Objective: This paper aims to construct engineered exosomes (enExos) by membrane surface modification technology, and use enExos to mediate the removal of epidermal growth factor receptor (EGFR)-rich tumor exosomes on the membrane surface by macrophages. Methods: The exosomes displaying the chemokine 8 (CXCL8) on the membrane surface were obtained by surface display technology. Meanwhile, the EGFR nucleic acid aptamer was modified on the CXCL8-expressing exosomes to prepare enExos. The size and potential of enExos were analyzed by nanoparticle tracking analysis and nanoparticle size potentiometer. Cell viability was measured by CCK-8 assay. The specific binding of enExos to EGFR-expressing tumor exosomes was observed by transmission electron microscopy. Finally, the phagocytosis of tumor exosomes with high EGFR expression by enExos-targeted chemotactic macrophages was detected by fluorescence imaging and flow cytometry. Results: The enExos with EGFR and CXCL8 displayed on the membrane surface were successfully constructed. enExos can specifically recognize and capture tumor exosomes with high EGFR expression, and at the same time use their chemokine CXCL8 to specifically target the macrophage membrane. Surface chemokine receptors CXCR1/CXCR2 stimulate the capture and clearance of tumor exosomes by macrophages. Conclusion: enExos promote the clearance of specific tumor exosomes, laying a foundation for the subsequent in-depth study of their role in inhibiting cancer metastasis, and they are expected to provide new research directions for cancer metastasis therapy.
Objective: The purpose is to provide a stable expression site with clear information for constructing a Chinese hamster ovary cell (CHO) line stably expressing recombinant protein by site-specific integration and shortening the research and development timeline. Methods: The CHO-K1-1d2 cell line with potential stable expression site randomly integrated with Zsgreen1 gene by lentivirus was continuously subcultured to verify the stability of expression; the integration site of lentiviral vector was analyzed by chromosome walking, and the editability of the site was verified by CRISPR/Cas9 technology. Results: 100% of CHO-K1-1d2 cells could emit green fluorescence and the fluorescence intensity was stable in the process of continuous adherent culture for 20 generations and suspension culture for 50 generations, indicating that Zsgreen1 protein could be stably expressed. The sequence results in chromosome walking analysis showed that the antiviral vectors were integrated between bases 1 159 463 and 1 159 467 of the CHO cell genome NW-003614092.1. The sequence results after co-transfection of sgRNA and Cas9 plasmid in CHO-K1 cells showed that this site can be edited by CRISPR/Cas9 technology. Conclusion: There is a stable expression site in CHO cell genome NW-003614092.1, which has clear information and can be edited by CRISPR/Cas9 technology.
Objective: The preparation of 4-vinyl derivatives by biological decarboxylation has many advantages and promising prospects. In this study, the enzymatic properties of Bacillus amyloliquefaciens Q-426 phenolic acid decarboxylase (BaPAD-Q-426) were studied in detail to provide theoretical basis for its future application. Methods: In this study, the phenolic acid decarboxylase gene was cloned from Bacillus amyloliquefaciens. Using pET-28a (+) as vector, the recombinant plasmid was transformed into E.coli BL21 (DE3), to achieve high expression of BaPAD-Q-426. It was purified by Ni-NTA affinity chromatography, and the enzymatic properties were identified. Results: BaPAD-Q-426 maintained good pH stability in the range of pH 7.0~9.0, and the optimum pH was 8.0. The enzyme maintained high enzyme activity in the range of 25~40℃, and the optimum temperature was 35℃. After holding at 4℃ for 30 minutes, the enzyme still maintained more than 95% enzyme activity. K+ significantly promoted the enzyme activity of BaPAD-Q-426 with an increase of 60%. This enzyme was well tolerated in petroleum ethers and retained more than 50% of the enzyme activity in the presence of 40% petroleum ethers. The optimum substrate of BaPAD-Q-426 was ferulic acid, and its enzyme activity reached 19.5 IU/mL. Conclusion: Compared with phenolic acid decarboxylases from other sources, BAPAD-Q-426 has better stability at low temperature and has the strongest catalytic decarboxylation of ferulic acid in weakly alkaline environment.
Objective: To establish a high-throughput platform for drug discovery targeting receptor binding domain (RBD) of SARS-CoV-2, a surface display system was designed and constructed to deliver functional RBD to the surface of Pichia pastoris. Methods: Four anchor molecules were fused to RBD, and then were transformed into Pichia pastoris by using electroporation. The surface display efficiency of RBD was measured using flow cytometry, and the affinity of RBD binding to the ACE2 receptor was further determined. Results: RBD-Sed1p system exhibited the highest surface display efficiency of 70%. The binding affinity to ACE2 of RBD displayed on the cellular surface (KD=30.42 nmol/L) was close to that of RBD in solution (KD=16.00 nmol/L). Conclusion: A surface display system of RBD was successfully developed in Pichia pastoris, which can be used for high-throughput screening and evaluation of anti-COVID-19 drugs.
Objective: The PiggyBac (PB) transposon is a mobile genetic element that transposes between vectors and chromosomes using a “cut and paste” mechanism. Transposon elements and transposase expression cassettes were integrated into an expression vector to construct an easy-to-use two-in-one PB transposon system. Methods: The transposon elements and transposase expression cassette required for the PiggyBac transposon system were obtained by polymerase chain reaction (PCR), and the transposase expression cassette was recombined with the original pUC18 vector using T4 DNA ligase. Gibson homologous recombination technology was used to combine transposon elements with recombination vector to construct a two-in-one PB transposon system. The system was tested for its efficacy and reliability using enhanced green fluorescent protein (EGFP) and functional damage suppressor protein (DSUP). Results: EGFP was visible and bright in all puromycin-resistant cells. In addition, a cell line stably expressing functional DSUP was obtained through the two-in-one PB transposon system, demonstrating that the foreign gene can be efficiently integrated into the genomic DNA and expressed. Conclusion: A new two-in-one PB transposition system was successfully constructed, which made the establishment of stable expression cell lines more convenient and economical.
Malignant tumor is one of the major diseases that threaten human life, and drug therapy is a common method. Currently, precision treatment has become a trend of tumor therapy. To achieve effective and precise drug therapy for malignant tumors, the drug screening models are very important points. Tumor organoids are three-dimensional cell models which have emerged in recent years. They have the advantages of retaining the characteristics and heterogeneity of parental tumors during long-term culture, with high success rate of culture and short culture cycle, and they can be used for high-throughput drug screening. They have been used for drug screening, predicting patients’ response of drug therapy, and providing guidance for personalized drug treatment. The progress of tumor organoids in drug screening and personalized drug treatment, and possible challenges were introduced.
The Chinese hamster ovary cells(CHO) cell expression system has become a widely used host expression system for the production of glycoproteins due to its high-density culture, high expression characteristics and relatively complete protein glycosylation modification system. Different CHO cell lines and various functional cell lines have been generated to date to meet the needs of large-scale production of glycoproteins and other experimental purposes. In recent years, with the development and application of genetic engineering, protein engineering, cell engineering, fermentation regulation and other technologies, breakthroughs have been made in the yield and degree of glycosylation of glycoproteins produced by CHO cells. However, with the increasing demand for glycoproteins in the biological product market, how to obtain a large quantity of glycoproteins with uniform mixture has also become an urgent problem to be solved. This review introduces the application of CHO and the research progress on gene construction and expression, enzyme engineering, cell lines, molecular chaperones, additives and physical conditions that affect the yield of expressed exogenous proteins and the degree of glycosylation modification in CHO cells. Combined with literature analysis, four directions for future CHO cell research are predicted: the development of new engineered CHO cell lines, a stable CHO expression system, synergistic strategies, and multi-omics applications. It is expected that the yield and quality of glycoproteins expressed by CHO cells can be improved in the future to meet the needs of clinical studies and research.
Monoclonal antibody disulfide bonds reduction has been a common issue in biopharmaceutical process, which could produce low-molecular weight fragments, affect product quality, and lead to decreased protein purity and stability. Moreover, it could also affect the safety and effectiveness of drugs. Antibody disulfide bonds reduction is a reversible redox reaction caused by intracellular thioredoxin system and glutathione system, and is related to the specific production process. In recent years, with the development of antibody drugs and mammalian cell culture scale, disulfide bonds reduction is observed more frequently. To solve this problem, scientists have been constantly developing mitigation strategies to ensure the product quality. In this paper, the antibody disulfide bonds structure, cause of disulfide bonds reduction and influencing factors in manufacturing process were summarized. It is focused on the prevention methods in the manufacturing process. Additionally, several feasible process analysis techniques are listed so as to provide reference for the further development of monoclonal antibody drugs in manufacturing.
Odd-chain fatty acids (OCFAs) are widely distributed in nature, while their level is low. OCFA has huge application potential in the fields of medicine, health, and industry. The current methods of obtaining OCFA are mainly included in the extraction and chemical synthesis, which limits its application due to the higher-cost and the lower-efficiency. Microbial fermentation is one of the most promising strategies for large-scale industrial production. This article briefly discusses the scope of application of OCFA, summarizes the microorganisms that can naturally synthesize OCFA, introduces in detail the related metabolic pathways involved in microbial synthesis of OCFA, and reviews the current strategies of genetic engineering and fermentation regulation for improving OCFA production. Taken together, this summary aims to provide a more systematic and comprehensive theoretical basis for improving OCFA production of microorganism by synthetic biology strategies.
In recent years, synthetic biology has made great progress in the design and construction of artificial life systems with the help of engineering ideas. In particular, the development and application of “cell factories” have brought profound changes to the synthesis of natural products. Cyclic lipopeptides are new natural surfactants. They can also be used as antibiotics because of their special structures and functions. At present, Bacillus are the most ideal microbial chassis for cyclic lipopeptides synthesis. Therefore, many researchers are devoted to improving the performance of Bacillus as cyclic lipopeptides cell factories through synthetic biology techniques. In this paper, the non-ribosomal peptide synthesis pathway of cyclic lipopeptides synthesized by Bacillus is first reviewed. Second, the regulatory factors related to the synthesis of cyclic lipopeptides are introduced. Third, the research progress of cyclic lipopeptides synthesis under the guidance of synthetic biology are summarized from the aspects including the selection of chassis cells, the development of gene editing tools, the optimization of synthesis pathway and the optimization of fermentation process. Finally, the possible challenges in cyclic lipopeptides synthesis are discussed and the future research trends are prospected. These will provide reference for the development of efficient cyclic lipopeptides cell factories.
Objective: The purpose is to reveal the research and technology development and patent layout of the 3D bioprinting industry, in order to provide competitive intelligence service for relevant institutions and data support for the development of the industry. Methods: Based on industry research and technology decomposition in the field of 3D bioprinting, we constructed queries to obtain data, and quantitatively analyzed relevant patents in multiple dimensions. Results: The development of 3D bioprinting industry can be divided into gestation period, budding period and high-speed development period; this industry has a low concentration and is in the stage of decentralized competition; applicants mostly choose partners based on geographical factors, and the partners are mostly different types of institutions; Chinese applicants account for nearly 50% of the world’s patent applications, but the average frequency of patent citations of American applicants is still far higher than that of China; American applicants pay more attention to overseas markets. Conclusion: A scale effect has not yet existed in 3D bioprinting industry, so it is necessary to integrate industry resources and build industrial clusters. Both China and the United States have dominant positions in this field, but China urgently needs to strengthen the layout of overseas patents. Taking into account the number and quality of patents, the patent competitiveness of US applicants is still higher than that of China, so China needs to strengthen the cultivation of core patents.
The explosive growth of global data has become an important engine for the development of the digital economy. However, traditional data storage media are limited by power consumption, volume and cost, and cannot meet the ever-increasing demand for data storage. The new storage method using deoxyribonucleic acid (DNA) molecule as storage medium has attracted great attention at home and abroad. Major countries in the world have carried out top-level planning for its research and deployed a series of important scientific research plans. However, DNA data storage is a new interdisciplinary research field, and its development “source” and “flow” still need to be deeply analyzed. To solve this problem, this paper explores the development “source” of DNA data storage from the perspective of fusion of information, semiconductor and synthetic biology, and analyzes and summarizes the development plan of DNA data storage in major countries and regions in the world in recent years. We present the layout of scientific research projects at home and abroad, particularly, the basic research program promoted by the Alliance for Semiconductor Synthetic Biology, the application-oriented intensive research program promoted by Defense Advanced Research Projects Agency (DARPA) and Intelligence Advanced Research Projects Activity (IARPA), the Horizon 2020 Program of the European Union, and the major research and development program of China. By comparison, it can be observed that the United States mainly adopts the government-led, application-oriented research mode, while the European Union and China follow up in time during the 13th Five-Year Plan period. During the 14th Five-year Plan period, China has set up the national key research and development program of “Fusion of the Biological Technology and Information Technology (BT and IT Fusion)”, which is committed to promoting the development of DNA data storage and related fields, and realizing the development of DNA data storage to drive the development of biochemical instruments, and even biological economy and digital economy. This paper explores the “source” and “flow” of the development of DNA data storage, and provides a reference for researchers to identify the “real” problems that really limit the development of this field, and also provides a reference for science and technology management departments to identify the international development trend in DNA data storage.
The National Natural Science Foundation of China has led and supported the basic research on microbiome for years. To a certain extent, the funding of the National Natural Science Foundation of China can reflect the research priorities and development trends in China’ s frontier science and technology fields. Based on the multi-dimensional analysis and comprehensive review of the funding of the National Natural Science Foundation of China during 2010-2021, the funding situation, intensity, and the main research groups of research on microbiome were revealed by analysis of the year, amount, type, and institution distribution of the funded projects. At the same time, the impact of the Natural Science Foundation of China on the scientific research influence and scientific research level of microbiome were discussed, in order to provide the basis for the management to formulate a reasonable discipline development plan.
