Expression and in vitro activity of a neutralizing antibody against West Nile virus that reduces antibody-dependent enhancement / 中华微生物学和免疫学杂志

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.

Establishment of an in vivo neutralization model based on H5N1 pseudovirus / 中华微生物学和免疫学杂志

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.

Preparation and identification of a broad-spectrum antibody targeting hemagglutinin protein of influenza A virus / 中华微生物学和免疫学杂志

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.

Preparation and identification of antibodies targeting influenza A virus N1 subtype neuraminidase / 中华微生物学和免疫学杂志

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.

Assessing Hemocompatibility of Transfusion Sets for Single Use / 中国输血杂志

【Objective】 To investigate the blood compatibility of transfusion sets for single use, the loss, damage and activation of blood components after passage through the transfusion sets. 【Methods】 Transfusion sets (sample A and B) were assessed by comparing samples of blood component taken prior to and after passage through. The following makers of damage/activation were evaluated: Red blood cells (RBCs)-supernatant free hemoglobin (FHb) and potassium(K+ ) amount; platelet concentrates (PCs)-pH, hypotonic shock response (HSR), supernatant lactate dehydrogenase(LDH), CD62P; fresh frozen plasma(FFP) – prothrombin fragments 1 and 2 (F1+ 2), fibrinopeptide A (FPA), coagulation factor Ⅻa (FⅫa), Thrombin-antithrombin complex (TAT). 【Results】 After passing through two types of transfusion sets, the loss of RBCs, PCs and FFP were less than 5%. There is no statistic difference for the change of FHb and K+ of RBCs(P>0.05). There were no statistics for pH, LDH(U/L) and CD62P(%) after platelet concentrates passing through both transfusion sets. There was no statistics for the HSR after passing through the sample A, while there was a small but statistically significant increase in the HSR of sample B (37.17±6.49 vs 40.75±6.24, P<0.05). After FFP passing through the transfusion giving sets, there were no statistical difference for F1+ 2, FPA, FⅫa and TAT (P>0.05). 【Conclusion】 Two types of transfusion sets caused negligible effects on RBCs, platelet concentrates and FFP.

Axl-targeting drugs in tumor therapy:research advances / 国际药学研究杂志

Axl belongs to the family of receptor tyrosine kinase. Axl and its ligand Gas6 are aberrantly over-expressed in numerous human cancers. The binding of Gas6 and Axl could activate multiple signaling pathways,which take part in the development and progression of cancer,including cell survival,migration,invasion,and angiogenesis. In recent years,Axl and Gas6 have been considered as a promising novel target for cancer therapeutics. Several Axl inhibitors,including small-molecule tyrosine kinase inhibi?tors and anti-Axl monoclonal antibodies are currently under research. In this paper ,we review the development of Axl-targeting drugs in tumor therapy.

Effect of amifostine on proliferation and differentiation of human megakaryocyte Dami cells / 中国药理学与毒理学杂志

OBJECTIVE To investigate the effect of amifostine(Amf)on the differentiation of human megakaryocyte cell line-Dami. METHODS Dami cells were treated with Amf 0.01-5.0 mmol · L-1 for 12 d. Dami cells were counted every day for the growth curve:only cells with a diameter>20μm. The platelet demarcation membrane system was observed by transmission electron microscopy. The expression of CD33,CD34,CD41a and DNA ploidy was detected by flow cytometry. RESULTS Amf 0.1-1.0 mmol · L-1 promoted the differentiation of Dami cells ,but inhibited their proliferation at a concentration>1.0 mmol · L-1. When these cells were treated with Amf 1.0 mmol · L-1 for 12 d,the platelet demarcation membrane system was observed,the percentage of cells with a diameter >20 μm was increased by 24.6%(P1.0 mmol·L-1).

Evaluation and applications of antibody variable stability / 军事医学

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 .

Establishment and identification of stable cell line secreting humen IgE Cε-4 protein / 中华微生物学和免疫学杂志

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.

INHIBITION OF OXIDIZED LIPOPROTEINS INDUCED APOPTOSIS OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS BY TEA POLYPHENOLS / 营养学报

Objective: To study the inhibition and mechanism of tea polyphenols (TP) on apoptosis induced by oxidized low density lipoprotein (ox-LDL) in human umbilical vein endothelial cells (HUVEC). Method: HUVEC cells were administered with TP (25 ?g/ml), ox-LDL (200 ?g/ml)+TP (25 ?g/ml), ox-LDL (200 ?g/ml) respectively, cells with equal volume of solvent as control. Cell viability was determined by MTT assay, apoptosis by acridine orange fluorescence dyeing, and expressions of Bcl-2、Bax and caspase-3 were analyzed by Western blotting. Results: Ox-LDL could inhibit HUVEC cell proliferation. After treated with both TP and ox-LDL , the cell proliferation increased obviously and showed significant difference from the cells treated with ox-LDL only (P