Sheng wu gong cheng xue bao = Chinese journal of biotechnology最新文献

Antimicrobial peptides (AMPs) are small molecular peptides widely existing in the innate immunity of organisms, serving as the first line of defense. Natural AMPs possess various biological activities and are difficult to develop drug resistance. However, they are easily broken down by digestive enzymes in the body. In recent years, increasing methods have been reported to enhance the stability of AMPs, including incorporation of unnatural amino acids, chemical modifications, strategic avoidance of enzyme cleavage sites, cyclization, and nano peptide design. This review summarizes the methods for improving the stability of AMPs against protease degradation, aiming to provide references for further research in this field.

To screen and identify the key host proteins interacting with the non-structural protein 15 (Nsp15) of porcine epidemic diarrhea virus (PEDV). The IP/pull-down assay and mass spectrometry were employed to screen and identify the host proteins interacting with Nsp15. The interaction between the host protein and Nsp15 was studied by co-immunoprecipitation and laser scanning confocal microscopy. Finally, Western blotting and RT-qPCR were employed to examine the interaction between SLC25a3 and PEDV. The recombinant eukaryotic expression vector pcDNA3.1(+)-Flag-Nsp15 was successfully constructed, and the host protein SLC25a3 interacting with PEDV Nsp15 was screened out. An interaction existed between SLC25a3 and Nsp15, and SLC25a3 significantly inhibited PEDV replication in a dose-dependent manner. SLC25a3 inhibits PEDV replication. The results of this study provide a basis for deciphering the role and mechanism of SLC25a3 in the host immune response to PEDV infection.

This study aimed to explore the roles of microRNAs (miRNAs) in the post-transcriptional regulation of cocaine- and amphetamine-regulated transcript (CART) peptide in the bovine hypothalamus and to screen key regulatory miRNAs. Targetscan was used to predict the potential miRNAs binding to CART 3' untranslated regions (3'UTR). Bioinformatics analysis predicted 7 miRNA binding sites in the bovine CART 3'UTR, which were bta-miR-377, bta-miR-331-3p, bta-miR-491, bta-miR-493, bta-miR-758, bta-miR-877, and bta-miR-381, respectively. Reverse transcription-PCR (RT-PCR) was carried out to determine the endogenous expression of CART and target miRNAs in the bovine hypothalamus. All the 7 target miRNAs and CART were endogenously expressed in the bovine hypothalamus. The dual-luciferase reporter gene assay was employed to detect the targeted binding relationship between CART 3'UTR and target miRNAs obtained from bioinformatics analysis. The dual-luciferase reporter gene assay confirmed that the 3'UTR of CART had a targeted binding relationship with the 7 target miRNAs. Cell experiments were conducted to examine the effects of target miRNAs on the messenger RNA (mRNA) and protein levels of exogenous CART and screen for key regulatory miRNAs. The results of cell experiments showed that the 7 miRNAs downregulated the mRNA level of CART, with bta-miR-491 demonstrating the strongest downregulating effect. Bta-miR-377, bta-miR-331-3p, bta-miR-491, bta-miR-493, and bta-miR-381 downregulated the protein level of CART, with bta-miR-381 exerting the strongest downregulating effect. Animal experiments were conducted to explore the effects of key regulatory miRNAs on the mRNA and protein levels of CART in the hypothalamus and the CART concentration in the serum. The results from animal experiments showed that miR-491 and miR-381 regulated the endogenous expression of CART in the hypothalamus and the concentration in the serum by binding to the CART 3'UTR. These results suggest that miR-491 and miR-381 are the main miRNAs regulating CART expression in the bovine hypothalamus, which can affect serum CART concentration by modulating endogenous CART expression.

Bluetongue virus (BTV) usually infects sheep, cattle, deer and other domesticated and wild ruminants through the bite of the vector insects, Culicoide, causing bluetongue (BT). BT in subtropical and even temperate regions poses a serious threat to the development and international trade of the livestock industry. This article introduced the structure and cellular invasion, and summarized the mechanisms of anti-BTV immune response of host cells and antagonism of host cell innate immune response by the non-structural proteins (e.g., NS3 and NS4) and structural proteins (e.g., VP3 and VP4) of BTV. This review provided a basis for understanding the antagonism mechanisms of BTV against the interferon (IFN) immune response in the host cell and the pathogenesis of BTV as well as for developing novel vaccines against this virus.

The purpose of this study is to construct a muscle-specific synthetic promoter library, screen out muscle-specific promoters with high activity, analyze the relationship between element composition and activity of highly active promoters, and provide a theoretical basis for artificial synthesis of promoters. In this study, 19 promoter fragments derived from muscle-specific elements, conserved elements, and viral regulatory sequences were selected and randomLy connected to construct a muscle-specific synthetic promoter library. The luciferase plasmids pCMV-Luc and pSPs-Luc were constructed and transfected into the myoblast cell line C2C12. The activities of the synthesized promoters were evaluated by the luciferase activity assay. Two non-muscle-derived cell lines HeLa and 3T3 were used to verify the muscle specificity of the highly active promoters. The sequences of promoters with high activity, good muscle specificity, and correct sequences were analyzed to explore the relationship between the element composition and activity of promoters. We successfully constructed a muscle-specific promoter library and screened out 321 effective synthetic promoter plasmids. Among them, the activity of SP-301 promoter was 5.63 times that of CMV. The 15 promoters with high activity were muscle-specific. In the promoters with high activity and correct sequences, there was a relationship between their element composition and activity. Muscle-specific elements accounted for a high proportion in the promoters, while they had weak correlations with the promoter activity, being tissue-specific determinants. Viral elements accounted for no less than 20% in highly active promoters, which may be the key elements for the promoter activity. The content of conserved elements was proportional to the promoter activity. This study lays a theoretical foundation for the synthesis of tissue-specific efficient promoters and provides a new idea for the construction and application of in-situ gene delivery systems.

This work aims to explore the effect of glycolysis on the replication of porcine reproductive and respiratory syndrome virus (PRRSV) in porcine alveolar macrophages (PAMs). The changes of glucose metabolism, PRRSV protein levels, PRRSV titers, and the relative expression levels of genes and proteins in PAMs were analyzed by ELISA, qPCR, virus titration, and Western blotting after PRRSV infection. The effect of hypoxia-inducible factor-1α (HIF-1α) on PRRSV replication was subsequently assessed by specific siRNAs targeting to HIF-1α. The results showed that PRRSV infection enhanced glycolysis, elevated the levels of glucose uptake and lactate in the supernatant (P<0.05 and 0.01, respectively), reduced ATP production (P<0.05), and up-regulated the expression of hexokinase 2 (HK2), 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3), and pyruvate kinase isozyme type M2 (PKM2) in PAMs (P<0.05 and 0.01, respectively). Glycolysis inhibitors down-regulated the expression of PRRSV proteins and reduced virus titers (P<0.01). The knockdown of HIF-1α by siRNAs inhibited glycolysis and lowered PRRSV titers (P<0.05). In addition, the interferon pathways inhibited by PRRSV infection were reversed by the inhibition of glycolysis. These findings may facilitate further investigation of the role of glycolysis in PRRSV replication.

Porcine deltacoronavirus (PDCoV) is a major pathogen causing fatal diarrhea in suckling piglets, and there is currently a lack of effective vaccines and drugs to prevent and control the virus. The nonstructural protein 13 (NSP13) serves as a virus-coded helicase and is considered to be a crucial target for antiviral drugs, making it imperative to investigate the helicase activity of NSP13. In this study, the NSP13 gene of PDCoV was synthesized and integrated into the prokaryotic expression vector pET-28a to construct the recombinant plasmid pET-28a-NSP13. NSP13 was successfully expressed in BL21 (DE3) and subsequently purified. The study also verified the helicase activity of the purified NSP13 and explored the factors that influence this activity. The results indicated that NSP13 from PDCoV was effectively expressed in the prokaryotic system and exhibited helicase activity, capable of unwinding double-stranded DNA with a tail at the 5' end. Additionally, NSP13 demonstrated an annealing function by promoting the complementary pairing of single-stranded nucleotide chains to form double strands. The helicase activity of NSP13 was affected by metal ions, but Mg²⁺concentrations in the range of 0.5-6.0 mmol/L had no significant effect on helicase activity of NSP13. When the solution pH was in the range of 4-9, there was no difference in helicase activity. ATP concentrations in the range of 0.25-6.00 mmol/L had a weak effect on helicase activity, and NSP13 concentration ≥80 nmol/L inhibited the helicase activity. We obtained the NSP13 of PDCoV and investigated its helicase activity. These findings provided a theoretical foundation for the further research on the regulatory mechanism of NSP13 in PDCoV replication and the development of anti-coronaviral drugs.

The structures and activities of enzymes are influenced by pH of the environment. Understanding and distinguishing the adaptation mechanisms of enzymes to extreme pH values is of great significance for elucidating the molecular mechanisms and promoting the industrial applications of enzymes. In this study, the ESM-2 protein language model was used to encode the secreted microbial proteins with the optimal performance above pH 9 and below pH 5, which yielded 47 725 high-pH protein sequences and 66 079 low-pH protein sequences, respectively. A deep learning model was constructed to identify protein acid-base tolerance based on amino acid sequences. The model showcased significantly higher accuracy than other methods, with the overall accuracy of 94.8%, precision of 91.8%, and a recall rate of 93.4% on the test set. Furthermore, we built a website (https://enzymepred.biodesign.ac.cn), which enabled users to predict the acid-base tolerance by submitting the protein sequences of enzymes. This study has accelerated the application of enzymes in various fields, including biotechnology, pharmaceuticals, and chemicals. It provides a powerful tool for the rapid screening and optimization of industrial enzymes.

The probiotic strain Escherichia coli Nissle 1917 (EcN) with high biocompatibility and susceptibility to genetic modification is often applied in bacterial therapies for cancer. However, most studies have used plasmids as vectors to construct engineering strains from EcN. Plasmid-based expression systems suffer from genetic instability, and they need antibiotic selective pressure to maintain high copy number. This study aimed to employ EcN for synthesizing the photosensitizer 5-aminolevulinic acid (5-ALA). Firstly, the key genes of 5-ALA synthesis, hemA^M and hemL, were integrated into the EcN genome by the phage integration technique. Then, chemically inducible chromosomal evolution (CIChE) was adopted to increase the copy number of hemA^M and hemL and thus improved the stable synthesis of 5-ALA. The in vitro cell experiments verified that the constructed engineering strain can deliver stably synthesized 5-ALA to tumor cells and inhibit their growth. This study provided a basis for applying the engineering strains of EcN in the photodynamic therapy for tumors.

Arsenic (As) is a common toxic pollution element. The microorganism-mediated transformation of arsenic forms is an important part in the biogeochemical cycle of As. In the various microbial metabolic processes involving As, the coupling reduction of As has a great impact on the environment and is a process that is easily overlooked. From the biogeochemical cycle of As, this review introduces the microorganism-mediated methane oxidation, anaerobic ammonium oxidation, and iron (Fe)-sulfur (S) oxidation coupled with As reduction. Organic matter, pH, and redox potential are the main factors affecting the coupling reduction. After the coupling reduction, the toxicity and migration of As are greatly enhanced, which may increase the risk of As pollution. Therefore, it is of great significance to clarify the influences of carbon, nitrogen, Fe, S and other elements on the coupling process and explore more microbial processes coupled with As reduction for the prevention and control of As pollution.