B cell maturation antigen (BCMA) is a receptor of B lymphocyte stimulator (BLyS). Human IgG1Fc fusion proteins with the extracellular domain of BCMA(eBCMA), also called decoy receptors, have beenused as a potential BLyS antagonists to block BLyS activities. In order to design novel BLyS antagonistpeptides, computer-aided homologue modeling was used to construct an eBCMA-Fc fusion protein based on thecrystal structures of BCMA and Fc fragmant. To ensure the activity of eBCMA not to be interfered by Fcfusion, the root mean square distance (RMSD) for eBCMA and Fc were calculated to be 0.036 nm and 0.064nm, respectively, based on molecular docking modeling. An eBCMA-Fc fusion gene was constructed andintroduced into E. coli for expression. As expected, the purified 36 kD eBCMA-Fc fusion protein was able tobind BLyS in vitro at a dosage-dependent manner and demonstrated an anti-proliferative activity induced byBLyS in Daudi cells. The results have provided useful information on the evaluation of computer modeling andthe in vitro biological activity for the design of potential BLyS antagonist peptides.

Objective To observe the effect of Jiawei Siwu Decoction for idiopathic thrombocytopenic purpura (ITP) on the effi‐cacy and immune aspects of the model mice .Methods We used guinea pig anti mouse platelet serum immune IT P model ,then con‐duct the corresponding drug treatment .The mice were observed in peripheral blood ,bone marrow changes of nuclear cell count ,ser‐um IL‐6 content and changes in the spleen index .Results Compared with the model group of physiological saline of peripheral blood WBC ,prednisone group and Chinese medicine group PLT count ,bone marrow nucleated cell counts were significantly elevated (P<0 .01) ,the content of IL‐6 in serum were increased significantly (P<0 .01) ,no difference between the two treatment groups . Thymus and spleen index increased too (P<0 .05) .Conclusion Jiawei Siwu Decoction may be related to the regulation of cellular immunity ,increased serum IL‐6 content ,improve bone marrow hematopoietic microenvironment to achieve therapeutic effects in the ITP mice .

Objective To establish a stable cell line secreting human IgE Cε2-4 protein, and in-vestigate the binding capacity of receptor FcεR Ⅰ Methods The E24 gene was derived from SKO-O07 cell line, and was then cloned into pcDNA3.1 (+) (signal peptides were synthesized and fused at the 5'-end of E24 gene) or pCMV-L vector. After transient transfection into 293T cell, the secreted F24 protein was ana-lyzed by sandwich ELISA. The best vector was chosen to be transfected into CHO cells with LipofectAMI-NETM 2000 reagent. After being selected by G418 and subcloned three times by limited-dilution method, two stable cell lines were established. E24 gene was amplified by RT-PCR, and the E24 protein in the superna-tant was identified by ELISA. Besides, the binding capacity of FceR ⅠⅡ was analyzed by flow cytometry method. Results Three mammalian expression vector SP-E24-F3. 1, SP lI-E24-P3.1 and E24-PL were constructed and transient transfected to 293T cells. The output of E24 protein in the supernatant were 19.1, 19.4 and 8.7 μg/ml, respectively. Then the vector SP IX-E24-P3.1 was transfected into CHO cells. Final-ly, two single clones secreting E24 protein were stably obtained. The output of E24 were all at least 25 μg/ml. RT-PCR could detect the E24 gene from one of the two clones. Furthermore, flow cytometry results showed that E24 could bind the receptor in a dose-dependent manner. Conclusion Two stable cell line se- creting E24 protein were obtained, while E24 could specifically bind FcεR Ⅰ.

Objective To design and express a novel peptide based on ricin toxin antibody in E. coli, and to evaluate its biological activity. Methods Based on the crystal structure of ricin toxin A chain (RTA) and the RTA-rRNA interact in the complex model, the steric conformation of RTA was theoretical modeled and its functional domain was preliminarily determined. The humanized single-domain RTA antibody was designed rationally by computer-guidod molecular design method. Its coding sequence was ob- tained by overlapping extension PCR, and cloned into the pET-32a vector. The fusion protein was then ex-pressed in E. coli BL21 (DE3), identified by Western blot, and purified with Ni-NTA agarose. The binding and neutralizing activity of this novel peptide for riein was evaluated by competitive ELlSA assay and MTT assay. Results A recombinant human single-domain antibody expressing a polypeptide against RTA in the CDR3 loop was designed. The fusion protein was successfully expressed in E. coll. The purified protein can bind to ricin, and neutralize its activity in SP2/0 viability assay. Conclusion The success of the novel pep-tide based on riein toxin antibody provides a novel method to develop new generation of ricin antagonists.

Objective:To explore the factors affecting the antiviral treatment efficacy of acquired immunodeficiency syndrome (AIDS) patients.Methods:A total of 107 patients diagnosed with human immunodeficiency virus (HIV) infection in the clinic of Beihai People′s Hospital from January 2016 to June 2018 were selected.The patients were divided into two groups according to whether they voluntarily accepted traditional Chinese medicine treatment, including treatment group who received highly active anti-retroviral therapy (HAART) and traditional Chinese medicine prescription of Ping Gan Jie Du (42 cases), and control group who were only treated with HAART (65 cases). The virological and immunological responses were compared between the two groups at 48 weeks of treatment. The interleukin-28B (IL-28B) rs12979860 genotypes were measured by using the direct sequencing of polymerase chain reaction products. Logistic regression was used to analyze the influencing factors of antiviral efficacy in AIDS patients.Comparison between groups was performed by independent sample t test、matched sample t test or chi-square test. Results:At week 48 of treatment, 41 (97.62%) of the 42 patients in the HAART plus Ping Gan Jie Du group obtained virological response, while 58 (89.23%) of the 65 patients in the HAART group alone acquired virological response, which was not significantly different ( χ2=0.100, P>0.05). The numbers of CD4 + T lymphocytes increased at week 48 of treatment in the HAART plus Ping Gan Jie Du group and HAART group were (244.32±101.83)/μL and (211.56±112.50)/μL, respectively. The was no statistically significant difference ( t=1.522, P>0.05). Among the 92 patients with IL-28B CC genotype, 88 (95.65%) acquired virological response, while 11 of the 15 patients with non-IL-28B CC genotype acquired virological response, which was not significantly different ( χ2=0.394, P>0.05). And CD4 + T lymphocytes in patients with IL-28B CC genotype increased ((229.72±101.17)/μL), which was higher than that without IL-28B CC genotype ((173.40±89.64)/μL), with statistically significant difference ( t=2.028, P=0.045). Multivariate logistic regression analysis showed that baseline CD4 + T lymphocyte count≤200/μL, IL-28B CC genotype, and treatment plan including protease inhibitor were helpful to improve the antiviral efficacy. Conclusion:Baseline CD4 + T lymphocyte count ≤200/μL, IL-28B CC genotype, and protease inhibitor in HAART regimen are the influencing factors of antiviral efficacy in AIDS patients.

A three-dimensional (3D) graphic model of a single-chain Fv (scFv) which was derived from an anti-human placental acidic isoferritin (PAF) monoclonal antibody (Mab) was constructed by a homologous protein-predicting computer algorithm on Silicon graphic computer station.The structure, surface static electricity and hydrophobicity of scFv were investigated. Computer graphic modelling indicated that all regions of scFv including the linker, variable regions of the heavy (VH) and light (VL) chains were suitable. The VH region and the VL region were involved in composing the "hydrophobic pocket". The linker was drifted away VH and VL regions. The complementarity determining regions (CDRs) of VH and VL regions surrounded the "hydrophobic pocket". This study provides a theory basis for improving antibody affinity, investigating antibody structure and analyzing the functions of VH and VL regions in antibody activity.

A three-dimensional (3D) graphic model of a single-chain Fv (scFv) which was derived from an anti-human placental acidic isoferritin (PAF) monoclonal antibody (Mab) was constructed by a homologous protein-predicting computer algorithm on Silicon graphic computer station.The structure, surface static electricity and hydrophobicity of scFv were investigated. Computer graphic modelling indicated that all regions of scFv including the linker, variable regions of the heavy (VH) and light (VL) chains were suitable. The VH region and the VL region were involved in composing the "hydrophobic pocket". The linker was drifted away VH and VL regions. The complementarity determining regions (CDRs) of VH and VL regions surrounded the "hydrophobic pocket". This study provides a theory basis for improving antibody affinity, investigating antibody structure and analyzing the functions of VH and VL regions in antibody activity.

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.

Objective To study the intrinsic relationships between the binding energy of the antibody light and heavy chains and the conformational characteristics , physical and chemical properties , and to establish a corresponding mathemat-ical model and evaluate the thermal stability of the antibody molecules , which contribute to the antibody design , optimiza-tion and affinity maturation .Methods Based on bioinformatics and computational biology methods , the antibody′s structur-al information with the crystal diffraction data was analyzed .The conformational character of the variable domain of the antibody was studied using distance geometry and computer graphics technology .With the aid of the intermolecular hydrogen bond formation theory and the reaction free energy theory , the dynamic structure and energy characteristics be-tween the heavy and light chain variable regions of the antibody were studied .Furthermore , using nonlinear fitting and regression analysis, a mathematical model was set up .Results According to simulation and statistic analysis , there was a linear relationship between the binding energy and the number of the intermolecular hydrogen bonding , Van der Waals interaction of the heavy and light chains of the antibody .There was polynomial correlation between the binding energy and the physicochemical properties of the antibody .Using the frequency of amino acid position and the established model , the humanized anti-ricin antibody , which could not obtain the stable engineering cell line , was evaluated and optimized .The stable engineering cell line of the humanized anti-ricin antibody was obtained in the experiment .Conclusion The self structure of the antibody variable region ( conformation and physicochemical properties ) has much effect on its stability . The antibody stability can be improved by structural optimization .

Objective To develop a human Tim-3 specific monoclonal antibody and evaluate its biological activity and possible use in clinical diseases associated with dysregulated Tim-3 expression .Methods The BALB/c mice were immu-nized by conventional method, and positive clones were used to develop anti-human Tim-3 antibody, the binding and neutralization activities of which in vitro and in vivo were investigated.Results ①A monoclonal antibody (clone L3D) which could specifically bind to human Tim-3 protein in ELISA assay was obtained and the subtype of the monoclonal antibody was IgG2a .②Flow cytometry indicated that the monoclonal antibody could bind to Tim-3 expressed in human U937 cells.This antibody also showed a cross activity to mice′Tim-3.③The monoclonal antibody inhibited the apoptosis of THP1 cells induced by Gal-9, the ligand of Tim-3.④Injection of Tim-3 antibody exacerbated sepsis in mice as marked by the decreased survival rate and increased expression of pro-inflammatory cytokines .Conclusion An anti-human Tim-3 monoclonal antibody is successfully obtained.The excellent binding and neutralization activities of this antibody enable it to be widely used in clinical diseases associated with deregulated Tim-3 expression .