Effect of IgG dimer concentration on the binding ability of IgG Fc fragment in IVIG to THP-1 cell surface receptors / 中国输血杂志

【Objective】 To investigate the effect of immunoglobulin G (IgG) dimer concentration of intravenous immunoglobulin (IVIG) on the binding ability of IgG Fc fragment to THP-1 cell surface receptors. 【Methods】 Firstly, protein purification and high performance liquid chromatography (HPLC) were used to prepare different concentrations of IgG dimers. After that, IgG dimer was added to IVIG to prepare IVIG containing different concentrations of IgG dimer. Finally, based on the method established in our laboratory, we analyzed the effect of IgG dimer concentration in IVIG on the binding ability of IgG Fc fragment to THP-1 cell surface receptors. 【Results】 When the concentration of IgG dimer in IVIG was 1.11%-10.30%, its binding ability to Fc receptors on the surface of THP-1 cell was 97.67%-135.33%, and this binding ability was positively correlated with the concentration of IgG dimer. When the IgG dimer concentration exceeded 13.22%, the binding ability had no correlation with the IgG dimer concentration. 【Conclusion】 A certain concentration of IgG dimer can promote the binding ability of the IgG Fc fragment in IVIG to receptors on the surface of THP-1 cells, which needs further verification from animal experiments and clinical data.

Preparation of CD73/PD-L1 bispecific antibody and evaluation of its antitumor function in vitro / 中国生物制品学杂志

@#Objective To prepare bispecific antibody targeting cluster of differentiation 73(CD73) and programmed cell death-ligand 1(PD-L1),and evaluate its binding ability and killing ability in vitro.Methods Using genetic engineering method,PD-L1 single-chain fragment variable(scFv) was inserted into the hinge region of CD73 monoclonal antibody to construct anti-CD73/PD-L1 bispecific antibody(BS-21),which was screened by CHO GS expression system to obtain highly expressed cell line.After purified by Protein A and molecular sieve,the purity of antibody was detected by size exclusion chromatography-high performance liquid chromatography(SEC-HPLC),the binding ability of antibody in vitro was detected by flow cytometry,and the killing ability in vitro was detected by using peripheral blood mononuclear cell(PBMC) to kill Calu 1 lung cancer cells in vitro.Results High-yield cell lines were obtained by pressure screening.A bispecific antibody BS-21 with a purity of 99.6% was obtained by purification,which bound to CD73 and PD-L1 molecules simultaneously.Compared with anti CD73 and anti PD-L1 groups,BS-21 group significantly increased the killing rate of immune cells to Calu 1 tumor cells(F=30.36,each P<0.001).Conclusion Bispecific antibody BS-21 reduced the immunosuppressive effect of CD73 and PD-Ll on immune cells simultaneously,and showed good anti-tumor function.

Effect of electroacupuncture on expression of CREB and its ability to bind to synaptic proteins in amygdala and hippocampus of rats with post-traumatic stress disorder / 针刺研究

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the expression of cAMP-response element binding protein (CREB, a key protein for BDNF-TrkB signaling) and it's blinding ability to synaptic key protein in the amygdala and hippocampus of rats with post-traumatic stress disorder (PTSD), so as to lay a foundation for further study of the interaction mechanism between BDNF-TrkB signaling and synaptic plasticity. METHODS: Twenty-four male SD rats were randomly divided into blank, model and electroacupuncture (EA) groups, with 8 rats in each group. The PTSD model was established by psychological stress (bondage) and physiological stress (forced swimming and anesthesia). After modeling, EA (2 Hz/100 Hz, 1 mA) was applied to "Baihui"(GV20) "Shenting"(GB24) and bilateral "Shenshu"(BL23) for 20 min, once daily for 21 days. The behavioral changes (spontaneous locomotor within 30 min and contextual fear conditioning tests in 7 days) were detected by using a spontaneous locomotor detection box, and a conditioned fear response test chamber, respectively. The expression of CREB was detected by immunohistochemistry and Western blot, separately. The binding abilities of CREB to synaptic proteins (post synaptic density 95 [PSD95], synaptophysin [SYN] and growth-associated protein 43 [GAP43]) were verified by chromatin-immunoprecipitation (CHIP) technique. RESULTS: After modeling, the spontaneous locomotor distance, the expression levels of CREB and the binding ability of CREB to PSD95 protein in the amygdala and hippocampus were significantly decreased (P0.05). CONCLUSION: EA can improve the motor activity in PTSD rats, which may be associated with its effect in increasing the binding ability of CREB to the synaptic key protein PSD95 to regulate the interaction between the synaptic plasticity and BDNF-TrkB signaling pathway of the amygdala and hippocampus.

Effect of electroacupuncture on expression of CREB and its ability to bind to synaptic proteins in rats with post-traumatic stress disorder

Objective: To observe the effects of electroacupuncture on the expression of CREB and its ability to blind to synaptic key proteins in rats with post-traumatic stress disorder (PTSD), thus to lay the foundation for further study of the mechanism of electroacupuncture treatment of PTSD (i.e., electroacupuncture promotes the interaction between synaptic plasticity and BDNF-TRKB signaling pathway). Methods: SD male rats were randomly divided into blank group, model group and electroacupuncture treatment group, with 8 rats in each group. The model of PTSD was established by single continuous stress method (this modeling method is dominated by forced drowning, and tropical disasters are also mostly related to drowning). After modeling, the treatment group received electroacupuncture therapy, followed by retaining the needles for 20 min. The treatment was conducted once daily for 21 d. The behavioral changes were evaluated by spontaneous activity and conditional fear reaction. The expression of CREB was detected by immunohistochemistry and Western Blot. The binding ability of CREB to synaptic protein was verified by CHIP. Results: Compared with the model group, the spontaneous activity distance of the treatment group rats increased (P<0.01) and the percentage of stiffness time decreased (P<0.01), the expression of CREB protein in the treatment group was significantly increased (P<0.01), the binding ability of CREB to PSD95 protein was increased in treatment group (P<0.05). Conclusions: Electroacupuncture regulates the interaction mechanism between synaptic plasticity and BDNFTrkB signaling pathway of the rat model of PTSD, most likely by increasing the binding ability of CREB to the key synaptic protein-PSD95.

Single B cell sorting-based amplification and functional identification of HIV-1 monoclonal antibody genes / 中华微生物学和免疫学杂志

Objective@#To amplify and identify monoclonal antibody genes from HIV-1-infected patients.@*Methods@#Single cell sorting was used to isolate antigen-specific single B cells. Sequence Identity Matrix and the international ImMunoGeneTics information system were used to analyze antibody variable region genes. Binding abilities were detected by enzyme linked immunosorbent assay. Neutralizing activities were tested by TZM-bl/pseudovirus assay.@*Results@#The heavy and light chain genes of four, seven, and eleven antibodies were amplified and sequenced from three HIV-1-infected patients, respectively. They were derived from various germline genes with flexible CDR3 lengths and somatic mutations. A1 and B3 antibodies bound to HIV-1 clade B, CRF01_AE, and CRF07_BC antigens. The half maximal inhibitory concentration values of A1 and B3 against MW965 virus were 0.04 μg/ml and 37.34 μg/ml.@*Conclusion@#In this study, we acquired a lot of monoclonal antibody genes and two HIV-1 monoclonal binding and neutralizing antibodies, which would provide basic data for further research on monoclonal antibody identification.

Streptococcus pneumoniae StkP kinase: binding ability with β-lactam antibiotics and correlation with drug resistance / 中华微生物学和免疫学杂志

Objective To investigate the correlation between Streptococcus pneumoniae (S.pneumoniae) StkP kinase and drug resistance and to analyze the binding ability of StkP extracellular region (EC-StkP) to β-lactam antibiotics.Methods A stkP gene knockout (ΔstkP) mutant was constructed from S.pneumoniae strain ATCC6306 by insertional inactivation method.E-test was performed to detect the minimum inhibitory concentrations (MIC) of penicillin (PCN) and cefotaxime (CTX) against ΔstkP mutant and its wild-type strain.Bioinformatic softwares were used to predict the EC-StkP of S.pneumonia strain ATCC6306,to generate the three-dimensional structure model of EC-StkP and to analyze the correlation between the structure and functions of EC-StkP.PCR was performed to amplify the extracellular segment of stkP (EC-stkP) gene and the product of it was sequenced after T-A cloning.A prokaryotic expression system of EC-stkP gene was constructed.SDS-PAGE in combination with a gel image analysis system was used to detect the expression of the recombinant EC-StkP (EC-rStkP).The expressed EC-rStkP was extracted by Ni-NTA affinity chromatography.The binding abilities of EC-rStkP to PCN and CTX were detected by isothermal titration calorimetry (VT-ITC) and surface plasmon resonance (Biacore).Results S.pneumonia strain ATCC6306 was sensitive to PCN (MIC=0.06 μg/ml) and CTX (MIC=0.12 μg/ml),but its ΔstkP mutant was resistant to the two antibiotics (PCN MIC=16 μg/ml,CTX MIC=32 μg/ml).The 295 aa segment was predicted as the extracellular region at C-end of StkP of S.pneumoniae strain ATCC6306,containing four penicillin-binding proteins and Ser/Thr kinase-associated (PASTA) domains.The cloned EC-stkP segment and the EC-stkP segment in GenBank shared 99.6% similarity in nucleotide sequence and 100% in amino acid sequence.The constructed prokaryotic expression system for EC-stkP gene expressed EC-rStkP in soluble form.Both PCN and CTX could bind to EC-rStkP and CTX was better than PCN in term of binding ability.Conclusion The stkP gene of S.pneumonia is closely related to drug resistance and the encoded protein,Ser/Thr kinase StkP,can recognize and bind to β-lactam antibiotics.

Study on Fused Expression of the IgG-binding Domain of Streptococcal Protein G and Its IgG-binding Activity / 中国生物工程杂志

The IgG binding domain of Streptococcal Protein G which can selectively immobilizes the Fc regions of immunoglobulin G(IgG) is a kind of good material for oriented immobilization of antibodies in antibody microarrays.Here,genetically engineered three glutathione S-transferase(GST) fused proteins,bearing one,two and three B-Domains respectively(GST-GBx).The IgG-bindding ability of GST-GBx was investigated by ELISA.The data revealed that when the B-domain's quantity of GST-GBx is identical,the GST-GB3 is the most efficient protein among three GST-GBx protein both the capacity and sensibility of binding IgG.The GST-GB2 is the next one and GST-GB1 is the least one.Thus,the GST-GB3 has significantly predominance in comparison to GST-GB2 and GST-GB1.