17α-hydroxyprogesterone (17α-OH-PROG) is a key intermediate in steroidal hormone drugs, and its biosynthesis is mainly catalyzed by cytochrome monooxygenase (CYP17). In this process, cytochrome P450 reductases (CPRs) are important electron transport chain partners of cytochrome P450 enzymes, which directly affect the catalytic efficiency of CYP17. In order to study the effect of different CPRs on the activity of 17α-hydroxylase, the expression vector pPIC3.5k-hCYP17 was constructed with human 17α-hydroxylase gene, and the recombinant Pichia pastoris strain was obtained. Then, three CPRs from different sources were screened, the expression vector pPICZX-CPR was constructed, and then 17α-hydroxylase and CPR co-expression strains were obtained, and the transformation of progesterone was carried out using the recombinant P.pastoris. Finally, the transformation products were analyzed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). The results showed that the successfully recombinant strain showed 17α-hydroxylase activity, which was able to catalyze progesterone to produce the target product 17α-OH-PROG and the by-product 16α-hydroxyprogesterone (16α-OH-PROG). Furthermore, compared with strain containing only 17α-hydroxylase, the yield of 17α-OH-PROG all improved when CPR and 17α-hydroxylase were co-expressed in P.pastoris. Of the three CPRs tested, co-expression of hCPR and CYP17 showed the highest progesterone transformation ability, and the yield of 17α-OH-PROG increased by 42%. The above results indicated that the suitable CPR co-expressed with 17α-hydroxylase resulted in the increase of 17α-OH-PROG production. This study provides a promising strategy for the production of 17α-OH-PROG catalyzed by CYP17, which is of great significance for the industrial production of steroid.
Objective: WIND (WOUND INDUCED DEDIFFERENTIATION) are important transcription factors belonging to ERF/AP2 (ETHYLENE RESPONSE FACTOR/ APETALA 2) family. The genes were first found to bind to ethylene response element GCC-BOX and dehydration response element DRE in Arabidopsis thaliana to respond to drought signals and regulate ethylene levels. Recent studies have found that WIND genes also play a key role in plant wound signal response, callus formation and adventitious shoot production. Previous studies have explored the mechanisms of the WIND gene controlling callus formation and adventitious shoot regeneration in Arabidopsis thaliana. However, the functions of WIND in woody plants remain unclear. The functions of WIND genes in wound signal response and adventitious shoot regeneration will be explored in Populus euphratica. A theoretical basis for solving the regeneration problem of P. euphratica at the molecular level will be provided. Methods: Gene cloning, qRT-PCR and transgenic phenotype analysis were used. Results: Two WIND genes of P.euphratica were cloned as PeWIND1 and PeWIND2, respectively. The coding regions were 1 050 bp and 1 032 bp, encoding 349 and 343 amino acids, respectively. Subcellular localization analysis showed that both genes were functional in the nucleus. Gene expression analysis showed that PeWIND1 and PeWIND2 expressed in roots, stems, leaves, and calli. The highest expression level was found in calli. The time course expression analysis showed that the expression levels of PeWIND1 and PeWIND2 were first increased and then decreased within 24 h after wound stimulation, and peaked at 1 h after wound stimulation. Phenotypic statistics of transgenic plants showed that the regeneration ability of adventitious shoots was enhanced after overexpression of PeWIND1 and PeWIND2. Conclusion: PeWIND1 and PeWIND2 responded to wound signal and the expression levels of PeWIND1 and PeWIND2 were first increased and then decreased within 24 h after wound stimulation. PeWIND1 and PeWIND2 promoted adventitious shoot regeneration from stems of poplars.
The bioenzymes were loaded on the γ-Al2O3 spherical carrier by adsorption method, and the bioenzymes/γ-Al2O3 and the carrier were characterized by scanning electron microscopy (SEM), specific surface area analysis (BET), Fourier infrared spectroscopy (FT-IR) and circular dichroism (CD). The results showed that the bioenzymes were adsorbed on the carriers. The prepared bioenzyme/γ-Al2O3 catalyzed oxidative desulfurization of real diesel fuel, and the effects of reaction temperature, reaction flow rate and enzyme solution concentration on the desulfurization effect of real diesel fuel were investigated, and qualitative and quantitative analyses of the desulfurization effect were performed; further response surface design optimization of the desulfurization process conditions was carried out to find out the optimal reaction conditions. The experimental results showed that the optimum desulfurization rate of 93.16% was derived from the reaction temperature of 49℃, reaction flow rate of 1.0 mL/min and enzyme solution concentration of 15.5% (enzyme loading of 28.13 g). Finally, the performance of the immobilized enzyme was investigated for repeated use, and the activity of the catalyst did not decrease significantly after seven uses, indicating that the immobilized enzyme is effective in catalyzing oxidative diesel desulfurization and has potential application value.
Objective: Preparation and enzymatic activity identification of sentrin-specific protease1 (SENP1) catalytic domain (SENP1C). Methods: The target genes were amplified by PCR from SENP1-pcDNA3.1 and EGFP-pcDNA3.1, and then cloned into pGEM-T vector. After enzyme digestion, the digested cDNAs were then subcloned into the prokaryotic expression vector pET-28a. Next, the positive recombinants were transfected into prokaryotic expression cells BL-21, which were then induced by isopropyl thiogalactoside (IPTG). The protein expression was identified by SDS-PAGE and coomassie brilliant blue staining. The extracted proteins were purified by Ni-NTA and dialysis treatment, and the protein purity was further checked by SDS-PAGE and coomassie brilliant blue staining. HT22 cells was pre-incubated with 1 μmol/L or 5 μmol/L Tat-EGFP for different times, and cell transfection was observed by fluorescence microscope. After pretreatment with 5 μmol/L Tat-SENP1 for 10 h, the SUMOylation of overall protein in HT22 cells was detected by immunoblot analysis. In addition, immunoprecipitation and immunoblotting were used to evaluate endogenous and exogenous Akt1-SUMO1 conjugations in HT22 cells or HT22 cells overexpressing Myc-Akt1 and HA-SUMO1. Results: Tat-SENP1C-pET-28a and Tat-EGFP-pET-28a prokaryotic expression recombinants were successfully constructed, which were efficiently induced to express target proteins by IPTG. The high purity target proteins were obtained by Ni-NTA and dialysis. After incubation with 5 μmol/L Tat-EGFP for 10 h, the penetration efficiency was higher in HT22 cells. Tat-SENP1C reduced the levels of total SUMOylation and endogenous and exogenous Akt1-SUMO1 conjugations significantly. Conclusion: Tat-SENP1C-pET-28a and Tat-EGFP-pET-28a prokaryotic expression recombinants were successfully constructed, and can be highly induced to express target proteins by IPTG. The purified Tat-SENP1C retains strong membrane penetration ability and enzymatic activity.
Living organisms such as cells isolated from human body, bacteria, and viruses can be engineered as live biotherapeutics, which can maintain biological activity, self-replicate, and express genes in a patient’s body. Compared with traditional drugs, engineered live biotherapeutics maintain a relatively long-time curative effect in vivo, express genes as expected to achieve versatility and homeostasis control, and have unique targeting and response capabilities. In recent years, the application of engineered live biotherapeutics in tumor immunotherapy has received widespread attention. CAR-T and other cell therapies and oncolytic virus therapies have performed good clinical effects, and engineered bacteria are also developing rapidly in clinical and preclinical research. Among all the types of engineered live biotherapeutics, human cells, bacteria, and viruses have different characteristics. Therefore, their design purposes and ideas are different. With the progress of synthetic biology technologies, engineered live biotherapeutics will have better safety and efficacy, and bring new opportunities for tumor therapy. Herein, the latest developments of engineered live biotherapeutics for tumor immunotherapy are reviewed, and the synthetic biology design and immunotherapy mechanisms are expounded.
Nucleic acid vaccine based on messenger RNA (mRNA) is a kind of mRNA technology emerging in recent years. mRNA vaccines have many advantages over traditional vaccines, which can be manufactured in a cell-free manner, enabling rapid, economical and efficient production. In addition, single mRNA vaccines can encode multiple antigens, enhance the immune response against certain pathogens, improve the efficiency of treatment process of diseases, and can target multiple microbial or viral variants in a single formulation. mRNA is seen as a revolutionary vaccine technology in COVID-19 prevention and control, which has been developed and successfully applied in record time. The mRNA vaccine is with poor stability, so the development and applications of novel delivery systems are essential. With the intensive study of pharmacology of mRNA vaccines, the clinical applications of mRNA vaccines enter into a new stage. Recently, mRNA technologies were used in the prevention and therapies of diseases, and some results were published. Here, the output of mRNA vaccines used in prevention and therapies of diseases was summarized, and the development of mRNA vaccines was also discussed.
Bispecific antibodies can simultaneously bind to two targets. Compared with monospecific antibodies, they have the advantages of high efficacy and less toxic and side effects, so they have become a research hotspot in recent years.However, since bispecific antibodies are composed of different heavy chains and light chains, and the expression of heavy and light chains is difficult to control at the same level, it is very easy to form by-products, which greatly increase the difficulty and cost of downstream purification.In recent years, several pharmaceutical companies have developed bispecific antibody preparation platforms, which have greatly improved the success rate of bispecific antibody assembly.However, various double-antibody molecular design strategies are not enough to completely prevent the production of by-products, so various chromatographic methods are needed to further remove the by-products of double-antibody molecules to improve product quality.This paper reviews several mainstream bispecific antibody design platforms in recent years, and systematically summarizes the chromatographic methods for removing homodimers, half antibodies and 3/4 antibodies and aggregates. The comprehensive information should be helpful to provide a theoretical basis for bispecific antibody purification.
Bone marrow mesenchymal stem cells are a kind of stem cells with multi-directional differentiation ability. In recent years, relevant studies have found that the exosomes secreted by bone marrow mesenchymal stem cells promote the repair of injured tendons and angiogenesis, inhibit oxidative stress response, protect neurons, promote cartilage regeneration, and regulate bone metabolism in addition to its other functions. Steroid-induced femoral head necrosis is a kind of aseptic necrosis of the femoral head caused by massive use of hormones. Its specific mechanism has not been clarified, and related signal pathways play a key role, such as Wnt/ β- catenin, RANKL-RANK, PTEN/AKT, PI3K/AKT and other pathways, and the activation of these signal pathways is closely related to exosomes. This article reviews the relevant research progress of exosomes from bone marrow mesenchymal stem cells and related signal pathways in steroid-induced femoral head necrosis, in order to have certain guiding significance for the prevention and treatment of steroid-induced femoral head necrosis and the development of related drugs.
Aromatic compounds including lots of species are in high demand and widely used in many areas. The synthesis of aromatic compounds by constructing microbial cell factories has unique advantages and industrial applications. Yeast chassis is often used to build cell factories due to its clear genetic background, well-performed gene manipulation tools, and mature industrial system. A series of advances has been made in the production of aromatic compounds by engineering yeast chassis, and several feasible solutions have been proposed to solve key problems. In view of the strategies and challenges of yeast synthesis of aromatic compounds, this paper systematically reviews and elaborates on the five aspects, namely, the aromatic compound synthetic pathway modification, utilization of diverse carbon sources and modification of transport systems, genome multi-target modification, special yeast chassis and microbial consortium construction, and applications of synthetic biology high-throughput technologies, and provides ideas for the construction and engineering of yeast chassis for producing aromatic compounds.
With the rise of green chemistry, research on the transformation and utilization of natural cellulose raw materials has been highly valued and widely applied. Cellulose degradation by cellulase has paved the way for fuel ethanol and biodiesel production. However, the high production cost of cellulase limits its industrial application. Trichoderma reesei is a cellulase-producing strain and produces abundant cellulase components. Comprehensive studies on the mechanism of cellulase induction and expression regulation of Trichoderma reesei are helpful to improve its cellulase production. In recent years great progress has been made on the induction process and regulation mechanism of cellulase production by Trichoderma reesei. This review summarizes recent progresses of the regulation of cellulase induction and gene expression by Trichoderma reesei, by first introducing the inducers (cellulose, cellobiose, sophorose, lactose, etc.) and their transporters, then summarizing the regulation function of several transcription factors, and moreover, the effects of chromosomal regulation, signaling pathway, and illumination conditions on cellulase induction are also introduced. Finally, the future research directions of cellulase induction in Trichoderma reesei was discussed, including exploring the nature and specific process of inducers, revealing the relationship between transcription factors and transcriptional regulatory network, searching for key functional proteins of signal transduction, and studying the effect of environmental factors on cellulase induction.
In vitro diagnostics plays an important role in the process of disease diagnosis and treatment, which is referred to as the doctor’s eye. In the context of the unprecedented increase in people’s attention to life and health caused by the COVID-19 pandemic, in vitro diagnostics has showed an explosive growth as an important part of the health industry. For the field of in vitro diagnostics, the developing status of the world and China was summarized. Particularly, the market situation, competition landscape, and national-made situation were analyzed. The problems in the current stage of in-vitro diagnostics industry in China were analyzed, and then the corresponding suggestions and countermeasures were put forward from some aspects, including making a breakthrough in original innovation, strengthening the guarantee of funds, building a complete industrial chain, and strengthening personnel training.
A new pattern of “two super companies, four strong companies, and differentiated development”has been formed basically in the global seed industry at present, and the seed industry giants are leading the research and development (R & D)of global crop breeding technology and industrial development. Study on technology R & D layout of multinational seed companies by in-depth analysis and mining of their crop breeding patents can be used as a reference for China to reasonably deploy crop breeding technology R & D and improve the layout and protection of intellectual property rights. The biological breeding R & D layout of the“two super and four strong”multinational seed companies was studied by analyzing their crop biological breeding patents applied for from 2015 to 2019 in Derwent Innovation (DI) database through text clustering method, and their focus of technology R & D was clarified by selecting key patents through measurement indicators and expert consultation. It was suggested that China should aim at the core field of biological breeding, strengthen the original innovation and integrated development of emerging cutting-edge technologies, enhance the exploration of new insect resistant genes and insect resistant mechanisms, intensify the collaborative protection and layout of intellectual property rights in the core technology chain and industrial chain of biological breeding, and improve the intellectual property protection level and intellectual property strategic awareness of globalization protection combinied with key layout.
Viruses are one of the main pathogens that endanger human health. The infectious diseases caused by virus infection and transmission seriously threaten human health. At present, viral diseases with high morbidity and low cure rate such as AIDS and viral hepatitis are still spreading around the whole world, and respiratory viruses such as influenza virus and corona virus are constantly mutating. Since 2019, the global epidemic caused by SARS-CoV-2 has brought severe challenges to the world, and there are still great uncertainties in the future course of the epidemic. Therefore, the development of safe and effective antiviral drugs has become an important means to deal with viral diseases. On the basis of summarizing the overall status of global antiviral drug research and development, this paper intends to analyze the progress of new drug research in key areas such as anti-HIV, hepatitis virus and SARS-CoV-2, and put forward suggestions to provide guidance and reference for the development of more efficient antiviral drugs in the future.
