Objective: Programmed cell death protein (PD-1) is a T cell immune checkpoint and an important target for tumor therapy. This article used CRISPR/Cas9 technology to repair the introduced mutations by non-homologous recombination, causing the frame shift of the gene protein reading frame and the loss of PD-1 function. Estabilishment of Pd-1 gene knockout mouse model provides the basis for in-depth exploration of Pd-1 gene function and mechanism. Methods: We designed and synthesized 2 pairs of sgRNA fragments for exons 2-4 of the Pd-1 gene, and transcribed them in vitro together with the Cas9 fragments encoding them. The two mRNAs were mixed into C57BL/6 mouse fertilized eggs by microinjection. F0 generation mice were obtained by PCR product sequencing and then mated with wild-type C57BL/6 mice to obtain F1 generation heterozygous mice. F1 generation mice were intercoursed to obtain F2 generation homozygous mouse strains (Pd-1-/-). After it was stimulated with concanavalin (ConA), PD-1 in Pd-1-/- mice was detected by Real-Time fluorescent quantitative PCR and flow cytometry at the mRNA and protein levels, respectively. The expression levels of IL-6, IFN-γ, IL12/IL23 and TNF-α in the serum of Pd-1-/- mice were detected by the ELISA method, and the mechanism of Pd-1 pathway in the regulation of T cell response and its countermeasures were preliminarily analyzed. Results: PCR and sequencing results showed that exons 2-4 of the Pd-1 gene in the mouse genome were successfully knocked out; Real-Time PCR experiments and flow cytometry results showed that the expression of PD-1 was significantly reduced in Pd-1-/- spleen, mesenteric lymph nodes, thymus and blood tissues compared with wild-type mice; the double-antibody sandwich ELISA test results showed that the expression of serum IL-6 and IFN-γ is up-regulated stimulated by ConA after Pd-1 gene was knocked out. Conclusion: The Pd-1 gene knockout mouse model has been successfully constructed. Preliminary analysis shows that Pd-1 deletion can upregulate the response of IL-6 and IFN-γ to ConA stimulation, increase the inflammatory response caused by ConA, and provide a new mouse model for the study of Pd-1 in vivo gene function and research ideas.
Objective: To construct the recombinant expression system of Cec4a, and to obtain the recombinant protein by induced expression and detect the antibacterial activity of the product. Methods: Based on the primers designed according to the sequence of Cec4a, the mature peptide part of Cec4a gene was amplified by PCR. Recombinant prokaryotic expression plasmid was constructed using prokaryotic expression vector (pCold-SUMO) and transformed into E. coli C41 (DE3) competent cells, which were induced by IPTG. His-SUMO labeled recombinant Cec4a fusion protein was purified by Ni-NTA affinity chromatography. The target protein was purified by Ni-NTA affinity chromatography after SUMO protease digestion. Acinetobacter baumannii (ATCC19606) was used to detect the antibacterial activity of the product. Results: pCold-SUMO-Cec4a prokaryotic expression plasmid was successfully constructed, and the sequencing analysis was consistent with the expected results. The expression level of Cec4a fusion protein was 42.8mg/L, and the MIC of purified Cec4a recombinant protein against Acinetobacter baumannii was 4 μg/mL. Conclusion: The recombinant Cec4a protein with antibacterial activity was successfully constructed and purified by Ni-NTA affinity chromatography. It lays a foundation for further study on the biological activity, the relationship between the structure and function of Cec4a.
Objective: In order to promote the enzymatic complete detoxification of zearalenone and the application in feed industry, we proposed to develop a new hydrolase exhibiting higher activity to α-ZOL. Methods: A novel zearalenone hydrolase EaZHD sequence from Exophiala aquamarina CBS 119918 with a total length of 263 amino acids was obtained. The recombinant plasmid pET46-Eazhd was constructed and expressed in E. coli. Then EaZHD was purified with Ni-NTA affinity chromatography and DEAE ion exchange column. The enzymatic properties and activity analysis were evaluated with HPLC. Results: The results showed that the activity of EaZHD was 0.764 U/mg against ZEN and 1.529 U/mg against α-ZOL, which is 2-fold higher. The optimal pH of EaZHD was 8.6 and the optimal temperature was 40 °C. It had a better stability at alkaline pH. The thermal stability is better than ZHD101. Three amino acid residues around active site were identified to play a key role in the catalysis activity and the other two residues were found to affect the substrate specificity. Conclusion: The study provides the basis for the enzymatic detoxification of zearalenone in the feed and stock raising industries.
Objective: To establish a method for detecting the infectious titers of adeno-associated virus type 9 (AAV9) vector products based on the median tissue culture infective dose (TCID50). Methods: HEK-293 cells were co-infected with HSV1 containing rep and cap genes of AAV2 and gradient diluted AAV9 vector products. After 48 hours of culture, the AAV-specific inverted terminal repeats (ITR) were amplified by quantitative real-time PCR(qPCR), and the infectivity titer of the sample was calculated by the Kärber method based on the number of positive and negative infected wells. Results: The AAV9 vector product carrying the enhanced green fluorescent protein reporter gene was used to determine the optimal multiplicity of infection (MOI) of the helper virus HSV1-rc as 5. The infectious titer of AAV9-101 is 1.6×109 TCID50/mL. Conclusion: This method can detect the infectious titer of AAV9 vector products with repeatability.
Objective: Marek’s disease virus (MDV) is an alphaherpesvirus and is classified into three serotypes: MDV serotype 1 (MDV-1) which includes the pathogenic strains and their derivatives; MDV serotype 2 (MDV-2) which consists of non-oncogenic viruses; and MDV serotype 3 (MDV-3), which is also referred to as turkey herpesvirus 1 (HVT). All three MDV serotypes have been used as vaccines. CVI988 is a cell culture passage attenuated MDV-1 virus and the gold standard among MD vaccines. CVI988 could be used as a viral vector to express exogenous gene. MDV and Newcastle disease virus (NDV) co-infection in chickens is very popular in the field, but there are few candidates of recombinant vaccines to prevent the infection of both viruses at the same time. Methods: In this study, CVI988 expressing F gene was constructed by the bacterial artificial chromosome (BAC) technology and packaged into recombinant virus. After it was amplified by PCR, the F gene together with Kana gene fragment was integrated into CVI988 BAC by homologous recombination to generate CVI988 BAC-F. Subsequently, CVI988 BAC-F was transfected into chicken embryo fibroblast (CEF) cells by calcium phosphate to rescue recombinant virus, which was confirmed by Western blotting and indirect immunofluorescence assay (IFA) assays. Results: The results of virus growth curve and plaque area measurement showed that the insertion of F gene did not affect virus proliferation in vitro. Conclusion: We successfully constructed a recombinant CVI988 BAC virus, which provides a basis for development of novel vaccines to prevent and control co-infection of NDV and MDV.
Currently, with the severity of population aging, cardiovascular disease(CVD) brings about health problems and unbearable economic burdens. In ischemic illnesses caused by the damage of small-diameter blood vessels, blood vessel transplantation has become an effective solution to tackle this challenge. However, small-diameter blood vessels are currently in high demand. Therefore, it is pretty crucial to construct small-diameter tissue-engineered blood vessels (TEBV) using tissue engineering methods. With the advancement of tissue engineering and 3D printing technology, the research of vascular grafts has developed rapidly. At present, most of the vascular graft materials used for large-diameter vascular grafts are polyester and polytetrafluoroethylene (PTFE). However, it is not applicable for the fabrication of small-diameter TEBV, in which case a myriad of unavoidable problems may come alone, such as inflammation and thrombosis. At the same time, current TEBV has such limitations as insufficient mechanical properties, which seriously hinder the clinical translation of TEBV. Therefore, in this experiment, we independently synthesized methacrylated gelatin (GelMA) and RGD-modified sodium alginate (RGD-Alginate) combined to form a double cross-linking system. By adding xanthan gum, the printability of the system is guaranteed. We used coaxial printing to fabricate a tube-like structure. Hybrid material system was characterized by a low vacuum cryo-scanning electron microscope. We found honeycomb-like forms appear on the surface, indicating that oxygen and nutrients could be provided to the cells in the tube through penetration. As for the selection of materials, the sacrificial material in the inner layer is 25% Pluronic F127 dissolved in 2% calcium chloride (CaCl2), and the outer material is 4% RGD-Alginate+5% GelMA+2% Xanthan Gum. During the printing process, the extrusion pressure of the printer is related to the diameter of the selected coaxial nozzles. When the 18G/14G coaxial nozzle is applied, the printing pressure is 55 kPa, and the printing speed is 5 mm/s. The syringe pump is utilized to extrude the material of the outer layer, whose speed is 264 μL/min. In the printing procedure, we selected two nozzles with different diameters to effectively fabricate a tube matching the nozzle diameter. In addition, a device for detecting burst pressure was established, which uses constant extrusion of the syringe pump to provide stable pressure to the tested tube. It has been demonstrated that the burst pressure is 328 mmHg±14 mmHg, which is quite different from the burst pressure of natural blood vessels in vivo. At the same time, it is sufficient to bear the vascular pressure in the physiological state of the human body. Human umbilical vein endothelial cells (HUVECs) were perfused into the tube-like structure (cell concentration was 1×107/mL). Through the imaging characterization of the cell state in the tube-like structure, it was found that HUVECs can be stably attached to the inner wall of a fabricated tube-like structure.
Objective: The Pichia pastoris strain was engineered to heterologously synthesize cinnamic acid and ρ-coumaric acid,which were important intermediates of flavonoid biosynthetic pathway. The biosynthetic pathway of precursors aromatic amino acids was optimized to improve the production capacity of P. pastoris. Methods: Phenylalanine ammonia lyase from Rhodotorula glutinis was expressed in P. pastoris GS115 by ethanol induced artificial transcription system, and the key enzymes or their mutants in the biosynthetic pathway of intracellular aromatic amino acids were overexpressed in the recombinant strain. Results: Heterologous expression of phenylalanine ammonia lyase could convert L-phenylalanine and L-tyrosine produced by P. pastoris into cinnamic acid (38.8 mg/L) and ρ-coumaric acid (34.2 mg/L). Through overexpression of related enzymes, the yields of cinnamic acid and ρ-coumaric acid reached 124.1 mg/L and 302.0 mg/L, respectively. Conclusion: Cinnamic acid and ρ-coumaric acid were successfully synthesized by P. pastoris, and the biosynthetic pathway of intracellular aromatic amino acids was optimized. It shows that P. pastoris has the application potential to produce flavonoids, and it also lays a foundation for the heterologous synthesis of other aromatic amino acid derivatives or plant compounds in P. pastoris.
Bone marrow mesenchymal stem cells are a type of adult stem cells with self-replication and multi-differentiation potential. It can differentiate into osteoblasts, chondrocytes, adipocytes, etc. through specific induction, so it has become the ideal seed cell for the most studies on bone regenerative medicine and cell therapy. Current studies have confirmed that in the process of bone marrow mesenchymal stem cells repairing bone defects, the expression increases of chondrogenesis-related genes in bone marrow mesenchymal stem cells, and then differentiate into chondrocytes. Later, with the formation of osteoblasts and osteoclasts and the growth of blood vessels, the cartilage matrix is gradually degraded and replaced by bone matrix. It shows that chondrocytes are involved in the pre-repair process of bone defects, and the signal pathways and related factors that regulate cartilage formation not only regulate the differentiation of bone marrow mesenchymal stem cells into chondrocytes, at the same time, they also play an important role in the process of osteoblast differentiation. Therefore, this article summarizes the regulatory effects and current research status of the signal pathways and related factors that regulate cartilage formation in the bone differentiation of bone marrow mesenchymal stem cells, in order to provide a theoretical basis and research direction for the clinical search for better treatment of bone defects.
Field-Effect Transistor (FET) biosensors have received a lot of attention due to their high sensitivity, fast analysis speed, no labeling, small size and simple operation. They are widely used in the detection of DNA, protein, cell, ion and other biomarkers. In recent years, nanomaterials and microelectronics technologies improve the sensing performance of sensors in sensor design. Field-effect transistor biosensors are developing at an amazing speed in the direction of high sensitivity, miniaturization, rapidness and multi-function. To study the working principle of field-effect transistor biosensors, to explain the latest research progress and applications of field-effect transistor biosensors in the field of biomedical testing in recent years, and to discuss the countermeasures of field-effect transistor biosensors to overcome various shortcomings provide a reference for the development of the sensor in future biomedical testing, which is of great significance to promote its development and application.
Extracellular electron transfer is the process that electroactive microorganisms (EAMs) acquire energy from the environment by extending respiratory chains to external electron acceptors. Organism Shewanella oneidensis is widely used as a model to study emerging bioelectrochemical technologies, including microbial fuel cell (MFC), microbial electrosynthesis, as well as pollutant degradation in bioremediation. A previous study reported that almost all electrons generated by S. oneidensis were transmitted into acceptors relying on flavins, including flavin mononucleotide (FMN) and riboflavin (RF). What’s more, flavins-mediated extracellular electron transfer is the rate-limiting step in the process of electron transmission. However, flavins secreted by wild-type S. oneidensis are negligible, and the engineering modifications for S. oneidensis are limited, both of which seriously hinder the electrons transfer process, and enable the main bottleneck of restricting the electrons transmission. In this study, the regulatory factors of flavin synthesis were systematically demonstrated from the perspective of flavin biosynthesis and transcriptional regulation relying on the mechanisms of flavins-mediated electron transfer in S. oneidensis. Besides, the strategies utilizing metabolic engineering, synthetic biology and modification of electrode materials for improving flavins-mediated electron transfer in recent years were summarized. Further, it can be proposed that in the future, systematic research and expression tools can be utilized to accelerate the flavins-mediated extracellular electron transfer in EAMs.
Functional enzymes have been widely used in the fields of food, chemicals, medicine, etc. However, high temperature reduces the catalytic efficiency of enzymes. The protein engineering technology can be used as a key link to modify enzymes’ structure and function, and to obtain industrial enzymes with thermostability. Traditional directed evolution methods can only rely on random mutations for manual screening with low efficiency. Rational design, as the main method of thermostability modification, can be used to predict potential mutation sites with various computer programs and software, but it requires deep understanding of the catalytic and thermal stability mechanism. For most natural enzymes, it is easy to obtain sequence and crystal structure, and is also an important basis for predicting function. This paper focuses on the modification strategies as follows: common mutation, the mutation based on amino acid preference, trunking of flexible regions, the optimization of intramolecular interactions, the modification of catalytic active regions and computer-aided design with the sequence and crystal structure analysis. These strategies have the advantages of high screening efficiency and modification accuracy, and strong practicability. And it also analyzes the thermostability modification cases of different enzymes, aiming to provide an effective reference for the selection of modification strategies, and also give theoretical support for the heat resistance research of industrial enzymes.
As a petroleum distillate fuel with high calorific value and low consumption rate, diesel can be matched with the use standard of high-power machinery. It accounts for an increasing proportion in traditional energy. It is widely used in the operation and production of various large-scale equipment. With the increase of diesel consumption, the pollution problem of diesel has begun to drawn attention. Sulfur, as the main pollutant, has higher requirements in the new diesel standard. It is necessary to explore the desulfurization methods and innovate the technology. Traditional hydrodesulfurization has a number of limitations, so a variety of non hydrodesulfurization methods have been developed for desulfurization research, aiming to develop a green desulfurization method with high efficiency and environmental friendliness. This paper mainly summarizes the advantages and disadvantages of various conventional desulfurization methods. The research status and the latest progress of enzyme catalyzed oxidative desulfurization at home and abroad were reviewed. The reaction mechanism and specific research examples of various desulfurization methods were discussed. On this basis, the various methods were summarized and analyzed, and the author’s point of view and the prospect of new desulfurization methods in the future were put forward.
Bioeconomy is the sum of all economic activities that use renewable natural resources to produce food, energy, biotechnology products and services in a sustainable way. Bioeconomy is the fourth wave of human economic and social development after agricultural economy, industrial economy and information economy. This paper outlines the current situation of global bioeconomy development, sorts out the bioeconomic strategic layout of the world’s major economies, and sums up the four main directions for future development of the bioeconomy. Through analysis of the investment data of three key areas of bio-industry, it predicts the future investment prospects of the bio-industry, and puts forward some suggestions on investment of bio-industry in China. The purpose of this paper is to provide reference information for the development and value investment of the bio-industry for the participants and related parties, in order to promote the transfer and transformation of scientific and technological achievements, technology capital docking and development of the bio-industry, and make contributions to the sustainable development of China’s economy and society.
Biotechnology base platforms are important foundation for biotechnology research, production and services, and meanwhile, policies related to biotechnology base platforms play an important role in guiding and promoting the development of biotechnology base platforms in China. This article takes the biotechnology base platform policies formulated by the Central Committee of the Communist Party of China, the State Council and the Ministry of Science and Technology and other government sectors in the past ten years as study object. Through textual analysis, this article summarizes the value connotation of China’s biotechnology base platform policies, and discusses the construction law, so as to provide reference for the innovation and development of the base platform.
