With their unique advantages of high affinity and high specificity, antibody drugs have played an increasingly important role in the diagnosis and treatment of malignant tumors, autoimmune diseases, and infectious diseases, becoming the focus of international innovative drug research. Since the outbreak of COVID-19, many research institutions and enterprises at home and abroad are in accelerated development of antibody drugs. In this situation, a systematic analysis of the status and trends of antibody drugs, the research progress of new antibody drugs for SARS-CoV-2, and the opportunities, challenges and suggestions for current antibody drug innovation in China is of great significance to promote the independent innovation of antibody drugs in China.
Objective: To investigate the effect of S100A6 in microenvironment on promoting angiogenesis via macrophages and the underlying mechanism. Methods: (1)Macrophages were treated by recombinant proteins GST-hS100A6: ①Conditioned medium was collected (named A6-Mφ-CM) to resuspend human umbilical vein endothelial cells (HUVEC), and the effect on angiogenesis was examined by in vitro endothelial capillary formation assay. ②The mRNA and protein levels of CD163, CCL2, IL-6, VEGFA in macrophages were evaluated by Real-time PCR and Western blot. And JAK2 and STAT3 and their phosphorylation levels in macrophages were detected by Western blot. ③ The migration ability of macrophages was detected by Transwell migration assay. (2)The change of macrophage migration ability was detected by Transwell assay after pre-treatment of JAK2 inhibitor XL019. Results: (1) A6-Mφ-CM had obvious effects on promoting angiogenesis,the number of branches and branch length of the A6-Mφ-CM group was significantly higher than that of the GST-Mφ-CM group (P<0.05; P<0.05), and was also significantly higher than that of GST-hS100A6 group, suggesting that S100A6 treated macrophages can promote angiogenesis, while S100A6 has no direct pro-angiogenic effect. (2) After treatment with GST-hS100A6, the mRNA and protein levels of CD163, CCL2, IL-6 and VEGFA in macrophages were up-regulated compared with GST group (P<0.05; P<0.05; P<0.05; P<0.05), suggesting that S100A6 induced polarization of macrophages into a pro-angiogenic phenotype. (3) The number macrophage migrated in GST-hS100A6 group was 1.4-fold as much as that in GST group (P<0.01), suggesting that S100A6 can enhance macrophage migration. (4) GST-hS100A6 upregulated the levels of JAK2, STAT3 and their phosphorylation proteins in macrophages, indicating that JAK2/STAT3 pathway of macrophages was activated. (5) The effect of GST-hS100A6 on promoting macrophage migration was partly inhibited by JAK2 inhibitor XL019(P<0.01). Conclusion: S100A6 in the microenvironment can recruit macrophages by activating JAK2/STAT3 signaling, and further induce macrophage into a pro-angiogenic phenotype to promote angiogenesis indirectly.
Objective: Helicobacter pylori candidate vaccine was constructed based on yeast surface display technology and its immunogenicity was further analyzed. Methods: Surface displayed S.cerevisiae EBY100/pYD1-VacA was constructed that vacA gene of Helicobacter pylori as used a research subject. S.cerevisiae EBY100/pYD1-VacA was detected by Western blot analysis,immunofluorescence assay and flow cytometry assay. BALB/c mice of SPF grade were administrated orally with that PBS and S.cerevisiae EBY100/pYD1 were used as the control groups, and S.cerevisiae EBY100/pYD1-VacA was used as the experimental group. VacA-specific sera IgG and secretory IgA titers were determined by ELISA assay. Results: The VacA antigen protein was successfully displayed on the surface of S.cerevisiae EBY100. S.cerevisiae EBY100/pYD1-VacA could produce a higher VacA specific antibodies. Conclusion: Surface displayed yeast can be used as a delivery vector of Helicobacter pylori candidate vaccine. Meanwhile, it will provide new ideas for developing bacteria or virus vaccines.
Tau is one of the microtubule-associated proteins. Its physiological function is to promote microtubule assembly and maintain microtubule stability. The phosphorylated Tau easily aggregated and subsequently caused a series of tauopathies. Of them, Alzheimer's disease is a common type of tauopathies. Six subtypes of Tau are also found in human brain because of different mRNA splicing of the same gene. They are respectively composed of 352, 381, 383, 410, 412 and 441 residues. The molecular weight of the six subtypes is among 48-67kDa, the R1, R2, R3 and R4 are the microtubule binding domains, which can bind to microtubule and maintain microtubule stability. Compared with the full-length Tau, the truncated Tau isoforms, such as R1-R4, are more likely to aggregate and have stronger aggregation kinetics. For example, the microtubule binding domain of R3 and R4 and the following 10 amino acid at the C-terminal of R4 sequence are the core peptide of the paired helical fibrils (PHFs), which has been proven to drive Tau aggregation. And its fibrillary 3D structure was also identified using micro-electron diffraction of Cryo-electron microscope. Herein, the core fragment 306-378 (Tau306-378) of Tau was heterogeneously expressed in Escherichia coli, and the purified Tau306-378 was obtained by Ni+ chelated affinity chromatography. The final production yield was about 10.35mg/L. Then, the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot proved that Tau306-378 was expressed successfully. Moreover, the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was further used to confirm the molecular weight of Tau306-378. It was proved that most of the Tau306-378 were in the form of monomers. In addition, a small portion of dimers were also found in the protein sample, which is caused by the formation of disulfide bond among two monomers. In order to explorethe effect of induction temperature on the production of the target protein, four different induction temperatures (16℃, 25℃, 30℃ and 37℃) were investigated. The results showed that the expression of Tau306-378 was the highest at 30℃, followed by 25℃ and 16℃. However, the expression of Tau306-378 was the lowest at 37℃. The optimal inducing condition of Tau306-378 were identified: inducing at 30℃ for 16-20h with 0.5mmol/L of isopropyl-β-D-thiogalactopyranoside. Finally, thioflavin T fluorescence staining experiments were performed to probe its fibrillogenesis. The results of thioflavin T fluorescence experiments showed that the trend of aggregation dynamics presents a typical S-shaped curve. That is, the recombinant Tau306-378 has good aggregation characteristics, which could be used to study the aggregation characteristics and toxicity of Tau in vitro, as well as in the screening of various inhibitors against Tau fibrillogenesis.
Maltose and glucose have significant effects on the production of curdlan by fermentation of Alcaligenes faecalis. The chemostat culture and steady-state carbon balanced metabolic flux analysis were applied to evaluate the effect of the substrates on curdlan biosynthesis in detail. Results demonstrated that the intracellular metabolism of A. faecalis were significantly different under the substrates of maltose and glucose as the carbon substrate at the dilution rate of 0.1h -1. The relative metabolic flux analysis showed the curdlan yield reached 53.8% under maltose source, which was more than 45.8% higher than that of glucose (36.9%). At the same time, the absolute metabolic flux of the HMP pathway increased more than 40% than that of glucose, and enhanced the supply rate of NADPH. The higher NADPH supply level promotes the flux ratio of curdlan biosynthesis, which depends on NADPH cofactors, and increases the molar conversion rate of curdlan from carbon source substrate. Moreover, the metabolic flux distribution results also showed that the ED pathway distribution and energy supply are also the key factors affecting the curdlan biosynthesis efficiency of A. faecalis. The lower residual glucose concentration with maltose as carbon source substrate could relieve the inhibition on curdlan synthesis, and could achieve higher flux ratio of ATP supply for promoting the curdlan biosynthesis efficiency.
Objective: To explore an effective purification procedure of recombinant HBcAg virus like particles (VLPs) that are prone to aggregation. Methods: The expression of recombinant HBcAg protein was induced by IPTG in cultured E. coli. The centrifugation precipitates of bacteria after ultrasonic fragmentation were resuspended and dissolved in PBS buffer with different concentrations of urea. The VLPs behavior was analyzed and identified by density gradient centrifugation and electron microscopy. The precipitate solution was purified by Sepharose 4 FF gel filtration chromatography under the selected urea conditions, and the purified target protein was further desalinate to remove urea with PBS containing 30% sorbitol. The entire process was analyzed by SDS-PAGE and electron microscopy. Results: The ultrasonic precipitates resuspensed with PBS buffer containing 1mol/L urea was effective dissolve the aggregated VLPs, which showed the behavior of typical HBcAg VLPs in sucrose density gradient centrifugation, and morphology and structure of the particales were complete by electron microscope. VLPs were further purified after 1mol/L urea gel filtration. In the process of urea removal, PBS containing 30% sorbitol was used as the mobile phase, which effectively avoided the reaggregation of VLPs after urea removal. Conclusion: The combined application of urea and sorbitol provides an effective solution for the purification and preparation of VLPs with aggregation phenomenon.
Objective: To construct an E. coli strain engineered for expressing TNF-α Fab' antibody, and to design an efficient and practical strategy to promote the expression of soluble Fab' antibody in the periplasmic space. Methods: First, the strategies were selected as changing different expression vectors, changing the order of light and heavy chains, changing signal peptides, co-expressing molecular chaperones (Skp), disulfide bond synthetase (Dsbc), peptidyl coenzyme cis-trans isomerase (PPIB), disulfide isomerase (hPDI), and nuclease to evaluate the improvement of Fab' antibody expression. Second, the expressed Fab' antibody was purified. High-purity Fab' antibodies were obtained through a three-step purification of periplasmic extraction, including Q anion exchange column purification, phenyl column capture, and Protein L column affinity purification. Finally, the affinity of the purified Fab' antibody was determined. Results: The best strategy to increase the expression of correctly assembled Fab' antibodies suggested the target protein was constructed into pET-30a vector; the heavy chain was in the front and the light chain was in the rear; light and heavy chains used different signal peptides, and co-expression of hPDI. The Fab' antibody concentration in the periplasmic extract reached 588.0mg/L, and the yield after purification was up to 28.2mg/L in the fermentation broth. The total recovery rate was 32.0% and the purity of Fab' was 90.9%. The affinity of Fab' antibody was (5.8±3.0)×10 -9mol/L, and in vitro activity IC50 was (5.2±2.4)×10 -11mol/L. Conclusion: Through the optimization of the molecular construction, an engineered E. coli strain with high level of soluble Fab' antibody expression was obtained.
After immobilization, the stability and environmental tolerance of the free enzyme were optimized, which can improve the utilization rate of enzymes and reduce the production costs in diverse fields such as food, medicine, chemical industry, environment and leather, and thus possesses great application potentials. The utilization of novel crosslinking agents in immobilized enzyme process has greatly advanced the study of immobilized enzymes.The optimized crosslinking and immobilization conditions were obtained by using the new crosslinking agent polyethylene glycol diglycidyl ether (PEGDGE), which was immobilized with the amino carrier LX-1000HA, and by using single factor and orthogonal experiment optimization: crosslinking temperature 30℃, crosslinking time 2h, crosslinking agent concentration 0.75%, pH 7.0, enzyme amount 800U, carrier volume 0.5g, immobilized time 2h, immobilized temperature 45℃. Using the optimized immobilization process, the immobilized enzyme LX-1000HA-PEGDGE-CRL was prepared to achieve 160.81U/g which was about twice of the activities of immobilized enzyme LX-1000HA-GA-CRL(obtained by LX-1000HA and glutaraldehyde crosslinked lipase) and LX-1000EA-PEGDGE-CRL(obtained by short-chain amino carrier LX-1000EA and PEGDGE crosslinked lipase). The optimal reaction temperature of immobilized enzyme LX-1000HA-PEGDGE-CRL was found to be 15℃ higher than that of free enzyme; the immobilized enzyme could still remain 70% of its original activity after incubation at 70℃ for 3h; the immobilized enzyme could remain about 65% of its original activity after recycling for 6 times; the immobilized enzyme also behaved good tolerance to acid and alkali condition and good storage stability, the immobilized enzyme could remain about 70% of the initial enzyme activity after storage at 4℃ for 30 days. After the comparation of immobilized enzyme LX-1000HA-PEGDGE-CRL with free enzyme, immobilized enzyme LX-1000HA-GA-CRL and immobilized enzyme LX-1000EA-PEGDGE-CRL, the immobilized enzyme LX-1000HA-PEGDGE-CRL behaved better application effect in thermo-tolerance and reusability.
Natural hydrogels refers to hydrogels, as a kind of raw materials, are derived from natural biological materials. Because this kind of natural polymers contains natural components of life structures, and its biological and chemical properties are similar to those of natural tissues,it receives special attentions. Natural hydrogels are considered to be excellent biomimetic matrix materials in bone tissue engineering due to their high degree of similarity to extracellular matrix. Due to the poor mechanical properties and weak osteogenic induction properties, the natural hydrogels are usually modified by introducing other materials or bioactive factors to obtain a more suitable composite biomaterial for bone tissue engineering applications. The application of natural hydrogel——based biomaterials in bone tissue engineering are summarized. Also summaries the different application forms of injectable hydrogels, porous hydrogel scaffold, and 3D bioprinting hydrogel scaffold in recent years, in order to provide a reference for the application of such natural hydrogel-based biomaterials in future bone tissue engineering.
Bacteriophage is widely used in many fields, so in the process of preparing phage, the different techniques or several techniques to obtain phage preparations with different purity to be used. The techniques commonly used to separate and purify phage are precipitation, filtration and centrifugation. In recent years, the application of chromatographic, field-flow fractionation techniques and electrophoresis techniques provides a new direction for obtaining phage preparations.
Fish has been used as an important model for melanoma research. In recent years, great progress has been made in the study of fish melanin due to its melanoma model with uniform pathological characteristics and ease of reproduction. Melanocytes are the most important pigment cells in fish. The eumelanin has an important effect on the surface coloring and life activities of fish. The synthesis process mainly involves the catalytic action of the tyrosine gene family. Pheomelanin was previously thought to be non-existent in fish, but recent research has yielded different insights. With the extensive study of fish melanin synthesis, its synthetic regulation mechanism has also been studied in depth. In the signal pathway that regulates melanin synthesis, microphthalmia-associated transcription factor (Mitf) is a key regulator of various signaling pathways, which is affected by upstream α-Msh/Mc1r, Wnt/β-Catenin, and Scf/c-Kit and other signaling pathways mediate the downstream tyrosine gene family and regulate the synthesis of melanin. The synthesis of fish melanin and related signaling pathways be reviewed, with a view to providing a reference for the basic theory of fish melanin synthesis and aquaculture, and also to help understand the origin and regulation of human melanin synthesis, and to overcome melanoma.
Filamentous fungi, which with excellent protein secretion and environmental adaptability, are widely used in protein expression fields. In recent decades, the expression efficiency of filamentous fungi including Aspergillus sp., Trichoderma sp. and Penicillium sp. has been continuously increased by means of mutagenesis,culture optimization and genetic modification. In order to promote the development of filamentous fungal protein expression system,filamentous fungi expression hosts, protein expression elements and their modification strategies were reviewed . At the same time, the disadvantage in the development of filamentous fungal expression system were discussed to provide references and insights for the study of novel filamentous fungi expression systems.
Surfactin is a kind of cyclic lipopeptide biosurfactant mainly synthesized by many Bacillus subtilis strains. Surfactin is well-accepted as one of the most powerful biosurfactants, presents excellent surface activities, can significantly reduce the surface tension force of water to below 27mN/m at a concentration of 10-30mg/L. In addition, surfactin has great biological activities like: anti-fungal, anti-virus, anti-tumor, insecticidal and anti-mycoplasma, and thus has of great applications potential in the fields of medicine, agriculture, food, cosmetics and petroleum industries. However, its real industrial applications were limited by the high production cost and lack of specific application yields. Many efforts have been done towards the improvement of surfactin production. The recent progresses on surfactin production, and its applications as an antibacterial agent have been reviewed.
As the core of modern biological industry, biomanufacturing covers the value chain from biological resources to biotechnology to bio-industry, and embodies the application of modern biotechnology in many industrial fields such as medicine, agriculture, energy, materials, chemical industry and environmental protection, which plays an important role in promoting the sustainable development of the economy and society. At present, biomanufacturing has become one of the key areas of the technology industry layout in the world's major developed economies, attracting a large amount of public investment and social capital, forming a billion-dollar investment boom.Investigates and statistics 201 financing events of 150 biomanufacturing-related enterprises in the world during 2018-2019, combs the development environment and financing status quo at home and abroad in the biomanufacturing industry, and makes suggestions for the development of China's biomanufacturing industry, with a view to guiding the transformation of scientific and technological achievements, technology capital docking and industry prosperity and development. To contribute to the sustainable development of China's economy and society.
