranges of frequencies of CD56^dimNK, IFNAR2⁺ NK and NKp46⁺ NK cells were on the rise, while the frequency of NKp46^high NK cells decreased after the recovery from AHB, and the varied ranges of CD56^brightNK and IFNAR2MFI, NKp46MFI decreased. In AHB, HBVDNA loads were positively correlated with ALT levels. Before and after the recovery of AHB: ΔHBV DNA and ΔALT, Δ NK/LY (%) were positively correlated; ΔALT and ΔNKp46^highNK/NK(%), ΔNKp46MFI, ΔIFNAR2MFI were positively correlated. Conclusions In CHB immune active phase, the activity of peripheral blood NK cells was too weak to remove the virus, but NK cells play an important role in eliminating the viruses and mediating liver tissue inflammation in AHB.

Objective: To investigate the differences in function of plasmacytoid dendritic cells (pDC) and CD4⁺ T helper cells (CD4⁺ Th cells) between acute hepatitis B (AHB) and chronic hepatitis B (CHB). Methods: In this study, patients with AHB and those with CHB in immune active (IA) phase were enrolled. The frequencies of pDC, CD86⁺ pDC, CD4⁺ T cells and their subsets, surface functional molecules were detected respectively among patients with chronic HBV infection in IA phase, patients with AHB, those recovered from AHB. Meanwhile, their correlations with ALT, HBV DNA and HBV markers were analyzed. Results: The ALT level in AHB was significantly higher than that in IA, and inflammation was more obvious in AHB. Between IA and AHB, CD86⁺ pDC frequency and the mean fluorescence intensity of functional molecule CD86 (CD86MFI) were higher in IA than those in AHB, but the frequency of CD4⁺ T cells in AHB was higher than that in IA. For patients who got over AHB, the frequency of CD86⁺ pDC increased; Th1 were on the rise, while the frequencies of CD4⁺ T and Th2 decreased after the recovery of AHB, and Th2 / Th1 ratio decreased..In AHB, HBVDNA loads were positively correlated with ALT levels and Th2 frequencies. Conclusions In CHB immune active phase, CD86⁺ pDC with stimulating function played an important role, but the cellular immune response of CD4⁺ T cells decreased. In AHB inflammatory stage, CD4⁺ T cells played a strong cellular immune response, which result ed in viral clearance. Th2 cells regulation of CD4⁺ T cells played a dominant role, which was involved in the inflammatory response, and the cytotoxic role of Th1 cells during the recovery period was dominant, playing a strong cellular immune response, then the virus were completely eliminated.