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OBJECTIVE To evaluate the effectiveness， safety， economy， innovation， suitability and accessibility of recombinant Mycobacterium tuberculosis fusion protein （EC）， and to provide evidence for selecting skin detection methods for tuberculosis infection diagnosis and auxiliary diagnosis of tuberculosis. METHODS The effectiveness and safety of EC compared with purified protein derivative of tuberculin （TB-PPD） were analyzed by the method of systematic review. Cost minimization analysis， cost-effectiveness analysis and cost-utility analysis were used to evaluate the short-term economy of EC compared with TB-PPD， and cost-utility analysis was used to evaluate the long-term economy. The evaluation dimensions of innovation， suitability and accessibility were determined by systematic review and improved Delphi expert consultation， and the comprehensive score of EC and TB-PPD in each dimension were calculated by the weight of each indicator. RESULTS The scores of effectiveness， safety， economy， innovation and suitability of EC were all higher than those of TB-PPD. The affordability scores of the two drugs were consistent， while the availability score of EC was lower than those of TB-PPD. After considering dimensions and index weight， the scores of effectiveness， safety， economy， innovation， suitability， accessibility and the comprehensive score of EC were all higher than those of TB-PPD. CONCLUSIONS Compared with TB-PPD， EC performs better in all dimensions of effectiveness， safety， economy， innovation， suitability and accessibility. However， it is worth noting that EC should further improve its availability in the dimension of accessibility.
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Background Neurotransmitter secretion disorder induced by chronic manganese poisoning has always been one of the important causes of body injury, but the mechanism of neurotransmitter secretion disorder caused by manganese is not clear at present. Objective To investigate the effects of presynaptic membrane intracellular protein 13-1 (Munc13-1) and synapse fusion protein binding protein 18-1 (Munc18-1) on dopamine secretion dysfunction induced by manganese chloride (MnCl2) in human neuroblastoma (SH-SY5Y) cells. Methods A SH-SY5Y cell model induced by MnCl2 was established. Cell viability was measured by MTT assay. Four experimental groups were set up: control group and low-, medium-, and high-dose manganese groups (0, 100, 200, and 400 μmol·L−1 MnCl2). They were treated with corresponding doses of MnCl2 for 24 h. The secretion of dopamine was measured by enzyme-linked immunosorbent assay. The mRNA expression of Syntaxin-1 was detected by real-time quantitaive PCR. Total cell proteins were extracted, and the protein expression levels of Munc13-1, Munc18-1, and Syntaxin-1 were detected by Western blotting. The correlations of MnCl2 exposure and dopamine secretion with the protein expressions of Munc13-1 and Munc18-1 were also analyzed by Pearson correlation. Results Compared with the control group, the cell viability rate decreased gradually with the increase of manganese exposure concentration, and the difference between the medium- and the high-dose manganese groups was statistically significant (P<0.05). The concentration of dopamine in cell culture medium of all manganese exposure groups decreased with the increase of manganese concentration, and compared with the control group and the low-dose manganese group, the medium- and the high-dose manganese groups were statistically significant (P<0.05). The expression of Syntaxin-1 at mRNA or protein level did not change significantly among groups (P>0.05). Compared with the control group, the protein expression of Munc13-1 decreased and that of Munc18-1 increased with the increase of manganese concentration (P<0.05). Compared with the low-dose manganese group, the changes of Munc13-1 protein in the high-dose manganese group and Munc18-1 protein in the medium- and high-dose manganese groups had statistical significance (P<0.05). Compared with the medium-dose manganese group, the protein changes of Munc18-1 in the high-dose manganese group were statistically significant (P<0.05). The correlation analysis showed that MnCl2 dose was negatively correlated with Munc13-1 protein expression (r=−0.898, P<0.05), and positively correlated with Munc18-1 protein expression (r=0.678, P<0.05). Dopamine secretion was positively correlated with Munc13-1 protein expression (r=0.932, P<0.05), and negatively correlated with Munc18-1 protein expression (r=−0.817, P<0.05). Conclusion The inhibition of dopamine secretion in SH-SY5Y cells induced by manganese exposure is related to up-regulation of Munc18-1 and down-regulation of Munc13-1 expression levels, which may be one of the reasons for nerve injury caused by manganese.
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@#Objective To express the fusion protein ABD-Fc-IL-2 in eukaryotic cells and detect its biological activity. Methods The target gene SP-ABD-Fc-IL-2 was amplified by direct and overlapping PCR,and then ligated to vector pcDNA3. 1(+).The obtained recombinant plasmid pcDNA3. 1/SP-ABD-Fc-IL-2 was transiently transfected into CHO-S cells to express the fusion protein ABD-Fc-IL-2,which was purified by Protein A beads affinity chromatography. The specificity of the purified fusion protein was detected by Western blot,the biological activity was detected by CTLL-2/MTT cell proliferation colourimetry,and the interaction between ABD fragment and human serum albumin(HSA)was detected by pull down/Western blot. Results The recombinant plasmid pcDNA3. 1/SP-ABD-Fc-IL-2 was constructed correctly as identified by restriction analysis and sequencing. The purified fusion protein ABD-Fc-IL-2 showed a purity of 90% and bound specifically to mouse anti-IL-2 monoclonal antibody with the biological activity of 3. 29 × 108IU/mL. The ABD of fusion protein and HSA bound to each other. Conclusion The eukaryotic fusion protein ABD-Fc-IL-2 had high biological activity,which promoted the proliferation of CTLL-2 cells and maintained the binding ability of ABD fragment to HSA.
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@#Objective To optimize the expression of recombinant human growth hormone-Fc(rhGH-Fc)fusion protein in CHO cells in order to obtain better glycosylation ratio and lower content of highmannose.Methods CHO cells expressing rhGH-Fc were cultured in a 7 L bioreactor.The glycosylation modifications of rhGH-Fc were adjusted by improving the composition of feeding media(using three commercial media:Gly-1:EX-CELL Glycosylation Adjust,Gly-2:SHEFF-CHO PLUS PG ACF and Gly-3:EfficientFeed C + AGT Supplement & GlycanTune C + Total Feed),and the glycosylation type and proportion of the target proteins were analyzed by mass spectrometry.Results The G0F(main glycosylation types:G0,G1 and G2;F:fucose)of Gly-1,Gly-2 and Gly-3 were 32.89%,58.66% and 33.28%,the G1F were 31.39%,18.03%and 34.90%,and the G2F were 31.39%,18.03% and 34.90%,respectively.Gly-1 and Gly-3 made the target protein contain less G0F while more G2F;Gly-3 feeding scheme-showed less high mannose modification than the other two schemes.Conclusion Gly-1 medium changed the glycosylation modification from G0F to G1F and G2F,while Gly-2 medium changed that from G2F and G1F to G0F.However,Gly-3 medium changed the glycosylation modification from G0F to G1F and G2F,and the contentof high mannose was less than 5%,which may have a better effect on modifying glycosylation type and proportion of the target protein.
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【Objective】 To investigate the effects of the loss of exon 1 of TFE3 on nuclear localization of chimeric TFE3 protein in TFE3 translocation renal cell carcinoma (TFE3 tRCC). 【Methods】 The localization of TFE3 protein in TFE3 tRCC and clear cell renal cell carcinoma (ccRCC) were detected with immunochemistry. The exon retention of TFE3 gene in TFE3 tRCC was analyzed in databases and literatures. The plasmids containing TFE3 full-length and different-length of TFE3 exons which were constructed to pCDH-MCS-EGFP-Puro were transfected into HEK293T using Lipo FiterTM. The localization of EGFP protein in HEK293T cells were detected with confocal microscopy. The localization of TFE3 protein and truncated TFE3 protein were detected with Western blotting. The mRNA expression of the downstream genes of TFE3 protein were detected with q-PCR. 【Results】 Strong nuclear signal of TFE3 protein was observed in TFE3 tRCC, whereas TFE3 protein in ccRCC was mainly localized in cytoplasm. The results of fluorescence imaging and Western blotting showed that TFE3 full-length protein was expressed both in nucleus and cytoplasm, and the expression of truncated TFE3 protein was mainly localized in nucleus. The q-PCR analysis demonstrated that the deletion of exon 1 in TFE3 gene led to a higher transcriptional level of targeted genes of TFE3 protein. 【Conclusion】 The loss of exon 1 in TFE3 played a critical role in preventing TFE3 protein from entering the nucleus. In TFE3 tRCC, the loss of exon 1 in TFE3 gene leads to the nuclear localization of TFE3 fusion protein and activation of its downstream target genes. This mechanism promises to uncover the occurrence and development of TFE3 tRCC.
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【Objective】 To observe the penetration and biological activity of PTD4-Cu, Zn-SOD into human astrocytes and whether it can mitigate hypoxia damages. 【Methods】 ①Immunohistochemistry and fluorescence test: We labeled the Cu, Zn-SOD by a monoclonal antibody, combined it with the fluorescent secondary antibody labeled with fluorescein isothiocyanate (FITC) to observe the effect of transduced PTD4-Cu, Zn-SOD on the viability of human astrocytes. ② The experimental group: After hypoxic damage model, the cells were divided into three groups: blank control, group Cu, Zn-SOD, and group PTD4-Cu, Zn-SOD. Group blank was added with DMEM medium (excluding serum) as control; DMEM medium was added to the other two for one hour (excluding serum) with its fusion proteins (Cu, Zn-SOD and PTD4 -Cu, Zn-SOD) with the final concentration of 2 μmoL/L. After the intervention, we used SOD and MDA test kits to observe PTD4-Cu, Zn-SOD and Cu, Zn-SOD in astrocytes after fusion protein intervention. 【Results】 The PTD4-Cu, Zn-SOD fusion protein could have aggregation distribution in the nucleus by FITC fluorescently labeled. After the intervention, it could increase the SOD activity in astrocytes in group PTD4-Cu, Zn-SOD and group Cu, Zn-SOD compared with control group, but the SOD activity was more obvious in the fusion proteins PTD4-Cu, Zn-SOD group. And the dose of MDA was reduced in group PTD4-Cu, Zn-SOD compared with group Cu, Zn-SOD and control group. 【Conclusion】 PTD4-Cu, Zn-SOD fusion protein can transcellular membrane of human astrocytes. The fusion protein PTD4-Cu, Zn-SOD can increase the SOD activity and reduce the content of MDA by human astrocytes from hypoxia injury.
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@#Vaccines play an important role in the prevention and control of infectious diseases. Bacterial flagellin can activate Toll like receptor 5(TLR5) and NOD like receptor C4(NLRC4) in host cells,and has immune adjuvant effect. As a new immune adjuvant,flagellin can not only share with antigen protein,but also fuse with antigen protein,which can significantly improve the immune effect. At the same time,it has achieved good application effect in mucosal immunity and antitumor immunity,becoming a hot spot in the research and development of vaccine adjuvants. This review mainly discussed the research progress on application of bacterial flagellin as vaccine adjuvant,so as to provide new ideas for the development of flagellin adjuvant vaccine.
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@#Objective To develop and verify a whole-column image capillary isoelectric focusing(iCIEF) electrophoresis method to analyze the charge heterogeneity of recombinant human growth hormone Fc fusion protein(Fc-rhGH).Methods The iCIEF analysis method of Fc-rhGH was developed by optimizing the target protein concentration,cosolvent(urea)concentration and focusing time.The target protein was simultaneously analyzed by this method and traditional flat plate isoelectric focusing(IEF) electrophoresis,and the results were compared;The specificity,accuracy,precision,limit of quantitation(LOQ) and durability of the developed method were verified.Results The optimized method was using the mixed solution of 8 mol/L urea,0.35% methyl cellulose(MC),4% amphoteric electrolyte and 0.5% isoelectric point marker as the sample buffer,and the focusing condition was 1 500 V 1 min,3 000 V 5.5 min.IEF was not suitable for analyzing the charge heterogeneity of Fc-rhGH solution.Using the optimized iCIEF for analysis,the target protein was significantly different from the unrelated protein,and the baseline of blank reagent was stable;The recovery rate of accuracy verification was within 90%～110%,and the linear range was 0.25～0.75 mg/mL(50%～150% of the target loading volume);The RSD of each isomer pI in the repeatability verification was less than 0.3%,and the RSD of peak area percentage was less than 5%;The LOQ was 0.04 mg/ml.The sample storage time durability,amphoteric electrolyte pharmalyte 3-10 durability and MC durability of this method were good.Using this method to analyze the charge heterogeneity of Fc-rhGH physicochemical reference substance,eight charge heterogeneities of the reference substance were effectively separated,and the pI ranged from 5.9 to 6.4.Conclusion The developed iCIEF method had good specificity,accuracy,precision and durability,and was more suitable for efficient analysis of charge heterogeneity of Fc-rhGH than traditional flat plate IEF,which was of great significance for the quality control of Fc-rhGH and other Fc fusion proteins.
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@#Objective To express recombinant human interleukin-29-Fc(rhIL-29-Fc) fusion protein in human embryonic kidney 293-F(HEK293F) cells and analyze its anti-tumor activity in vitro.Methods The recombinant expression plasmid UCOE-IL-29-Fc was constructed and transiently transfected into HEK-293F cells.After expression and purification,rhIL-29-Fc fusion protein was obtained and identified by SDS-PAGE and Western blot;Female Japanese white rabbits were immunized with rhIL-29 and rhIL-29-Fc protein subcutaneously in the left ear respectively,2 rabbits in each group,0.5 mg per rabbit.Blood samples were collected from the vein of right ear,and the serum was separated.The half-life was measured by ELISA and the anti-proliferation effect of rhIL-29-Fc protein on human colon cancer HT-29,human colon cancer HCT-116,human Burkkit lymphoma Daudi,human non-small cell lung cancer NCI-H1975,human small cell lung cancer NCI-H209,human esophageal cancer EC109 and human pancreatic cancer PANC-1 cells in vitro was detected by CCK-8 assay,and the inhibitory concentration 50(IC_(50)) was calculated.Results The recombinant expression plasmid UCOE-IL-29-Fc was constructed correctly as identified by double digestion and sequencing.After transient transfection into HEK-293 cells for 6 d,the culture supernatant was harvested.The relative molecular mass of the purified rhIL-29-Fc fusion protein was consistent with the expectation.The protein showed a specific binding reaction with mouse anti-human IL-29 monoclonal antibody with a concentration of 1.5 mg/ml and a purity of 93%.RhIL-29-Fc protein had a half-life of 25 h and showed different inhibitory effects on the proliferation of 7 kinds of tumor cells,and the IC_(50) on different cells was also different.Conclusion The rhIL-29-Fc fusion protein was successfully expressed in HEK-293F cells,and the half-life of the fusion protein was 20 h longer than that of rhIL-29.According to the different anti-tumor proliferation activity in vitro and IC_(50) results on 7 kinds of tumor cells,it was found that the anti-tumor activity of rhIL-29-Fc fusion protein was higher than that of rhIL-29.This study laid a foundation of the development of IL-29 protein in the treatment of tumors.
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Objective:To construct a recombinant vaccine of Schistosoma japonicum (Sj) mediated by Enterococcus faecalis (Efs, rEfs-Sj26GST vaccine), and to study the expression of Sj26GST-GST fusion protein in the recombinant vaccine. Methods:The recombinant plasmid pGEX-Sj26GST was transformed into the susceptible strain Efs ATCC47077 by electroporation to construct rEfs-Sj26GST vaccine, and the plasmid was extracted for PCR identification. After induction of expression with isopropyl-beta-D-thiogalactopyranoside (IPTG), the products were analyzed and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot.Results:After PCR identification, a 676 bp fragment was amplified, which was consistent with the length of Sj26GST amplification fragment. SDS-PAGE analysis showed that the relative molecular mass was 52 × 10 3, which was consistent with the band of Sj26GST-GST fusion protein. Western blot results showed that the Sj26GST-GST fusion protein expressed by rEfs-Sj26GST vaccine could be specifically recognized by the serum of Sj infected patients. Conclusion:The rEfs-Sj26GST vaccine is successfully constructed, and the Sj26GST-GST fusion protein expressed by recombinant vaccine can be specifically recognized by the serum of Sj infected patients.
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Objective:To predict the inflammatory activity of patients with ankylosing spondylitis (AS) after 12 weeks treatment with recombinant human tumor necrosis factor-α receptor Ⅱ immunoglobulinG Fc fusion protein (rhTNFR:Fc) by Doppler ultrasonography at baseline.Methods:A total of 60 patients with AS were selected, and their general clinical characteristics before and after treatment were compared. Meanwhile, Doppler ultrasonography of the sacroiliac joint was performed to compare the Doppler parameters before and after treatment, and the correlation between baseline Doppler ultrasonography and clinical characteristics was analyzed, along with its diagnostic performance. The pre-treatment and post-treatment parameters were compared to the measured data followed by paired t-test for normal distribution, and the counting data were paired with Chi- square test. Pearson correlation test was used to analyze the correlation between pretreatment ultrasound parameters and pre-treatment disease activity. All statistical tests were bilateral, with a statistically significant difference of P<0.05. Results:After treatment, the overall score [(1.4±1.0) points vs (6.0±1.8) points, t=17.80, P<0.001], night pain score [(1.6±1.2) points vs (5.7±1.5) points, t=15.80, P<0.001], back pain score [(1.9±1.3) points vs (5.5±1.2) points, t=16.39, P<0.001], morning stiffness [(12±6) min vs (38±21) min points, t=8.93, P<0.001], Bath ankylosing spondylitis disease activity index (BASDAI) [(1.1±0.6) vs (4.6±1.3), t=12.41, P<0.001], ankylosing spondylitis disease activity score-C-reactive protein (ASDAS-CRP) [(1.0±0.4) points vs (3.7±0.9) points, t=22.01, P<0.001] and ASDAS-erythrocyte sedimentation rate (ESR) [(1.0±0.7) points vs (4.0±0.8) points, t=20.10, P<0.001] of patients with ankylosing spondylitis were lower than those before treatment, and the differences were statistically significant ( P<0.001). Compared with AS patients before treatment, the color blood flow grading score was significantly lower after treatment [(1.7±0.8) points vs (3.9±1.1) points, t= 12.86, P<0.001). The post-treatment proportion of AS patients with bilateral sacroiliac joint blood flow signal was 67% (40/60), which was lower than 87% (52/60) before treatment, but the difference was not statistically significant ( P=0.251). After treatment, the peak systolic velocity (PSV), pulsatile index (PI) and resistance index (RI) were significantly higher than those before treatment [(30±17) cm/s vs (19±8) cm/s, t=-5.42, P<0.001; (1.55±0.69) vs (1.00±0.45), t=0.45, P<0.001; (0.81±0.11) vs (0.55±0.14), t=11.20, P<0.001)]. The end diastolic velocity (EDV) before and after treatment had no statistical significant differences [(6.7±2.5) cm/s vs (6.3±1.9) cm/s, t=0.80, P=0.428]. Baseline Doppler ultrasound parameters and pre-treatment clinical indicators showed that PI and RI were negatively correlated with BASDAI ( r=-0.49, P=0.005; r=-0.51, P<0.001) , and blood flow grades were positively correlated with BASDAI ( r=0.46, P=0.028). However, there were no significant correlation between PSV, EDV and BASDAI ( r=-0.12, P=0.176; r=0.03, P=0.756). Baseline Doppler ultrasound parameters were correlated with ASDAS-CRP ( r=-0.45, P=0.012; r=0.29, P<0.048; r=-0.52, P<0.035; r=-0.76, P<0.001; r=0.61, P<0.001). There was no correlation between EDV and ASDAS-ESR ( r=0.30, P=0.110), the other ultrasound Doppler parameters were correlated with ASDAS-ESR ( r=-0.36, P<0.001; r=-0.54, P<0.001; r=-0.61, P=0.021; r=0.41, P=0.028). The receiver operating characteristic curve was drawn with the baseline RI value as a variable. According to the ASDAS-CRP value, the diagnostic threshold for determining the presence or absence of AS activity after 12 weeks of treatment was 0.49, with an area under the curve of 0.817, sensitivity of 88.1%, specificity of 61.1%, positive predictive value of 66.7%, and negative predictive value of 86.1%. Conclusion:Baseline Doppler ultrasound correlates well with clinical indicators, among which baseline RI values is a good predictor of inflammatory activity status after rhTNFR:Fc treatment.
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Ultrashort peptides have higher stability, tissue penetrability, biocompatibility, and less immunogenicity, and are widely applied in biology and medicine. GHK (glycyl-l-histidyl-l-lysine) and GQPR (glycyl-l-glutamyl-l-prolyl-l-arginine) can stimulate collagen renewal and inhibit collagen degradation. GHK and GQPR have been used in cosmetic anti-wrinkle skincare and make-up products. The most common approach for ultrashort peptide production is the solid-phase synthesis, which is eco-unfriendly due to heavy usage of organic chemical reagents during the manufacturing process. Here we report a new approach to the production of ultrashort peptides. Recombinant expression of ultrashort peptides is usually unfeasible because of the short amino acid sequences. A vector pET28a-Trxm harboring the thioredoxin gene was first constructed for subsequent fusion expression. The tandem repeats of GHK and GQPR genes were used as the templates for rolling circle amplification (RCA). The RCA reaction was tuned to incorporate noncanonical nucleotides 5-methylcytosine to obtain long DNA fragments. Gene sequences with various lengths were generated through double digestion of Acc65 Ⅰ and Apa Ⅰ. The resulting digestion products were gel recovered by size (from 500 bp to 1 500 bp) and cloned into pET28a-Trxm to obtain the recombinant vector pET28a-Trxm-(TRSP)n. The pET28a-Trxm-(TRSP)n was introduced into E. coli BL21(DE3) to generate a library of Trxm-(TRSP)n sequences with a controlled distribution of lengths. Through double digestion and sequencing, positive clones with tandem repeats n=1, 2, 3, 4, 6, 7, 8, 9 were obtained. Protein expression results showed protein bands with corresponding molecular weight, and the protein expression level decreased as the tandem repeats increased. The expression level of Trxm-(TRSP)1 achieved 50% of the total protein, while the expression level of Trxm-(TRSP)2 was 30% of the total protein. The crude extracts from cell pellets were further treated with enterokinase cleavage, and the supernatants containing (TRSP)1 were collected after ultrafiltration and then subjected to trypsin cleavage. HPLC analysis indicated that the ultrashort peptides GHK and GQPR were successfully obtained through two-step cleavage. This study may facilitate the commercial production of ultrashort peptides.
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Escherichia coli/metabolism , Peptides/chemistry , Amino Acid Sequence , Gene Library , Tandem Repeat SequencesABSTRACT
Abstract The present study deals with the computational design and analysis of a novel fusion protein based on a single chain variable fragment that binds to the extracellular domain of human epidermal growth factor receptor 2 (HER2) in breast cancer cells. Alpha luffin, a small ribosome inactivating protein (RIP), was attached to the anti-HER2 antibody fragment. I-TASSER modeling provided the full-length structure of the fusion protein. Molecular docking evaluated the molecular interactions of the complementarity-determining regions of designed fusion protein to HER2. Energy minimization and molecular dynamics simulations were conducted to refine the complexes. RMSD plot revealed reasonable stability of the fusion protein during the simulation. The free binding energy profile of complexes affirmed a favorable binding affinity of proteins in complex with HER2 using molecular mechanics Poisson-Boltzmann surface area (G-MMPBSA) algorithm. In general, this approach looks promising in the development of new fusion proteins in terms of immunotoxins with appropriate cytotoxicity.
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Acute leukemia (AL) is a kind of malignant clonal disease of hematopoietic stem cells. Rearrangement of mixed lineage leukemia (MLL) gene can be observed in about 5%-10% of AL patients. Currently, AL patients with MLL-rearrangements (MLL-r) lack effective treatment and are usually associated with poor prognoses. Recent studies have shown that many epigenetic regulators are directly or indirectly involved in the occurrence and development of AL carrying MLL-r (MLL), which provides a biological basis for the use of epigenetic regulation strategies to treat MLL. In this review, we start from the epigenetic regulation mechanism of MLL, and select representative drug targets to briefly analyze the relationship between each target and MLL and summarize the development progress of their inhibitors, hoping to provide reference for the subsequent research and development of drugs for the treatment of MLL.
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With the development of global economy, the dramatically increased production of polyethylene terephthalate (PET) plastics has led to a remarkably increased amount of plastic waste. PET waste can be treated by landfill, incineration, or biodegradation. While landfilling and incineration may cause secondary pollution, biodegradation has since received increased attentions due to its environmental friendliness. Recent studies have indicated that the carbohydrate binding module (CBM) can effectively enhance the binding of PET degrading enzymes to PET, and consequently increasing PET degradation rate. Here we constructed a fusion protein BaCBM2-Tfuc containing the BaCBM2 from Bacillus anthraci and the cutinase Tfuc from Thermobifida fusca, by megaprimer PCR of whole plasmids (MEGAWHOP). Notabaly, the PET film degradation efficiency (at 60 ℃) of BaCBM2-Tfuc was 2.8 times that of Tfuc. This study may provide technical support for constructing fusion proteins capable of efficiently degrading PET.
Subject(s)
Carbohydrates , Carboxylic Ester Hydrolases , Polyethylene Terephthalates , ThermobifidaABSTRACT
@#Psoriasis is an autoimmune disease characterized by chronic skin inflammation, and its etiology and pathogenesis have not been fully elucidated to date. In the previous study, rhIL23R-CHR/Fc fusion protein had been found to significantly relieve the symptoms of psoriasis mice and the pharmacological mechanism had been initially elucidated.In this study, we established a psoriasis cell model (Act-HaCaT) using TNF-α-activated human immortalized keratinocytes (HaCat).In our current study, the lncRNA that plays a key role in the regulation of Act-HaCaT function by the rhIL23R-CHR/Fc fusion protein was screened by transcriptome sequencing combined with qRT-PCR.The results showed that rhIL23R-CHR/Fc fusion protein significantly inhibited cell proliferation and inflammatory factor production in Act-HaCaT.lncRNA ENST00000522718 was obtained by screening, and knockdown of ENST00000522718 was found to significantly inhibit cell proliferation and inflammatory factor production.Our findings suggest that ENST00000522718 plays an important role in the pathological mechanism of psoriasis.
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Objective:To investigate the effects of a eukaryotic expression plasmid for IL-6 and B-cell activating factor (BAFF) fusion protein on the histopathological changes in salivary and lacrimal glands of non-obese diabetic (NOD) mice with Sj?gren′s syndrome and to elucidate the possible therapeutic mechanism of IL-6/BAFF fusion protein eukaryotic expression plasmid in NOD mice.Methods:The eukaryotic expression plasmid for IL-6/BAFF fusion protein was constructed. After transfecting CHO cells with the plasmid, the expression of IL-6/BAFF fusion protein was detected by Western blot. BALB/c mice were injected with the plasmid every two weeks for three times and the titers of anti-IL-6 and anti-BAFF antibodies were measured by ELISA. Twenty-one NOD mice were randomly divided into three groups (control group, empty vector group and therapy group) by numerical table method. The mice in the therapy group were injected with the IL-6/BAFF fusion protein eukaryotic expression plasmid once a week for six times and the mice in the empty vector group were injected with empty plasmid. The levels of anti-IL-6 and anti-BAFF antibodies as well as cytokines (IL-6, BAFF, INF-γ, IL-10 and IL-17A) in mouse serum samples were detected by ELISA. The proportions of Th17, Treg, Th1 and Th2 cells in mouse splenocytes were measured by flow cytometry. Focal lymphocyte infiltration and pathological changes in the lacrimal and salivary glands of mice were observed under light microscopy after HE staining.Results:The eukaryotic expression plasmid for IL-6/BAFF fusion protein increased the levels of anti-IL-6 and anti-BAFF antibodies in the serum of BALB/c mice ( P<0.05). The levels of anti-IL-6 and anti-BAFF antibodies in the serum of NOD mice in the therapy group increased ( P<0.01), while the expression of IL-6, BAFF, INF-γ, IL-10 and IL-17A in NOD mice in the therapy group was lower than that in the control group and the empty vector group ( P<0.05). The percentages of Treg and Th2 cells in the splenocytes of NOD mice increased after treatment ( P<0.05). Moreover, the eukaryotic expression plasmid for IL-6/BAFF fusion protein significantly improved the irregular size and morphology of glandular vesicles in the lacrimal and salivary glands, reduced the ductal dilatation and decreased the focal lymphocyte infiltration in NOD mice. Conclusions:The eukaryotic expression plasmid for IL-6/BAFF fusion protein induced the production of anti-IL-6 and anti-BAFF antibodies, decreased the expression of inflammatory cytokines, regulated the balance of Th17/Treg and Th1/Th2 cells, improved the irregular alveolar structure and ductal dilation in the lacrimal and salivary glands and reduced the focal lymphocyte infiltration in NOD mice. This study showed that eukaryotic expression plasmid for IL-6/BAFF fusion protein might serve as a potential target for therapeutic targeting of T and B cells.
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CD47 is widely expressed on the cell surface, and combines with signal regulatory protein α (SIRPα) to transmit the "don't eat me" signal, which plays a key role in self-recognition and tumor immune escape. Studies have shown that the high expression of CD47 in different hematologic neoplasms is associated with the occurrence, progression and poor prognosis of tumors. As a new immune checkpoint, CD47 is gradually becoming an effective target for tumor immunotherapy. and various related preclinical and clinical studies for hematologic neoplasms are underway. This article summarizes the application of CD47 in hematologic neoplasms, in order to provide references for the treatment.
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Objective:To develop an unnatural amino acid-labelled HiD-Hin47 fusion protein as a novel carrier.Methods:Twenty versions of the fusion protein were designed, each of which contained a different amino acid site replaced by an azide-bearing amino acid, N6-(2-azidoethoxy) (carbonyl)-L-Lysine (NAEK). These fusion proteins were constructed, expressed and purified, and the yield were evaluated by SDS-PAGE. Based on the highest protein yield, which was approximately 70% of the wild-type yield, the fusion protein with the unnatural amino acid in E677 site was selected. The pneumococcal polysaccharides of serotype 3 (F3) and serotype 6B (F6B) were coupled to the selected fusion protein through a "click" reaction. The conjugates were purified and compared in animal studies with other F3 and F6B conjugates that were coupled to CRM197, tetanus toxoid (TT) and HiD by conventional methods.Results:The immunogenicity of F3 conjugate using HiD-Hin47 as carrier (F3-HiD-Hin47) was slightly better than that of other F3 conjugates. F6B-HiD-Hin47 was significantly better than F6B-TT and F6B-HiD in terms of immunogenicity, but showed no significant difference with F6B-CRM197.Conclusions:NAEK-labelled HiD-Hin47 had the potential as a carrier for pneumococcal polysaccharide conjugate vaccine and was worthy of further study.
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Curcumin is exclusively isolated from Zingiberaceae plants with a broad spectrum of bioactivities. In the present study, we used the diketide-CoA synthase (DCS) and curcumin synthase (CURS) genes to construct a non-natural fusion gene encoding diketide-CoA synthase::curcumin synthase (DCS::CURS). This fusion protein, together with the acetyl coenzyme A carboxylase (ACC) and the 4-coumarate coenzyme A ligase (4CL), were introduced into Escherichia coli for the production of curcumin from ferulic acid. The process is divided into two stages, the growth stage using LB medium and the fermentation stage using the modified M9 medium. The yield of curcumin reached 386.8 mg/L by optimizing the induction of protein expression in the growth stage, and optimizing the inoculum volume, medium composition and fermentation time in the fermentation stage, as well as the addition of macroporous resin AB-8 into the second medium to attenuate the toxicity of the end product. The exploitation of the non-natural fusion protein DCS::CURS for the production of curcumin provides a new alternative to further promoting the production of curcumin and the related analogues.