Objective:To establish an antibody expression system to reduce the antibody-dependent enhancement (ADE) effect of target antibody.Methods:Site-directed mutagenesis was used to mutate the 234 and 235 sites of the Fc region of the mammalian cell antibody expression vector-L234A and L235A to establish the antibody expression vector pFRT-IgG1κ-FcM. An antibody Wt-WNV with significant ADE effect obtained in previous work was selected and expressed by the pFRT-IgG1κ-FcM system to obtain mutant antibody FcM-WNV. The binding ability of FcM-WNV to target antigen West Nile virus envelope protein-DⅢ (WNV E-DⅢ) was detected by ELISA, and the its binding ability to human high-affinity IgG Fc receptor hFcγRⅠ (hCD64 ) was analyzed by flow cytometry. The neutralizing activity of FcM-WNV in vitro was detected by pseudovirus infection of host cells (BHK21 and K562). Results:The expression levels of FcM-WNV and Wt-WNV were comparable, and FcM-WNV could recognize and bind to WNVE-DIII in a concentration-dependent manner. Compared with Wt-WNV, the binding ability of FcM-WNV to hCD64 was significantly weakened, showing a significant decrease in fluorescence intensity. Consistent with the previous experimental results, Wt-WNV at a concentration of 5 μg/ml significantly enhanced the infection of K562 by WNV pseudovirus, while FcM-WNV at a concentration of 5 μg/ml could effectively block pseudovirus infection in both K562 and BHK21 cells.Conclusions:The established antibody expression system can effectively reduce the ADE effect of the target antibody.

Objective:To establish an in vivo infection model of West Nile virus (WNV) pseudovirus and evaluate the neutralizing activity of antibody WNV-XH1.Methods:A stable cell line that can package the WNV pseudovirus was established in the early stage to prepare the pseudovirus supernatant. The supernatant was concentrated and infected BHK21 cells to detect the titer of the pseudovirus. After intraperitoneal injection of the pseudovirus into C57BL/J mice, bioluminescence imaging was performed to observe the infection status of the pseudovirus in the mice. After simultaneous infection, blood was collected and ELISA was used to detect NS1 levels in mouse serum. The in vivo functional activity of antibody WNV-XH1 was evaluated using the established mouse infection model.Results:Fluorescence was detected in C57BL/J mice infected with WNV pseudovirus, and the NS1 levels in the peripheral blood serum of mice infected with pseudovirus were significantly higher than those of non infected mice (1.453±0.09vs0.305±0.018). After intravenous administration of WNV-XH1 antibody before the attack, the fluorescence signal in the mice decreased and the serum NS1 level decreased (0.384±0.015).Conclusions:A successful in vivo infection model of WNV pseudovirus was established, and it was confirmed that the antibody WNV-XH1 had a protective effect against WNV pseudovirus infection in vivo.

Objective:To establish an in vivo infection model of H5N1 pseudovirus and to detect the neutralizing activity of FHA3 antibody using this model. Methods:Based on the sequence information of hemagglutinin (HA) and neuraminidase (NA) of A/Anhui/1/2005/H5N1 strain, two recombinant plasmids of pcDNA3.1-HA5 and pcDNA3.1-NA1 were constructed. The two plasmids and plasmid pNL4-3.Luc.R-E- were co-transfected into 293T cells to prepare H5N1 pseudovirus supernatant. The morphology of pseudovirus particles in the supernatant was observed by electron microscopy. MDCK cells were infected with the pseudovirus supernatant and the virus titer was detected. BALB/c mice were injected with the pseudovirus supernatant by intraperitoneal injection and subjected to bioluminescence imaging at 2, 5, 8, and 12 d after infection to detect the pseudovirus infection in vivo. The functional activity of FHA3 antibody in vivo was evaluated using the established mouse infection model. Results:The recombinant plasmids pcDNA3.1-HA5 and pcDNA3.1-NA1 were correctly constructed and could be used to prepare pseudovirus supernatants of high titer by co-transfecting 293T cells with the plasmid pNL4-3.Luc.R-E-. The virus particles were round under electron microscope. H5N1 pseudovirus-infected mice exhibits strong fluorescence signals, which were attenuated by FHA3 treatment before challenge.Conclusions:The in vivo infection model of H5N1 pseudovirus was successfully constructed and FHA3 antibody was proved to be protective against the pseudovirus infection.

Objective:To prepare and identify a broad-spectrum antibody FHA3 targeting influenza A virus hemagglutinin (HA).Methods:According to the single-chain antibody fragment (scFv) sequence, the heavy chain (VH) and light chain (VL) variable regions of FHA3 were amplified by PCR and a recombinant plasmid pFRT-IgG1κ-FHA3 was constructed by linking the expression vector pFRT-IgG1κ. FHA3 was expressed in the ExpiCHO system and purified by affinity purification. The binding activity of FHA3 to influenza A virus HA was detected by ELISA. The neutralizing activity of FHA3 was detected in vitro by infecting host cells with pseudovirus. Results:SDS-PAGE showed that high-purity FHA3 was obtained. FHA3 could bind to H1N1 HA, H2N2 HA, H3N2 HA, H5N1 HA, H7N9 HA and H9N2 HA in a concentration-dependent manner. FHA3 had good neutralizing activity in vitro that was it could effectively block the invasion of H5N1 and H7N9 pseudoviruses into target cells at a low concentration of 5 μg/ml and H1N1 pseudovirus at 0.012 5 μg/ml. Conclusions:A broad spectrum antibody targeting HA protein of influenza A virus with neutralizing activity in vitro was obtained.

Objective:To prepare and identify a functional antibody FNA1 targeting the neuraminidase (NA) of influenza A virus N1 subtype.Methods:According to single-chain antibody fragment (scFv) sequence, the heavy chain and light chain variable region sequences of FNA1 were synthesized, and the recombinant expression plasmid pFRT-IgG1κ-FNA1 was constructed by linking the expression vector pFRT-IgG1κ. The FNA1 antibody was expressed in ExpiCHO cells and purified using affinity purification technique. The binding ability of FNA1 to the target proteins, influenza A virus N1 subtype NA antigens, was detected by ELISA. Flow cytometry was performed to analyze the binding ability of FNA1 to the NA antigens expressed on the surface of cell membrane. The in vitro activity of FNA1 against NA was evaluated by infecting 293T cells with pseudovirus. Results:Protein electrophoresis showed that FNA1 with high purity was obtained. FNA1 specifically recognized and bound to N1 subtype NA antigens in a concentration-dependent manner. FNA1 could effectively block NA activity by binding to N1 subtype NA protein expressed on the surface of cell membrane, thus inhibiting the release of packaged pseudovirus from cell surface and further inhibiting target cell infection.Conclusions:An antibody FNA1 targeting influenza A virus N1 subtype NA with in vitro functional activity was obtained.

Objective To analyze the therapeutic effect and prognosis of peritoneal dialysis in patients with end-stage polycystic kidney disease.Methods A retrospective analysis was performed on patients with polycystic kidney disease who were treated with peritoneal dialysis for more than 3 months between July 2007 and September 2016 in the Second Hospital Affiliated to Soochow University.A total of 45 patients were enrolled in this study.Another 45 patients of non-diabetic nephropathy were selected as the control group matched by gender,age,and time of PD initiation.The information of the two groups such as general data,dialysis related complications,incidence of peritonitis,prognosis was recorded.Survival analysis was performed using the Kaplan-Meier method and Log-rank test.The risk factors affecting patients' survival were analyzed with Cox regression model.Results There were no significant difference in pre-dialysis age,sex ratio,blood pressure,urine volume,body weight,eGFR,biochemical data,and the proportion of hypertension and diabetes mellitus in the polycystic kidney group and control group.24 h ultra-filtration volume,4 h D/Pcr,Kt/V and Ccr between the two groups showed no significant difference (all P ＞ 0.05).The incidence of peritonitis and the time of the first peritonitis in the two groups respectively as one episode per 82.4 months vs one episode per 81.5 months,(35.8±22.8) months vs (34.5±20.9) months had no statistical difference.The ratio of hernia (6.6％ vs 2.2％),thoracic and abdominal leakage (4.4％ vs 2.2％),dialysate leakage (0 vs 0),catheter dysfunction (4.4％ vs 6.6％),exit-site infections (11.1％ vs 6.6％),tunnel infections (4.4％ vs 2.2％) and non PD related infections (11.1％ vs 13.3％) had no significant difference.The 1-year,3-year,5-year patient survival of two groups respectively were 95.2％ vs 93.3％,78.9％ vs 75.0％,67.6％ vs 64.9％ (P=0.475),and 5-year technique survival was 78.7％ vs 76.7％ (P=0.623),demonstrating no obvious difference.Cox regression analysis showed that age and serum albumin were risk factors for the survival of patients.Conclusions The effect and prognosis of peritoneal dialysis in patients with polyeystic kidney and non polyeystic kidney were similar.Peritoneal dialysis is not the contraindication of polycystic kidney.Peritoneal dialysis can be used as a routine renal replacement therapy in patients with polycystic kidney disease.

As a targeted drug,monoclonal antibodies have been successful in tumor therapy. Thus,antibodies and related products are the fastest-developing biological agents. There are currently more than 40 monoclonal antibodies in the market that have been approved by the FDA,half of which are used to treat cancer. In recent years,a new generation of antibody drugs,such as human anti?body,glyco-engineered antibody,bispecific antibody,antibody-drug conjugates and immune check?point blockade antibody,have successfully cured various malignant tumors. In this paper,the history of antibody treatment for cancer,and the development and prospect of anti-tumor antibodies have been reviewed.

Objective To examine whether Tim-3 plays a protective role in paraquat poisoning induced excessive immune response and tissue damage based on the critical roles of Tim-3 controlling inflammatory response.Methods A paraquat poisoning model was established in wild type and in Tim-3 transgenic C57BL/6 mice by intraperitoneal injection of paraquat (40 mg/kg) .In addition, C57BL/6 mice with paraquat poisoning were injected with Tim-3 soluble protein( sTim-3) or control protein to see the effect of Tim-3 blocking on the progression of paraquat poisoning.Samples were collected at 6 and 24 h after paraquat injection respectively and were examined for tissue damage, cytokine expression and paraquat metabolism.Results After paraquat poisoning, there was significantly attenuated tissue damage in the lungs and kidneys and decreased TNF-α,IL-6 and IL-1 beta expression in the PBMCs or in the serum from Tim-3 transgenic mice compared to wild type mice.The serum concentration of paraquat in Tim-3 transgenic mice was also significantly decreased.However, in sTim-3 treated paraquat poisoning mice, there was significantly increased cytokine expression and tissue damage compared to control protein treated mice.The in vitro data showed that Tim-3 signaling negatively regulated macrophages mediated inflammatory response.Conclusion Tim-3 plays a critical role in maintaining the homeostasis after paraquat poisoning. Further investigation on the regulatory roles of Tim-3 in inflammation will shed new light on the pathogenesis of paraquat poisoning and provide new therapeutic strategies.

The interleukin-12 (IL-12) family, including IL-12, IL-23, IL-27,and IL-35, is characterized by unique structures and molecular partners.This is the only family of heterodimeric cytokines, which endows them with a unique set of connections and functional interactions.They not only play an important role in the regulation of inflammation, but are closely related to various autoimmune diseases.Here we discuss the structural aspects of these cytokines and their effect on some autoimmune diseases.

Based on situation assessment of the core areas of biosecurity , this paper summarizes three typical features of biosecurity that make it different from traditional safety , abd proposes that biosecurity be regarded as a living-project of a country .A mature and powerful country has to possess sufficient capacity to ensure biosecurity .This study also analyzes the national biosecurity strategyies and practices of the United States ,which has established a national-level capacity building strategy and roadmap with clear objectives ,specified the responsibilities of each federal agency , and regulations linked up with strategic planning .Biosecurity in China is at the crossroads where crisis coexists with opportunity , making it urgent to establish the national biosecurity strategy .