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

Nanobodies (Nbs), the unique single-domain antibodies discovered in the species of Camelidae and sharks, are also known as the variable domain of the heavy chain of heavy-chain antibody (VHH). They offer strong antigen targeting and binding capabilities and overcome the drawbacks such as large size, low stability, high immunogenicity, and slow clearance of conventional antibodies. Nbs can be boosted by bioconjugation with toxins, enzymes, radioactive nucleotides, fluorophores, and other functional groups, demonstrating potential applications in the diagnosis and treatment of human and animal diseases. This article introduces the structures and characteristics of Nbs, the construction and screening of Nb libraries, and the strategies for affinity maturation and then reviews the current applications of Nbs in diagnosis and treatment, providing a reference for the development of diagnostic reagents and clinical therapies for infectious diseases.

In order to establish a stable in vitro culture platform for chicken small intestine three-dimensional (3D) organoids, in this study, crypt cells were collected from the small intestine of 18-day-old embryos of AA broilers. On the basis of the L-WRN conditioned medium, we optimized the culture conditions of chicken small intestinal organoids by adjusting the proportions of nicotinamide, N-acetylcysteine, LY2157299, CHIR99021, Jagged-1, FGF, and other cytokines to select the medium suitable for the long-term stable growth of the organoids. The optimization results showed that the addition of 1.5 µmol/L CHIR99021 significantly improved the organoid formation efficiency and organoid diameter. When 0.5 µmol/L Jagged-1 was added, a small amount of bud-like tissue appeared in organoids. After the addition of 50 ng/mL FGF-2, the rate of organoid germination was significantly increased. The 1.5 µmol/L CHIR99021, 0.5 µmol/L Jagged-1, and 50 ng/mL FGF-2 added in the medium can cooperate with each other to improve the formation and speed up the proliferation and differentiation of organoids, while improving the stemness maintenance of cells. The morphology, cell types, and culture characteristics of chicken small intestinal organoids were studied by HE staining, transmission electron microscopy, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), indirect immunofluorescence, and immunohistochemistry. The results showed that the 3D organoids of the chicken small intestine cultured in vitro were morphologically consistent with the chicken intestinal tissue and contained differentiated epithelial cells. In summary, we successfully established an in vitro culture system for chicken small intestinal organoids, providing a new method for the subsequent research on chicken intestinal physiology, pathology, and host-pathogen interaction mechanism and the development of relevant drugs.

This study developed ferritin-based nanoparticles carrying the African swine fever virus (ASFV) p30 protein and evaluated their immunogenicity, aiming to provide an experimental basis for the research on nanoparticle vaccines against ASFV. Initially, the gene sequences encoding the p30 protein and SpyTag were fused and inserted into the pCold-I vector to create the pCold-p30 plasmid. The gene sequences encoding SpyCatcher and ferritin were fused and then inserted into the pET-28a(+) vector to produce the pET-F-np plasmid. Both plasmids were expressed in Escherichia coli upon induction. Subsequently, the affinity chromatography-purified p30 protein was conjugated with ferritin in vitro, and the p30-ferritin (F-p30) nanoparticles were purified by size-exclusion chromatography. The morphology and structural integrity of F-p30 nanoparticles were examined by a particle size analyzer and transmission electron microscopy. Mice were immunized with F-p30 nanoparticles, and the humoral and cellular immune responses were assessed. The results showed that F-p30 nanoparticles were successfully prepared, with the particle size of approximately 20 nm. F-p30 nanoparticles were efficiently internalized by bone marrow-derived dendritic cells (BMDCs) cells in vitro. Compared with the p30 protein alone, F-p30 nanoparticles induced elevated levels of specific antibodies and cytokines in mice and stimulated the proliferation of follicular helper T cell (T_FH) and germinal center B cell (GCB) in lymph nodes as well as CD4⁺ and CD8⁺ T cells in the spleen. In conclusion, we successfully prepared F-p30 nanoparticles which significantly enhanced the immunogenicity of p30 protein, giving insights into the development of vaccines against ASFV.

Metallothionein (MT) plays a significant role in heavy metal removal, antioxidant defense, and immune regulation. The current predominant approach for obtaining natural MT is extraction from tissue, which often entails complex procedures resulting in limited yields. In recent years, researchers have adopted the strategy of fusing labels such as GST or His for the heterologous expression of MT. However, a challenge in industrial production arises from the subsequent removal of these labels, which often leads to a significant reduction in the yield. The fusion with elastin-like polypeptides (ELPs) offers a promising solution for achieving soluble expression of the target protein, while providing a simple and fast purification process. In this study, ELP was fused with MT, which significantly up-regulated the soluble expression of MT. The fusion protein ELP-MT with the purity above 97% was obtained efficiently and simply by inverse transition cycling (ITC). ELP-MT exhibited a remarkable 2,2'-azinobis(3-ethylbenzothiazoline-6- sulfonic acid) ammonium salt (ABTS) scavenging activity, with the half maximal inhibitory concentration (IC₅₀) of 0.77 μmol/L, which was 53.7 times that of the vitamin E derivative Trolox. In addition, the fusion protein demonstrated strong 1,1-diphenyl-2-trinitrohydrazine (DPPH) scavenging ability. Furthermore, ELP-MT had no toxicity to the proliferation and promoted the adhesion and migration of NIH/3T3 cells. All these results indicated that ELP-MT had good biocompatibility. We constructed the fusion protein ELP-MT combining the unique properties of MT and elastin, laying a technical foundation for the large-scale production of recombinant MT and facilitating the applications in food, health supplement, and cosmetic industries.

Protein phosphorylation plays a key role in Mycobacterium tuberculosis, the pathogen of tuberculosis, holding promise as a new target of anti-tuberculosis drugs. We used M. smegmatis, a close relative of M. tuberculosis, as a model organism to study the protein phosphorylation at different growth phases. We identified 573 phosphorylated peptides and 816 phosphorylated sites of 385 proteins in the M. smegmatis samples at both logarithmic and stationary phases, and then established a comprehensive dataset of phosphorylated proteins in M. smegmatis. By comparing the expression levels of phosphorylated proteins between the logarithmic and the stationary phase with the selected ion monitoring (SIM) strategy, we verified 68 upregulated proteins involved in cell division and protein translation, and 69 downregulated proteins mainly involved in the tricarboxylic acid cycle pathway. The differentially expressed phosphorylated proteins were significantly enriched in important cellular cycle events such as cell elongation and division. The findings of this study provide proteome evidence for elucidating the phosphorylation in both M. smegmatis and M. tuberculosis.

The small G-protein Rac1 is the main regulatory factor of the actin cytoskeleton. Rac1 cycles between the inactive GDP-bound form and the active GTP-bound form. Rac1 not only promotes viral replication and infection, but also regulates the actin cytoskeleton rearrangement, adhesion, and invasion of glioma cells. In addition, Rac1 is implicated in human diseases such as tumors and epilepsy. This article reviews the latest research on the small G-protein Rac1 in virology, cell biology, and human pathology. It is found that the existence of Rac1 is closely related to the replication and infection of viruses, that is, inhibiting the existence of Rac1 can effectively reduce the replication and transportation of viruses, providing new ideas for the development of various therapeutic drugs targeting Rac1.

Human epidermal growth factor (hEGF) can be applied in the treatment of surgical trauma (burns, scalds), tissue repair, skin moisturizing, beauty, skincare, etc. However, the low expression and high cost limit the application of hEGF. In order to improve the expression level of hEGF and reduce the production cost, considering the high expression of polyhedrin, this study fused a partial sequence of polyhedrin with hEGF and expressed the fused sequence by using a silkworm baculovirus expression vector system. In view of the small molecular weight of hEGF, we connected hEGF genes in series and optimized the codons to construct multiple fusion expression vectors by fusing different partial sequences of polyhedrin at the N-terminus. The results showed that through the above strategy, the protein expression level of hEGF was significantly increased. The expression vector containing three concatenated hEGF genes with optimized codons and fused with the sequence encoding 25 or 35 residues at the N-terminus of polyhedrin showed the highest expression level.

We knocked out the retinoic acid-inducible gene I (RIG-I) in HEK293 cells via CRISPR/Cas9 to reveal the effects of RIG-I knockout on the key factors in the type I interferon signaling pathway. Three single guide RNAs (sgRNAs) targeting RIG-I were designed, and the recombination vectors were constructed on the basis of the pX459 vector and used to transfect HEK293 cells, which were screened by puromycin subsequently. Furthermore, a mimic of virus, poly I: C, was used to transfect the cells screened out. RIG-I knockout was checked by sequencing, real-time quantitative PCR, Western blotting, and immunofluorescence assay. Meanwhile, the expression levels of key factors of type I interferon signaling pathway such as melanoma differentiation-associated gene 5 (MDA5), interferonβ1 (IFNβ1), and nuclear factor-kappa B p65 [NF-κB(p65)], as well as cell viability, were determined. The results showed that two HEK293 cell lines (S1 and S3) with RIG-I knockout were obtained, which exhibited lower mRNA and protein levels of RIG-I than the wild type HEK293 cells (P < 0.05). The mRNA levels of MDA5 and IFNβ1 in S1 and S3 cells and the protein level of NF-κB(p65) in S3 cells were lower than those in the wild type (P < 0.05). More extranuclear NF-κB(p65) protein was detected in S1 cells than in the wild type after transfection with poly I: C. Plus, the wild-type and S1 cells transfected with poly I: C for 48 h showcased reduced viability (P < 0.05), while S3 cells did not display the reduction in cell viability. In summary, the present study obtained two HEK293 cell lines with RIG-I knockout via CRISPR/Cas9, which provided a stable cell model for exploring the mechanism of type I interferon signaling pathway.

The transcription factors (TFs) in the NAC family are involved in regulating multiple biological processes, playing an important role in plant growth, development, and stress adaptation. Our previous studies have demonstrated that TaNAC14, a member of the NAC family in wheat (Triticum aestivum L.), positively regulates root growth and development and enhances the drought tolerance of wheat seedlings. In this study, we analyzed the physicochemical properties and structure and verified the subcellular localization and transcriptional activation activity of TaNAC14. The prokaryotic expression vector pET21a-HMT-TaNAC14 was constructed and transformed into Escherichia coli BL21 CodonPlus (DE3)-RIPL. The conditions for inducing the expression of the recombinant protein HMT-TaNAC14 were optimized. The solubility of the recombinant protein was analyzed, and the protein was purified by affinity chromatography on a Ni-nitrilotriacetic acid column. The results indicated that TaNAC14 had a conserved domain of the NAM family. It was located in the nucleus and had transcriptional activation activity. The optimal conditions for expression of the recombinant protein in E. coli were induction with 0.2mmol/L IPTG for 4 h. The recombinant protein mainly existed in the soluble form, and the target protein was obtained after purification. This study lays a foundation for the identification of target genes regulated by TaNAC14.

Proteins with specific functions and characteristics play a crucial role in biomedicine and nanotechnology. De novo protein design enables the customization of sequences to produce proteins with desired structures that do not exist in the nature. In recent years, with the rapid development of artificial intelligence (AI), deep learning-based generative models have increasingly become powerful tools, enabling the design of functional proteins with atomic-level precision. This article provides an overview of the evolution of de novo protein design, with focus on the latest algorithmic models, and then analyzes existing challenges such as low design success rates, insufficient accuracy, and dependence on experimental validation. Furthermore, this article discusses the future trends in protein design, aiming to provide insights for researchers and practitioners in this field.