Objective To investigate prokaryotic expression of the antigen sequence (amino acids 59-145) of mouse leukemia-related protein 16 (LRP16) protein and preparation of rabbit anti-mouse LRP16 polyclonal antibody. Methods The prokaryotic expression plasmid pLS962-LRP16 was constructed by the molecular cloning method and transferred into E.coli Rosetta to express LRP16 protein induced by IPTG. The recombinant protein was purified using Ni-NTA affinity columns followed by gel filtration chromatography. New Zealand white rabbits were immunized with the purified antigen to generate polyclonal antiserum, with antibody titer quantified by ELISA. Antigen-specific IgG was affinity-purified using Sepharose-coupled LRP16 and validated through Western blot and immunofluorescence assays. Results SDS-PAGE analysis confirmed insoluble expression of the LRP16 fusion protein as inclusion bodies. ELISA demonstrated exceptional antiserum titer (1:256 000). Western blot and immunofluorescence verified that the polyclonal antibody could specifically recognize endogenous LRP16 in murine tissues. Conclusion The prokaryotic expression of the LRP16 gene is successfully achieved, and the rabbit anti-mouse LRP16 polyclonal antibody exhibiting high specificity is prepared. This lays the foundation for further studies on the function of the LRP16 gene.
Animals ; Rabbits ; Mice ; Antibodies/immunology* ; Escherichia coli/metabolism* ; Enzyme-Linked Immunosorbent Assay ; Blotting, Western ; Antibody Specificity

Corticotomy is a clinical procedure to accelerate orthodontic tooth movement characterized by the regional acceleratory phenomenon (RAP). Despite its therapeutic effects, the surgical risk and unclear mechanism hamper the clinical application. Numerous evidences support macrophages as the key immune cells during bone remodeling. Our study discovered that the monocyte-derived macrophages primarily exhibited a pro-inflammatory phenotype that dominated bone remodeling in corticotomy by CX3CR1^CreERT2; R26^GFP lineage tracing system. Fluorescence staining, flow cytometry analysis, and western blot determined the significantly enhanced expression of binding immunoglobulin protein (BiP) and emphasized the activation of sensor activating transcription factor 6 (ATF6) in macrophages. Then, we verified that macrophage specific ATF6 deletion (ATF6^f/f; CX3CR1^CreERT2 mice) decreased the proportion of pro-inflammatory macrophages and therefore blocked the acceleration effect of corticotomy. In contrast, macrophage ATF6 overexpression exaggerated the acceleration of orthodontic tooth movement. In vitro experiments also proved that higher proportion of pro-inflammatory macrophages was positively correlated with higher expression of ATF6. At the mechanism level, RNA-seq and CUT&Tag analysis demonstrated that ATF6 modulated the macrophage-orchestrated inflammation through interacting with Tnfα promotor and augmenting its transcription. Additionally, molecular docking simulation and dual-luciferase reporter system indicated the possible binding sites outside of the traditional endoplasmic reticulum-stress response element (ERSE). Taken together, ATF6 may aggravate orthodontic bone remodeling by promoting Tnfα transcription in macrophages, suggesting that ATF6 may represent a promising therapeutic target for non-invasive accelerated orthodontics.
Animals ; Mice ; Macrophages/metabolism* ; Tumor Necrosis Factor-alpha/genetics* ; Tooth Movement Techniques/methods* ; Activating Transcription Factor 6/metabolism* ; Bone Remodeling ; Flow Cytometry ; Blotting, Western

Objective To evaluate the correlation between alterations in DNase1 and DNase1L3 enzyme activities and impairment of NET degradation in patients with sporadic SLE, and to investigate the underlying mechanism. Methods 46 sporadic SLE patients and 30 age- and sex-matched healthy individuals were recruited. Serum levels of DNase1, DNase1L3 and corresponding autoantibodies were detected by ELISA. DNase1 and DNase1L3 were isolated by immunoprecipitation; NETs and enzyme degradation activities were detected using a modified immunofluorescence. DNase1L3 secretion by PBMCs was analyzed by ELISPOT, Western blotting and reverse transcription PCR. Results Levels of H3-dsDNA and Ela-dsDNA complexes were significantly elevated in SLE patients. LDGs in SLE population was significantly higher than in the control group, and LDGs was positively correlated with H3-dsDNA and Ela-dsDNA NETs complexes. The ability of SLE patients to degrade NET in vitro was significantly lower than that of the control group. Degradation experiments of DNase1 and DNase1L3 in different proportions showed that the decrease in DNase1L3 activity was the primary contributor to the elevated NET residue level. The concentration of DNase1L3 autoantibodies in SLE patients was significantly elevated compared to the control group. In addition, the capacity of PBMCs to secrete DNase1L3 was significantly lower in the SLE patients compared to the control group. Conclusion Decreased secretion of DNase1L3 and the presence of relevant autoantibodies notably impede NET degradation in patients with SLE, offering new directions for the monitoring and treatment of SLE patients.
Humans ; Autoantibodies ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Extracellular Traps ; Lupus Erythematosus, Systemic

Plant bioreactor is a new production platform for expression of recombinant protein, which is one of the cores of molecular farming. In this study, the anti DYKDDDDK (FLAG) antibody was recombinantly expressed in tobacco (Nicotiana benthamiana) and purified. FLAG antibody with high affinity was obtained after immunizing mice for several times and its sequence was determined. Based on this, virus vectors expressing heavy chain (HC) and light chain (LC) inoculated into Nicotiana benthamiana leaves by using Agrobacterium-mediated delivery. Accumulation of the HC and LC was analyzed by SDS/PAGE followed by Western blotting probed with specific antibodies from 2 to 9 days postinfiltration (dpi). Accumulation of the FLAG antibody displayed at 3 dpi, and reached a maximum at 5 dpi. It was estimated that 66 mg of antibody per kilogram of fresh leaves could be obtained. After separation and purification, the antibody was concentrated to 1 mg/mL. The 1:10 000 diluted antibody can probe with 1 ng/mL FLAG fused antigen well, indicating the high affinity of the FLAG antibody produced in plants. In conclusion, the plant bioreactor is able to produce high affinity FLAG antibodies, with the characteristics of simplicity, low cost and highly added value, which contains enormous potential for the rapid and abundant biosynthesis of antibodies.
Animals ; Mice ; Antibodies ; Nicotiana/genetics* ; Agrobacterium/genetics* ; Bioreactors ; Blotting, Western

Objective To prepare and identify rabbit anti-cyclin dependent kinase 6 (CDK6) antibody. Methods The recombinant pET21a (+)/CDK6 was successfully constructed, then the recombinant plasmid was transformed into E.coli BL21 (DE3) competent cells and was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) for protein expression, which was detected by SDS-PAGE and Western blot analysis. The expressed protein was purified by nickel-chelating nitrilotriacetic acid (Ni-NTA) agarose and then analyzed by SDS-PAGE. Japanese white rabbits were immunized with purified CDK6 protein for many times every two weeks. The blood was collected at 0, 2, 4 and 6 weeks after immunization, and serum was separated from blood. The titer was detected by indirect ELISA. Western blot analysis, immunofluorescence assay and immunohistochemistry were employed to determine the specificity. Results High purity CDK6 protein and high specificity of rabbit anti-CDK6 antibody were successfully prepared. The titer of CDK6 rabbit serum antibody reached 1:30 000 after immunization, which could specifically recognize the CDK6 protein expressed in cervical cancer cell line and cervical cancer tissues. Conclusion The high titer and specificity of rabbit anti-CDK6 antibody is successfully prepared.
Animals ; Female ; Humans ; Rabbits ; Antibodies ; Antibody Specificity ; Blotting, Western ; Cyclin-Dependent Kinase 6 ; Enzyme-Linked Immunosorbent Assay ; Uterine Cervical Neoplasms

Objective To prepare rabbit polyclonal antibody specifically against human lactate dehydrogenase C4 (LDHC4). Methods Site-directed mutation was performed by PCR to generate the mutated LDHC gene, and the mutated gene was ligated into the pET-28a vector to form the pET-28a-LDHC recombinant expression vector. The recombinant vector was introduced into E. coli BL21 (DE3), and LDHC4 protein was obtained by induced expression. The recombinant protein was used as an antigen to immunize New Zealand rabbits, and the antiserum was obtained after three boosted immunizations. The titer of the antiserum against LDHC4 were detected by ELISA. Western blot was used to detect the specificity of the antiserum, and immunohistochemistry was used to detect the expression of LDHC4 in human triple-negative breast cancer tissue. Results A specific rabbit anti-human LDHC4 polyclonal antibody was obtained with an antibody titer of 1:51 200. The antibody can be used for Western blot and immunohistochemistry. Conclusion The specific rabbit anti-human LDHC4 polyclonal antibody is successfully prepared.
Humans ; Rabbits ; Animals ; Escherichia coli/genetics* ; Antibodies ; Enzyme-Linked Immunosorbent Assay ; L-Lactate Dehydrogenase/metabolism* ; Blotting, Western ; Antibody Specificity

OBJECTIVE: To investigate the changes in gray matter volume in depressive-like mice and explore the possible mechanism. METHODS: Twenty-four 6-week-old C57 mice were randomized equally into control group and model group, and the mice in the model group were subjected to chronic unpredictable mild stimulation (CUMS) for 35 days. Magnetic resonance imaging was performed to examine structural changes of the grey matter volume in depressive-like mice. The expression of brain-derived neurotrophic factor (BDNF) in the grey matter of the mice was detected using Western blotting and immunofluorescence staining. RESULTS: Compared with the control mice, the mice with CUMS showed significantly decreased central walking distance in the open field test (P < 0.05) and increased immobile time in forced swimming test (P < 0.05). Magnetic resonance imaging showed that the volume of the frontal cortex was significantly decreased in CUMS mice (P < 0.001, when the mass level was greater than or equal to 10 756, the FDRc was corrected with P=0.05). Western blotting showed that the expression of mature BDNF in the frontal cortex was significantly decreased in CUMS mice (P < 0.05), and its expression began to decrease after the exposure to CUMS as shown by immunofluorescence staining. The volume of different clusters obtained by voxel-based morphometry (VBM) analysis was correlated with the expression level of mature BDNF detected by Western blotting (P < 0.05). CONCLUSION The decrease of frontal cortex volume after CUMS is related with the reduction of mature BDNF expression in the frontal cortex.
Animals ; Mice ; Blotting, Western ; Brain-Derived Neurotrophic Factor ; Cerebral Cortex ; Depression/physiopathology* ; Frontal Lobe/pathology*

OBJECTIVE: To investigate the prognostic value of death-associated protein 5 (DAP5) in gastric cancer (GC) and its regulatory effect on aerobic glycolysis in GC cells. METHODS: We analyzed DAP5 expression levels in GC and adjacent tissues and its association with survival outcomes of GC patients using public databases. We collected paired samples of GC and adjacent tissues from 102 patients undergoing radical resection of GC in our hospital from June, 2012 to July, 2017, and analyzed the correlation of DAP5 expression level detected immunohistochemically with the clinicopathological parameters of the patients. Cox regression analysis, Kaplan-Meier analysis, and ROC curves were used to explore the independent risk factors and the predictive value of DAP5 expression for 5-year survival of the patients. In the cell experiments, we observed the changes in aerobic glycolysis in MGC-803 cells following lentivirus-mediated DAP5 knockdown or overexpression by measuring glucose uptake and cellular lactate level and using qRT-PCR and Western blotting. RESULTS: Analysis using the public databases showed that DAP5 was highly expressed in GC and correlated with tumor progression and poor survival outcomes of the patients (P < 0.05). In the clinical samples, DAP5 expression was significantly higher in GC than in the adjacent tissues (3.19±0.60 vs 1.00±0.12; t=36.863, P < 0.01), and a high expression of DAP5 was associated with a reduced 5-year survival rate of the patients (17.6% vs 72.5%; χ²=29.921, P < 0.05). A high DAP5 expression, T3-4, N2-3, and CEA≥5 ng/mL were identified as independent risk factors affecting 5-year survival outcomes of GC (P < 0.05), for which DAP5 expression showed a prediction sensitivity, specificity and accuracy of 73.2%, 80.4% and 79.0%, respectively. In MGC-803 cells, DAP5 knockdown significantly reduced glucose uptake, lactate level and the expressions of GLUT1, HK2 and LDHA, and DAP5 overexpression produced the opposite effects (P < 0.05). CONCLUSION A high expression of DAP5 in GC, which enhances cellular aerobic glycolysis to promote cancer progression, is correlated with a poor survival outcome and may serve as a biomarker for evaluating long-term prognosis of GC patients.
Humans ; Stomach Neoplasms ; Blotting, Western ; Databases, Factual ; Glucose ; Lactates

c-Myc protein encoded by c-Myc (cellular-myelocytomatosis viral oncogene) gene regulates the related gene expression through the Wnt/β-catenin signaling pathway, and has received extensive attention in recent years. The purpose of this study was to express Helicoverpa armigera c-Myc gene (Ha-c-Myc) by using prokaryotic expression system, prepare the polyclonal antibody, examine the spatio-temporal expression profile of Ha-c-Myc, and investigate the possible function of Ha-c-Myc in regulating H. armigera sterol carrier protein-2 (SCP-2) gene expression. The Ha-c-Myc gene was amplified by PCR and cloned into a prokaryotic expression plasmid pET-32a(+). The recombinant plasmid pET-32a-Ha-c-Myc was transformed into Escherichia coli BL21. IPTG was used to induce the expression of the recombinant protein. Protein was purified by Ni²⁺-NTA column and used to immunize New Zealand rabbits for preparing the polyclonal antibody. The Ha-c-Myc expression levels in different developmental stages (egg, larva, prepupa, pupa, and adult) of H. armigera and different tissues (midgut, fat body, head, and epidermis) of the prepupa were determined by real-time quantitative reverse transcription PCR (qRT-PCR). Ha-c-Myc siRNA was synthesized and transfected into H. armigera Ha cells. The relative mRNA levels of Ha-c-Myc and HaSCP-2 in Ha cells were detected by qRT-PCR. Results showed that the pET-32a-Ha-c-Myc recombinant plasmid was constructed. The soluble Ha-c-Myc protein of about 65 kDa was expressed in E. coli. The polyclonal antibody was prepared. Western blotting analysis suggested that the antibody had high specificity. Enzyme linked immunosorbent assay (ELISA) showed that the titer of the antibody was high. Ha-c-Myc gene expressed at all developmental stages, with high levels in the early and late instars of larva, and the prepupal stage. Tissue expression profiles revealed that Ha-c-Myc expressed in various tissues of prepupa, with high expression level in the midgut, but low levels in the epidermis and fat body. RNAi results showed that the knockdown of Ha-c-Myc expression significantly affected transcription of HaSCP-2, leading to a 50% reduction in HaSCP-2 mRNA expression level. In conclusion, the Ha-c-Myc was expressed through a prokaryotic expression system, and the polyclonal anti-Ha-c-Myc antibody was obtained. Ha-c-Myc may promote the expression of HaSCP-2 and play an important role in the lipid metabolism of H. armigera. These results may facilitate further study on the potential role and function mechanism of Ha-c-Myc in H. armigera and provide experimental data for exploring new targets of green pesticides.
Animals ; Rabbits ; Escherichia coli/metabolism* ; Enzyme-Linked Immunosorbent Assay ; Moths/genetics* ; Blotting, Western ; Larva/genetics* ; Isoantibodies/metabolism* ; Antibody Specificity

Objective To investigate antigen optimization of Shisa like protein 1 (SHISAL1) for preparing mouse anti-human SHISAL1 polyclonal antibody and to identify the specificity of the prepared antibody. Methods Bioinformatics was employed to predict the antigenic epitope region of SHISAL1 protein, and then a polypeptide composed of amino acid residues from the site of 28 to 97 of SHISAL1, termed SHISAL1-N, was selected as the antigen. The coding region of SHISAL1-N was cloned by molecular cloning technique, and then it was inserted into pET-28a to generate pET28a-SHISAL1-N recombinant plasmid. The two recombinant plasmids pET28a-SHISAL1-N and pET28a-SHISAL1 were transformed into BL21 (DE3) bacteria and induced to express by IPTG. The two proteins were purified and immunized to female Kunming mice, respectively. The specificities and sensitivities of the acquired antibodies were detected by Western blot analysis, immunoprecipitation and immunofluorescent cytochemical staining. Results pET28a-SHISAL1-N recombinant plasmid was successfully constructed, and the two fused proteins, SHISAL1 and SHISAL1-N, were induced to express. Moreover, two types of SHISAL1 mouse polyclonal antibodies, derived from SHISAL1-N and SHISAL1 antigens, were obtained. Western blot results showed that the antibody prepared from SHISAL1 antigen was less specific and sensitive compared with the antibody prepared from SHISAL1-N antigen which could specifically identify different endogenous SHISAL1 protein. Immunoprecipitation results showed that SHISAL1-N antibody could specifically pull down SHIISAL1 protein in hepatocellular carcinoma cells and immunofluorescence results demonstrated that SHISAL1-N antibody could specifically bind to SHISAL1 protein in the cytoplasm. Conclusion We have optimized the SHISAL1 antigen and prepared the mouse anti-human SHISAL1 polyclonal antibodies successfully, which can be used for Western blot analysis, immunoprecipitation and immunofluorescence cytochemical staining.
Animals ; Female ; Humans ; Mice ; Antibodies ; Antibody Specificity ; Blotting, Western ; Cloning, Molecular ; Epitopes/genetics*