Development of novel monoclonal antibodies for blocking NF-κB activation induced by CD2v protein in African swine fever virus.

Background: CD2v, a critical outer envelope glycoprotein of the African swine fever virus (ASFV), plays a central role in the hemadsorption phenomenon during ASFV infection and is recognized as an essential immunoprotective protein. Monoclonal antibodies (mAbs) targeting CD2v have demonstrated promise in both diagnosing and combating African swine fever (ASF). The objective of this study was to develop specific monoclonal antibodies against CD2v. Methods: In this investigation, Recombinant CD2v was expressed in eukaryotic cells, and murine mAbs were generated through meticulous screening and hybridoma cloning. Various techniques, including indirect enzyme-linked immunosorbent assay (ELISA), western blotting, immunofluorescence assay (IFA), and bio-layer interferometry (BLI), were employed to characterize the mAbs. Epitope mapping was conducted using truncation mutants and epitope peptide mapping. Results: An optimal antibody pair for a highly sensitive sandwich ELISA was identified, and the antigenic structures recognized by the mAbs were elucidated. Two linear epitopes highly conserved in ASFV genotype II strains, particularly in Chinese endemic strains, were identified, along with a unique glycosylated epitope. Three mAbs, 2B25, 3G25, and 8G1, effectively blocked CD2v-induced NF-κB activation. Conclusions: This study provides valuable insights into the antigenic structure of ASFV CD2v. The mAbs obtained in this study hold great potential for use in the development of ASF diagnostic strategies, and the identified epitopes may contribute to vaccine development against ASFV.

Expression and Purification of Mammalian NMDA Receptor Protein for Functional Characterization.

N-methyl-D-aspartate (NMDA) receptors are critical for brain function and serve as drug targets for the treatment of neurological and psychiatric disorders. They typically form the tetrameric assembly of GluN1-GluN2 (2A to 2D) subtypes, with their diverse three-dimensional conformations linked with the physiologically relevant function in vivo. Purified proteins of tetrameric assembled NMDA receptors have broad applications in the structural elucidation, hybridoma technology for antibody production, and high-throughput drug screening. However, obtaining sufficient quantity and monodisperse NMDA receptor protein is still technically challenging. Here, we summarize a paradigm for the expression and purification of diverse NMDA receptor subtypes, with detailed descriptions on screening constructs by fluorescence size-exclusion chromatography (FSEC), generation of recombinant baculovirus, expression in the eukaryotic expression system, protein purification by affinity chromatography and size-exclusion chromatography (SEC), biochemical and functional validation assays.

Establishment of an Indirect ELISA Method for the Detection of the Bovine Rotavirus VP6 Protein.

The objective of this study was to develop an indirect ELISA utilizing a polyclonal antibody against bovine rotavirus (BRV) VP6 protein. To achieve this, pcDNA3.1-VP6, a recombinant eukaryotic expression plasmid, was constructed based on the sequence of the conserved BRV gene VP6 and was transfected into CHO-K1 cells using the transient transfection method. The VP6 protein was purified as the coating antigen using nickel ion affinity chromatography, and an indirect ELISA was subsequently established. The study found that the optimal concentration of coating for the VP6 protein was 1 µg/mL. The optimal blocking solution was 3% skim milk, and the blocking time was 120 min. The secondary antibody was diluted to 1:4000, and the incubation time for the secondary antibody was 30 min. A positive result was indicated when the serum OD450 was greater than or equal to 0.357. The coefficients of variation were less than 10% both within and between batches, indicating the good reproducibility of the method. The study found that the test result was positive when the serum dilution was 217, indicating the high sensitivity of the method. A total of 24 positive sera and 40 negative sera were tested using the well-established ELISA. The study also established an indirect ELISA assay with good specificity and sensitivity for the detection of antibodies to bovine rotavirus. Overall, the results suggest that the indirect ELISA method developed in this study is an effective test for detecting such antibodies.

Expression, purification, and functional verification of recombinant human glycoprotein hormone beta 5/alpha 2 fusion protein in CHO-S cells / 中国药理学通报

Aim To express and purify recombinant hCGH-CTP fusion protein in high-density suspension culture of Chinese hamster ovary cells (CHO-S), and to verify the lipid accumulation effect of rhCGH-CTP on 3T3-L1 mature adipocytes. Methods The recombinant protein expression vector (pcDNA3. 1-rhCGH-CTP) was constructed, achieved by fusing the human glycoprotein hormone beta 5/alpha 2 cDNA with CTP Linker. The expression plasmid was transiently transfected into the suspended CHO-S to express rhCGH-CTP protein and then purified, and the protein biological activity was verified. Intervention with 3T3-L1 mature adipocyte cells for 24 h was performed to detect the changes of intracellular triglyceride (TG) level. Results Western blot results showed that rhCGH-CTP protein was successfully expressed in CHO-S cells, and the yield was up to 715. 4 mg • L~ . The secreted protein was purified by AKTA pure system with higher purity that was up to 90% as identified by SDS-PAGE. In addition, the intracellular cAMP content of mature adipocytes with high expression of TSHR gene significantly increased after intervention with different concentrations of rhCGH-CTP protein by ELISA kit, indicating that rhCGH-CTP protein had biological activity. Oil red 0 staining showed that compared with the control group, the lipid content of mature adipocytes in the intervention groups with different concentrations of rhCGH-CTP protein significantly decreased (P < 0. 05) . Conclusions The rhCGH-CTP protein has been successfully expressed and purified with biological activity, and effectively reduce TG. This research provides an important theoretical basis for further revealing the physiological role of CGH protein and its potential application in clinical practice.

Generation of Porcine Angiogenin 4-Expressing Pichia pastoris and Its Protection against Intestinal Inflammatory Injury.

Antimicrobial peptides have been extensively studied as potential alternatives to antibiotics. Porcine angiogenin 4 (pANG4) is a novel antimicrobial peptide in the angiogenin (ANG) family, which may have a regulatory effect on intestinal microflora. The object of present study is obtained pANG4 protein by heterologous expression, so as to explore the biological function of recombinant pANG4 (rpANG4). The pANG4 was expressed in Pichia pastoris (P. pastoris) and anti-inflammatory effects were investigated in intestinal porcine epithelial cell line-J2 (IPEC-J2) and mice. Purified rpANG4 had bacteriostatic activity and did not cause hemolysis or cytotoxicity at concentrations below 128 µg/mL. Purified rpANG4 increased the activity of IPEC-J2 and reduced apoptosis in vitro. rpANG4 reduced the pro-inflammatory gene expression and upregulated tight junction protein gene expression during inflammation. rpANG4 alleviated lipopolysaccharide (LPS)-induced liver and spleen damage, intestinal inflammation, jejunal apoptosis genes' expression, and improved immune function in an in vivo mice model. rpANG4 increased tight junction protein gene expression in jejunum, thereby improving the jejunum intestinal barrier function. In conclusion, rpANG4 had antibacterial activity, inhibited intestinal inflammation, improved intestinal barrier function, and alleviated liver and spleen damage. The current study contributes to the development of antibiotic substitutes and the improvement of animal health.

[Bovine viral diarrhea virus Erns protein expressed in Chinese hamster ovary cells and its immunogenicity analysis].

The aim of this study was to produce Erns protein of bovine viral diarrhea virus (BVDV) by using suspensively cultured CHO cells expression system and to analyze the immunogenicity of the purified Erns protein. In this study, the recombinant eukaryotic expression plasmid pcDNA3.1-BVDV-Erns was constructed based on the gene sequence of BVDV-1 NADL strain. The Erns protein was secreted and expressed in cells supernatant after transfecting the recombinant expression plasmid pcDNA3.1-BVDV-Erns into CHO cells. The expression and purification of the Erns protein was analyzed by SDS-PAGE, the reactivity was determined with anti-His monoclonal antibodies and BVDV positive serum with Western blotting. Immunogenicity analysis of the Erns protein was determined after immunizing New Zealand white rabbits, and the serum antibodies were tested by indirect ELISA (iELISA) and indirect immunofluorescence (IFA). The serum neutralizing titer of the immunized rabbits was determined by virus neutralization test. The concentration of the purified Erns protein was up to 0.886 mg/mL by BCA protein quantification kit. The results showed that the Erns protein could be detected with anti-His monoclonal antibodies and anti-BVDV sera. Serum antibodies could be detected by iELISA on the 7th day post-prime immunization, and the antibody level was maintained at a high titer until the 28th day post-immunization. The antibody titer was 1:128 000. Furthermore, the expression of the Erns protein in BVDV-infected MDBK cells could be detected with immunized rabbits sera by IFA. Moreover, antigen-specific neutralizing antibodies of 2.71 log10 was induced in rabbits. In this study, purified BVDV Erns protein was successfully produced using CHO suspension culture system, and the recombinant protein was proved to have a good immunogenicity, which may facilitate the development of BVD diagnosis method and novel subunit vaccine.

Effects of different concentrations of androgens on KAP24.1 gene expression in Hetian sheep and Karakul sheep.

The purpose of the experiment was to clone and eukaryotic expression of hair follicle keratin associated protein 24.1 (KAP24.1), study the effect of different concentrations of androgen on protein expression, and compare KAP24.1 gene in skin and hair follicles of different breeds of sheep expression, explore KAP24.1 Expression difference of gene among local sheep breeds in southern Xinjiang and its effect on wool quality. The body-side hair follicles of Plain-type Hetian sheep, Mountain-type Hetian sheep and Karakul sheep were used as experimental materials, and the KAP24.1 gene sequence of sheep in GenBank (accession number: JX112014.1) was used as the reference to design primers. The KAP24.1 gene was amplified by PCR, and the pMD19-T-KAP24.1 cloning plasmid was constructed. After double digestion and identification, the pEGFP-N1-KAP24.1 eukaryotic recombinant expression plasmid was constructed. After PCR and double digestion and identification, sequencing and sequence analysis were performed, and the expression was transfected into Hela cells. SDS-PAGE and Western blotting were used to detect the expression levels of androgen at different concentrations. The expression of KAP24.1 gene in different sheep skin follicles was detected by real-time fluorescent quantitative PCR. Three sheep KAP24.1 were cloned The CDS region sequence of gene is 759 bp, encoding 252 amino acids, all of which are unstable hydrophobic proteins.The results of similarity comparison showed that compared with the reference gene, the gene sequence similarity of Mountain-type Hetian sheep and Karakul Sheep was 99.47%, and that of Plain-type Hetian sheep was 99.34%. Phylogenetic tree analysis showed that the three sheep had the closest genetic relationship with Capra hircus and the furthest genetic relationship with Cervus canadensis.The secondary structure of KAP24.1 was mainly composed of random coil.PEGFP-N1-KAP24.1 was successfully constructed eukaryotic recombinant expression plasmid was successfully transfected into HeLa cells to obtain 58 kDa KAP24.1 recombinant protein. When the concentration of androgen is 10-8 mol / L, the protein expression is the highest. The expression of KAP24.1 gene in skin and hair follicles of Mountain-type Hetian sheep was significantly different from that of plain-type Hetian sheep (P < 0.05), and there was significant difference between Mountain-type Hetian sheep and Karakul Sheep (P < 0.05). The expression of Karakul Sheep was significantly higher than that of Plain-type Hetian sheep (P < 0.05). The 759 bp CDS sequence of KAP24.1 gene in sheep was cloned, and PEGFP-N1-KAP24.1 was constructed eukaryotic recombinant expression plasmid to obtain 58 kDa KAP24.1 recombinant protein. When the concentration of androgen was 10-8mol / L, the protein expression was the highest, and KAP24.1 gene was expressed in the skin and hair follicles of three sheep breeds, and the expression of Mountain-type Hetian sheep was the highest.

High expression of antimicrobial peptides cathelicidin-BF in Pichia pastoris and verification of its activity.

Antibacterial peptides are endogenous polypeptides produced by multicellular organisms to protect the host against pathogenic microbes, they show broad spectrum antimicrobial activities against various microorganisms and possess low propensity for developing resistance. The purpose of this study is to develop recombinant antibacterial peptide cathelicidin-BF by genetic engineering and protein engineering technology, and study its antibacterial activity in vitro and in vivo, so as to provide reference for the production and application of recombinant antibacterial peptide cathelicidin-BF. In this study, on account of Pichia pastoris eukaryotic expression system, we expressed and prepared antibacterial peptide cathelicidin-BF. Then, the minimum inhibitory concentration of antibacterial peptide cathelicidin-BF and the comparison with the antibacterial activity of antibiotics were determined through the antibacterial experiment in vitro. Chickens as infection model were used to verify the antibacterial peptide activity in vivo. The results show that the bacteriostatic ability of antibacterial peptide cathelicidin-BF is similar to that of antibiotics in certain concentration, and can reach the treatment level of antibiotics. Although the mode of administration of antibacterial peptide is still limited, this study can provide reference for the future research of antibacterial peptide.

Antibacterial and immunoregulatory activity of an antimicrobial peptide hepcidin in loach (Misgurnus anguillicaudatus).

Antimicrobial peptides (AMPs) are members of humoral immunity and particpate in resisting microbial invasion. In this study, an AMP gene hepcidin was obtained from the oriental loach Misgurnus anguillicaudatus and named Ma-Hep. This Ma-Hep encodes a peptide of 90 amino acids, with a predicted active peptide segment (Ma-sHep) of 25 amino acids at C terminus. Stimulation by a bacterial pathogen Aeromonas hydrophila resulted in significant up-regulation of Ma-Hep transcripts in loach midgut, head kidney, and gill. Ma-Hep and Ma-sHep proteins were expressed in Pichia pastoris and their antibacterial activity was examined. Results showed that Ma-sHep possessed stronger antibacterial activity against various Gram-positive and Gram-negative bacteria, compared to Ma-Hep. Scanning electron microscopy showed that Ma-sHep might kill bacteria by destroying bacterial cell membranes. Moreover, we found that Ma-sHep had an inhibitory effect on blood cell apoptosis induced by A. hydrophila and facilitated the bacterial phagocytosis and clearance in loach. Histopathological analysis indicated Ma-sHep could protect liver and gut of loach from bacterial infection. Ma-sHep has high thermal stability and PH stability, which is conducive to further feed addition. Feed supplemented with Ma-sHep expressing yeast improved the intestinal flora of loach by increasing the dominant bacteria and decreasing the harmful bacteria. Feed supplemented with Ma-sHep expressing yeast also regulated the expression of inflammatory related factors in various tissues of loach and reduced the mortality of loach upon bacterial infection. These findings show that the antibacterial peptide Ma-sHep is involved in the antibacterial defense of loach and can be used as a candidate for new antimicrobial agents in aquaculture.

Cloning and expression of the mitochondrial cytochrome c oxidase subunit II gene in Sitophilus zeamais and interaction mechanism with allyl isothiocyanate.

In the United States, allyl isothiocyanate (AITC) has been registered as an insecticide, bactericide, and nematicide. And it has been confirmed that AITC has significant insecticidal activities against four stored product pests including Sitophilus zeamais Mostchulky (Coleoptera: Curculionidae). This study aimed to verify the mechanism of action of AITC on cytochrome c oxidase core subunits II in S. zeamais. Enzyme - catalyzed reactions and Fourier transform infrared spectrometer (FTIR) analysis revealed that the expressed COX II proteins could competitively bind and inhibit the activity of COX II. Furthermore, molecular docking results showed that a sulfur atom of AITC could form a 2.9 Å hydrogen bond with Ile-30, having a binding energy of -2.46 kcal/mol.

Characterization and expression of highly active recombinant human glucuronyl C5-epimerase in mammalian cells.

Heparin, a highly sulfated and epimerized form of heparan sulfate, is a linear polysaccharide with anticoagulant activity widely used in the clinic to prevent and treat thrombotic diseases. However, there are several noteworthy drawbacks associated with animal-sourced heparin during the preparation process. The in vitro enzymatic synthesis of heparin has become a promising substitute for animal-derived heparin. The synthesis of bioengineered heparin involves recombinant expression and preparation of polymerases, sulfotransferases, and an epimerase. D-glucuronyl C5-epimerase (HSepi) catalyzes D-glucuronic acids immediately adjacent to N-sulfo-glucosamine units to L-iduronic acid. Preparation of recombinant HSepi with high activity and production yield for in vitro heparin synthesis has not been resolved as of now. The findings of this study indicate that the catalytic activity of HSepi is regulated using post-translational modifications, including N-linked glycosylation and disulfide bond formation. Further mutation studies suggest that tyrosine residues, such as Tyr168, Tyr222, Tyr500, Tyr560, and Tyr578, are crucial in maintaining HSepi activity. A high-yield expression strategy was established using the lentiviral-based transduction system to produce recombinant HSepi (HSepi589) with a specific activity of up to 1.6 IU/mg. Together, this study contributes to the preparation of highly active HSepi for the enzymatic synthesis of heparins by providing additional insights into the catalytic activity of HSepi.

Eukaryotic Expression of the Cytochrome c Oxidase Subunit I of Sitophilus zeamais and Its Interaction with Allyl Isothiocyanate.

Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) is a destructive pest of stored grains around the world. Allyl isothiocyanate (AITC) was shown to have good bioactivity in the control of S. zeamais. In this study, the interaction of AITC on cytochrome c oxidase core subunits I (COX I) and their binding mechanism were determined using spectroscopic, isothermal titration calorimetry and molecular docking techniques. The results indicate the binding constant (Ka) of AITC and COX I was 6.742 × 103 L/mol. Analysis of spectroscopic revealed that the binding of COX I to reduced Cyt c induced conformational changes of reduced Cyt c, while AITC could competitively bind and inhibit the activity of the COX I protein. Moreover, molecular docking results suggested a sulfur atom in the AITC structure could form a hydrogen bond having a length of 3.3 Å with the Gly- 27 of COX I.

Evaluation of biological activity of an eukaryotic expressed anti-H5N1-M1 cell entry single molecule antibody / 中国生物制品学杂志

@#Objective To evaluate the biological activity of a eukaryotic expressed anti-H5N1-M1 cell entry single molecule antibody(TAT-ScFv-mFc). Methods The immune binding activity and affinity of TAT-ScFv-mFc to H5N1-M1 protein were detected by Western blot and localized surface plasmon resonance(LSPR)respectively;The inhibitory effect of TAT-ScFv-mFc on influenza virus H1N1 was detected by CCK-8 assay;The membrane penetration ability of TAT-ScFv-mFc to MDCK cells was verified by immunofluorescence assay. A total of 30 female BALB/c mice were injected with TAT-ScFv-mFc via tail vein,200 μL per mouse. Blood samples were collected at 5,60,120,240 and 360 min after injection. Serum samples were separated and detected for the titers by ELISA,and the half-life of TAT-ScFv-mFc was calculated according to the half-life curve drawn by Origin 2021 software. Results TAT-ScFv-mFc showed specific binding to H5N1-M1 protein with a binding rate constant of 6. 67 × 10~4[1/（M*s）]. The survival rate of MDCK cells infected by H1N1 increased gradually with the increase of TAT-ScFv-mFc concentration in a dose-dependent manner,which obviously inhibited the replication of H1N1. TAT-ScFv-mFc penetrated the cell membrane of MDCK cells in a short time,entered the cell and bound to virus M1protein,thus inhibiting virus replication and assembly. The half-life of TAT-ScFv-mFc in mice was 212 min. Conclusion TAT-ScFv-mFc has good immune binding activity and affinity with H5N1-M1,can effectively inhibit the replication of H1N1,has good penetration ability to MDCK cell membrane,and has a long half-life in mice,which lays a foundation of the drug treatment,vaccine research and preventive treatment of H5N1 infection.

Construction of eukaryotic expression plasmid of human promyelocytic leukemia gene of six transcripts and subcellular location analysis of its recombinant proteins / 中国生物制品学杂志

@#Objective To construct eukaryotic expression plasmids of human promyelocytic leukaemia(hPML) gene of six transcripts and analyze the subcellular location of the recombinant proteins.Methods Primers were designed according to the hPML gene sequences registered in GenBank databases.Six transcripts of hPML gene fragments(hPML Ⅰ,Ⅱ,Ⅳ,Ⅴ,Ⅵ and Ⅶ) were amplified by RT-PCR,which were linked to the eukaryotic expression vector pCAGGS respectively.The obtained eukaryotic expression plasmids of six transcripts of hPML gene were transfected into 293T cells respectively and detected for their protein expression by Western blot,while transfected into Vero cells and detected for their subcellular location by indirect immunofluorescence assay(IFA).Results The target gene fragments of the six eukaryotic expression plasmids were consistent with the hPML gene sequences registered in GenBank.All the six recombinant proteins showed specific binding with Myc antibody,among which the recombinant protein hPML Ⅰ,Ⅱ,Ⅳ,Ⅴ and Ⅵ were located in the nucleus and cytoplasm,while the recombinant protein hPML Ⅶ was mainly located in the cytoplasm,rarely in the nucleus.Conclusion The eukaryotic expression plasmids of six transcripts of hPML gene all can be expressed correctly in mammalian cells,and the expressed recombinant proteins were located in nucleus and cytoplasm simultaneously or mainly in cytoplasm.This study provides an experimental basis for subsequent study on the antiviral and other biological functions of recombinant protein hPML.

Bovine viral diarrhea virus Erns protein expressed in Chinese hamster ovary cells and its immunogenicity analysis / 生物工程学报

The aim of this study was to produce Erns protein of bovine viral diarrhea virus (BVDV) by using suspensively cultured CHO cells expression system and to analyze the immunogenicity of the purified Erns protein. In this study, the recombinant eukaryotic expression plasmid pcDNA3.1-BVDV-Erns was constructed based on the gene sequence of BVDV-1 NADL strain. The Erns protein was secreted and expressed in cells supernatant after transfecting the recombinant expression plasmid pcDNA3.1-BVDV-Erns into CHO cells. The expression and purification of the Erns protein was analyzed by SDS-PAGE, the reactivity was determined with anti-His monoclonal antibodies and BVDV positive serum with Western blotting. Immunogenicity analysis of the Erns protein was determined after immunizing New Zealand white rabbits, and the serum antibodies were tested by indirect ELISA (iELISA) and indirect immunofluorescence (IFA). The serum neutralizing titer of the immunized rabbits was determined by virus neutralization test. The concentration of the purified Erns protein was up to 0.886 mg/mL by BCA protein quantification kit. The results showed that the Erns protein could be detected with anti-His monoclonal antibodies and anti-BVDV sera. Serum antibodies could be detected by iELISA on the 7th day post-prime immunization, and the antibody level was maintained at a high titer until the 28th day post-immunization. The antibody titer was 1:128 000. Furthermore, the expression of the Erns protein in BVDV-infected MDBK cells could be detected with immunized rabbits sera by IFA. Moreover, antigen-specific neutralizing antibodies of 2.71 log10 was induced in rabbits. In this study, purified BVDV Erns protein was successfully produced using CHO suspension culture system, and the recombinant protein was proved to have a good immunogenicity, which may facilitate the development of BVD diagnosis method and novel subunit vaccine.

Eukaryotic expression and biological activity identification of fusion protein ABD-Fc-IL-2 / 中国生物制品学杂志

@#Objective To express the fusion protein ABD-Fc-IL-2 in eukaryotic cells and detect its biological activity. Methods The target gene SP-ABD-Fc-IL-2 was amplified by direct and overlapping PCR,and then ligated to vector pcDNA3. 1(+).The obtained recombinant plasmid pcDNA3. 1/SP-ABD-Fc-IL-2 was transiently transfected into CHO-S cells to express the fusion protein ABD-Fc-IL-2,which was purified by Protein A beads affinity chromatography. The specificity of the purified fusion protein was detected by Western blot,the biological activity was detected by CTLL-2/MTT cell proliferation colourimetry,and the interaction between ABD fragment and human serum albumin(HSA)was detected by pull down/Western blot. Results The recombinant plasmid pcDNA3. 1/SP-ABD-Fc-IL-2 was constructed correctly as identified by restriction analysis and sequencing. The purified fusion protein ABD-Fc-IL-2 showed a purity of 90% and bound specifically to mouse anti-IL-2 monoclonal antibody with the biological activity of 3. 29 × 108IU/mL. The ABD of fusion protein and HSA bound to each other. Conclusion The eukaryotic fusion protein ABD-Fc-IL-2 had high biological activity,which promoted the proliferation of CTLL-2 cells and maintained the binding ability of ABD fragment to HSA.

Inhibition of mutant RAS-RAF interaction by mimicking structural and dynamic properties of phosphorylated RAS.

Undruggability of RAS proteins has necessitated alternative strategies for the development of effective inhibitors. In this respect, phosphorylation has recently come into prominence as this reversible post-translational modification attenuates sensitivity of RAS towards RAF. As such, in this study, we set out to unveil the impact of phosphorylation on dynamics of HRASWT and aim to invoke similar behavior in HRASG12D mutant by means of small therapeutic molecules. To this end, we performed molecular dynamics (MD) simulations using phosphorylated HRAS and showed that phosphorylation of Y32 distorted Switch I, hence the RAS/RAF interface. Consequently, we targeted Switch I in HRASG12D by means of approved therapeutic molecules and showed that the ligands enabled detachment of Switch I from the nucleotide-binding pocket. Moreover, we demonstrated that displacement of Switch I from the nucleotide-binding pocket was energetically more favorable in the presence of the ligand. Importantly, we verified computational findings in vitro where HRASG12D/RAF interaction was prevented by the ligand in HEK293T cells that expressed HRASG12D mutant protein. Therefore, these findings suggest that targeting Switch I, hence making Y32 accessible might open up new avenues in future drug discovery strategies that target mutant RAS proteins.

Structural Modelling Prediction of Recombinant Plasmodium falciparum K13-F446I and K13-C580Y Gene by AlphaFold Method and Heterologous Expression in Spodoptera frugiperda 9 Cells.

P. falciparum Kelch 13 (Pfk13) is an essential protein that contains BTB and Kelch-repeat propeller domains (KRPD), which was predicted to bind substrate during ubiquitin-dependent degradation pathway. However, the function of Pfk13 and the structural alterations associated with artemisinin resistance mutations remain unknown. Herein, we screened two proteins, namely Pfk13-F446I and Pfk13-C580Y, which are closely associated with artemisinin, for structural prediction analysis. The 389 amino acids from 1011 nt to 2178 nt of KRPD were cloned into pFastBacTM1. The recombinant plasmids were heterologously expressed in Spodoptera frugiperda 9 cells (SF9) and a ~44 kDa protein band was yielded by SDS-PAGE and Western Blot. A total of five structure models were generated and predicted by AlphaFold for each protein. The models predicted that Pfk13-F446I would be located in the central protein cavity, proximal to mutations in cysteine residues primarily in ß strands. Unlike Pfk13-F446I, the Pfk13-C580Y is located on the small channel that runs through the center of the K13 protein. Interestingly, the hydrogen bond between C580 and C533 in the wide type (WT) was not detected, suggesting that the hydrogen bond may be lost during the mutation. Besides, the Pfk13-F446I and Pfk13-C580Y mutation were found to add 11 and 9 hydrogen bonds variations that may lead to conformational change of the protein structure compared to WT, respectively. Future work should pay more attention to the binding characteristics of those mutations related with KPRD pockets and their binding substrates, which will further clarify the structure and function of Pfk13 and its mutant.

Involvement of galectin-9 from koi carp (Cyprinus carpio) in the immune response against Aeromonas veronii infection.

Galectins are ß-galactoside sugar binding proteins which function as important pattern recognition receptors (PRRs) in innate immunity. Here, we identified a galectin-9 gene from koi carp (Cyprinus carpio), named kGal-9. The ORF of kGal-9 is 963 bp in length, which encodes a polypeptide of 320 amino acids without either signal peptide. The predicted molecular weight is 36.25 kDa, and the isoelectric point is 8.3. Multiple sequence alignment showed that the putative kGal-9 contains two carbohydrate recognition domains (CRD), which are conserved in Galectin-9s. The phylogenetic tree showed that kGal-9 clustered to Galectin-9s from other teleosts, and shared the highest identity of 87.5% with Qihe crucian (Carassius auratus). kGal-9 mRNA was abundant in head kidney, gills, and gut, but low in liver and muscle. Further, the expression level of kGal-9 in the head kidney and liver increased significantly after Aeromonas veronii (abbreviated A.v) infection. Unexpectedly, kGal-9 showed a remarkable downregulation in the spleen at various time points post A.v infection. Intramuscular injection of pckGal-9 not merely reduced the bacterial load of spleen tissue, but also improved the survival rate of koi carp post A.v challenge. Besides, administration of pckGal-9 stimulated the expression of several immuno-related genes including proinflammatory cytokines (IL-1ß, IL-6), anti-inflammatory cytokine (IL-10), complement components (C4, C9), with fluctuation in spleen and head kidney. Taken together, the obtained results suggest that kGal-9 occupies an important role in innate immunity and defense against bacterial infection in koi carp.

Eukaryotic expression and immunogenicity of Ancylostoma ceylanicum calreticulin.

Ancylostoma ceylanicum is a zoonotic soil-derived nematode that parasitizes human and animal intestines, causing malnutrition and iron-deficiency anemia. Calreticulin is a multifunctional protein involved in all stages of parasitic infection. Studies have found that parasites can secret calreticulin to regulate the host's immune response. To explore the immunogenicity of the eukaryotic expression plasmid of Ancylostoma ceylanicum calreticulin (Ace-CRT), we constructed a recombinant Ace-CRT eukaryotic expression plasmid (pEGFP-N3-Ace-CRT). Successful expression of the target protein in Human Embryonic Kidney (HEK) 293 T cells was confirmed by indirect immunofluorescence and Western blot analysis. BALB/c mice were immunized with pEGFP-N3-Ace-CRT plasmid. Measuring IgG antibody levels in immunized mice sera by ELISA showed that the recombinant plasmid stimulated IgG antibody production in mice. Spleen lymphocytes were collected from vaccinated mice to determine the proportion of T cell subsets and the expression levels of cytokines. Flow cytometry revealed that the percentage of CD3 + CD4+ and CD3 + CD8+ T cells in mice spleen in the immunization group was significantly higher than that in the control group. Recombinant plasmid immunization increased IL-4, IL-10, IL-12, and IL-13 expression while decreasing IL-5, IL-6, and INF-γ in mice spleens. These results indicate that the eukaryotic plasmid constructed in this study had good immunogenicity and mainly induced a T helper 2 response in the host, laying a foundation for screening candidate molecules for anti-hookworm vaccines.