Recombinant Expression Plasmid Research Articles

Structure and Antigenicity of the Porcine Astrovirus 4 Capsid Spike

Porcine astrovirus 4 (PoAstV4) has been recently associated with respiratory disease in pigs. In order to understand the scope of PoAstV4 infections and to support the development of a vaccine to combat PoAstV4 disease in pigs, we designed and produced a recombinant PoAstV4 capsid spike protein for use as an antigen in serological assays and for potential future use as a vaccine antigen. Structural prediction of the full-length PoAstV4 capsid protein guided the design of the recombinant PoAstV4 capsid spike domain expression plasmid. The recombinant PoAstV4 capsid spike was expressed in Escherichia coli, purified by affinity and size-exclusion chromatography, and its crystal structure was determined at 1.85 Å resolution, enabling structural comparisons to other animal and human astrovirus capsid spike structures. The recombinant PoAstV4 capsid spike protein was also used as an antigen for the successful development of a serological assay to detect PoAstV4 antibodies, demonstrating that the recombinant PoAstV4 capsid spike retains antigenic epitopes found on the native PoAstV4 capsid. These studies lay a foundation for seroprevalence studies and the development of a PoAstV4 vaccine for swine.

Enhancement of Orthodontic Tooth Movement by Local Administration of Biofunctional Molecules: A Comprehensive Systematic Review.

Enhancement of orthodontic tooth movement (OTM) through local administration of biofunctional molecules has become increasingly significant, particularly for adult patients seeking esthetic and functional improvements. This comprehensive systematic review analyzes the efficacy of various biofunctional molecules in modulating OTM, focusing on the method of administration and its feasibility, especially considering the potential for topical application. A search across multiple databases yielded 36 original articles of experimental human and animal OTM models, which examined biofunctional molecules capable of interfering with the biochemical reactions that cause tooth movement during orthodontic therapy, accelerating the OTM rate through their influence on bone metabolism (Calcitriol, Prostaglandins, Recombinant human Relaxin, RANKL and RANKL expression plasmid, growth factors, PTH, osteocalcin, vitamin C and E, biocompatible reduced graphene oxide, exogenous thyroxine, sclerostin protein, a specific EP4 agonist (ONO-AE1-329), carrageenan, and herbal extracts). The results indicated a variable efficacy in accelerating OTM, with Calcitriol, Prostaglandins (PGE1 and PGE2), RANKL, growth factors, and PTH, among others, showing promising outcomes. PGE1, PGE2, and Calcitriol experiments had statistically significant outcomes in both human and animal studies and, while other molecules underwent only animal testing, they could be validated in the future for human use. Notably, only one of the animal studies explored topical administration, which also suggests a future research direction. This review concluded that while certain biofunctional molecules demonstrated potential for OTM enhancement, the evidence is not definitive. The development of suitable topical formulations for human use could offer a patient-friendly alternative to injections, emphasizing comfort and cost-effectiveness. Future research should focus on overcoming current methodological limitations and advancing translational research to confirm these biomolecules' efficacy and safety in clinical orthodontic practice.

Preparation and preliminary application of the polyclonal antibody against Toxoplasma gondii dense granule protein 24

To prepare and characterize the mouse polyclonal antibody against the dense granule protein 24 (GRA24) of Toxoplasma gondii, and explore its preliminary applications. The GRA24 coding sequences of different T. gondii strains were aligned using the MEGA-X software, and the dominant peptide of the GRA24 protein was analyzed with the Protean software. The base sequence encoding this peptide was amplified using PCR assay and ligated into the pET-28a vector, and the generated GRA24 truncated protein was transformed into Escherichia coli BL21. After induction by isopropyl-beta-D-thiogalactopyranoside (IPTG), the expression and purification of the recombinant GRA24 protein was analyzed using sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). BALB/c mice were immunized by subcutaneous injection with the purified recombinant GRA24 truncated protein to generate the polyclonal antibody, and the titer of the polyclonal antibody was measured using enzyme linked immunosorbent assay (ELISA). The specificity of the polyclonal antibody was tested using Western blotting, and the intracellular localization of the polyclonal antibody was investigated using immunofluorescence assay (IFA). SDS-PAGE showed successful construction of the recombinant expression plasmid, and Coomassie brilliant blue staining showed the generation of the high-purity recombinant GRA24 truncated protein. ELISA measured that the titer of the polyclonal antibody against the GRA24 truncated protein was higher than 1:208 400, and Western blotting showed that the polyclonal antibody was effective to recognize the endogenous GRA24 proteins of different T. gondii strains and specifically recognize the recombinant GRA24 truncated protein. Indirect IFA showed that the GRA24 protein secreted 16 hour following T. gondii invasion in host cells. The polyclonal antibody against the T. gondii GRA24 protein has been successfully prepared, which has a widespread applicability, high titers and a high specificity. This polyclonal antibody is available for Western blotting and IFA, which provides the basis for investigating the function of the GRA24 protein.

Isolation and functional characterization of (iso)eugenol O-methyltransferase (IEMT) gene in Asarum sieboldii

Methyleugenol is one of the main active constituents in the volatile oil of the traditional Chinese medicine Asari Radix et Rhizoma. It possesses various pharmacological effects such as analgesic, anesthetic, and anti-inflammatory properties. In biosynthesis, the initial precursor phenylalanine is finally converted into methyleugenol through a series of intermediate compounds including coniferyl acid, courmaryl acid, caffeic acid, ferulic acid/ferulic-CoA, coniferyl aldehyde, conferyl alcohol, cnfiferyl acetate, and eugenol/isoeugenol, which are produced through catalysis of a large number of enzymes. Eugenol O-methyltransferase(EOMT) is one of the key enzymes in the biosynthesis pathway, capable of methylating eugenol on the para-site hydroxyl group of the benzene ring, thereby generating methyleugenol. Here, an(iso)eugenol O-methyltransferase(IEMT) gene was cloned for the first time from Asarum siebo-ldii, holding an open reading frame that consisted of 1 113 bp and encoded a protein containing 370 amino acid residues. Bioinformatics analysis results showed that this protein was equipped with the characteristic structural domains of methyltransferases such as S-adenosylmethionine(SAM) binding sites and dimerization domains. The prokaryotic expression recombinant plasmid pET28a(+)-AsIEMT was constructed, and the candidate protein was induced and purified. In vitro enzyme assays confirmed that AsIEMT had dual functions. The enzyme could catalyze the production either of methyleugenol from eugenol or of methylisoeugenol from isoeugenol, although the latter was more prevalent. When isoeugenol was used as the substrate, the kinetics parameters K_m and V_(max) of catalytic reaction were(0.90±0.06) mmol·L~(-1) and(1.32±0.04)nmol·s~(-1)·mg~(-1), respectively. This study expanded our understandings of critical enzyme genes involved in phenylpropanoid metabolic pathways, and would facilitate the elucidation of quality formation mechanisms of the TCM Asari Radix et Rhizoma.

Co-immunization with DNA vaccines encoding yidR and IL-17 augments host immune response against Klebsiella pneumoniae infection in mouse model

ABSTRACT Klebsiella pneumoniae is an important gram-negative bacterium that causes severe respiratory and healthcare-associated infections. Although antibiotic therapy is applied to treat severe infections caused by K. pneumoniae, drug-resistant isolates pose a huge challenge to clinical practices owing to adverse reactions and the mismanagement of antibiotics. Several studies have attempted to develop vaccines against K. pneumoniae, but there are no licensed vaccines available for the control of K. pneumoniae infection. In the current study, we constructed a novel DNA vaccine, pVAX1-YidR, which encodes a highly conserved virulence factor YidR and a recombinant expression plasmid pVAX1-IL-17 encoding Interleukin-17 (IL-17) as a molecular adjuvant. Adaptive immune responses were assessed in immunized mice to compare the immunogenicity of the different vaccine schemes. The results showed that the targeted antigen gene was expressed in HEK293T cells using an immunofluorescence assay. Mice immunized with pVAX1-YidR elicited a high level of antibodies, induced strong cellular immune responses, and protected mice from K. pneumoniae challenge. Notably, co-immunization with pVAX1-YidR and pVAX1-IL-17 significantly augmented host adaptive immune responses and provided better protection against K. pneumoniae infections in vaccinated mice. Our study demonstrates that combined DNA vaccines and molecular adjuvants is a promising strategy to develop efficacious antibacterial vaccines against K. pneumoniae infections.

Expression, purification, and biological activity evaluation of cathepsin L in mammalian cells.

Cathepsin L (CTSL) could cleave and activate SARS-CoV-2 Spike protein to promote viral entry, making it a hopeful therapeutic target for COVID-19 prevention and treatment. So CTSL inhibitors are considered to be a promising strategy to SARS-CoV-2 infection. CTSL has previously been expressed in inclusion body in Escherichia coli. In order to prepare CTSL with high purity and activity in soluble active form, we transformed HEK-293T cells with a recombinant mammalian expression plasmid. CTSL was purified to a purity about 95%, found to migrate at approximately 43kDa and exhibited substrate specificity against Z-Phe-Arg-AMC with specific activity of no less than 85081 U/mg, characteristic of active CTSL. Although eukaryotic purified CTSL is commercially available, our study for the first time reported the details of the expression, purification, and characterization of active, recombinant CTSL in eukaryocyte system, which laid an experimental foundation for the establishment of high-throughput screening model for anti-coronavirus drugs targeting CTSL.

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.

A novel defensin-like peptide C-13326 possesses protective effect against multidrug-resistant Aeromonas schubertii in hybrid snakehead (Channa maculate ♀ × Channa argus ♂).

The purpose of this study was to investigate whether a defensin-like antimicrobial peptide (C-13326 peptide) identified in Hermetia illucens could possess protective effect against multidrug-resistant Aeromonas schubertii in hybrid snakehead (Channa maculate ♀ × Channa argus ♂). The cDNA of C-13326 peptide comprised 243 nucleotides encoding 80 amino acids, with six conserved cysteine residues and the classical CSαβ structure. The recombinant expression plasmid pPIC9K-C-13326 was constructed and transformed into GS115 Pichia pastoris, and the C-13326 peptide was expressed by induction with 1% methanol. The crude extract of C-13326 peptide was precipitated by ammonium sulfate, assayed by Braford method, detected by tricine-SDS-PAGE, evaluated by BandScan software and identified by liquid chromatography-mass spectrometry. The C-13326 peptide was shown to have inhibitory activity against the growth of multidrug-resistant A. schubertii DM210910 by using the minimum growth inhibitory concentration and Oxford cup method. In addition, scanning electron microscopy analysis suggested that C-13326 peptide inhibited the growth of A. schubertii DM210910 by damaging the bacterial cell membrane. To explore the role of peptide C-13326 invivo, hybrid snakehead was fed with peptide C-13326 as feed additives for 7 days. The results revealed that C-13326 peptide could significantly down-regulate the expression levels of IL-1β, IL-8, IL-12 and TNF-α (p < .05), and significantly improved the survival rate of hybrid snakehead after challenging with A. schubertii DM210910. Therefore, the C-13326 peptide is a promising antimicrobial agent for A. schubertii treatment in aquaculture.

Anti-infective immune functions of type IV interferon in grass carp ( Ctenopharyngodon idella): A novel antibacterial and antiviral interferon in lower vertebrates.

Type IV interferon (IFN-υ) is a recently discovered cytokine crucial for host defense against viral infections. However, the role and mechanisms of IFN-υ in bacterial infections remain unexplored. This study investigated the antibacterial and antiviral functions and mechanisms of grass carp ( Ctenopharyngodon idella) IFN-υ (CiIFN-υ) both in vivo and in vitro. The CiIFN-υ gene was first identified and characterized in grass carp. Subsequently, the immune expression of CiIFN-υ significantly increased following bacterial challenge, indicating its response to bacterial infections. The eukaryotic recombinant expression plasmid of CiIFN-υ was then constructed and transfected into fathead minnow (FHM) cells. Supernatants were collected and incubated with four bacterial strains, followed by plate spreading and colony counting. Results indicated that CiIFN-υ exhibited more potent antibacterial activity against gram-negative bacteria compared to gram-positive bacteria and aggregated gram-negative bacteria but not gram-positive bacteria. In vivo experiments further confirmed the antibacterial function, showing high survival rates, low tissue edema and damage, reduced tissue bacterial load, and elevated proinflammatory response at the early stages of bacterial infection. In addition, the antiviral function of CiIFN-υ was confirmed through in vitro and in vivo experiments, including crystal violet staining, survival rates, tissue viral burden, and RT-qPCR. This study highlights the antibacterial function and preliminary mechanism of IFN-υ, demonstrating that IFN-υ possesses dual functions against bacterial and viral infections.

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.

Recombinant porcine interferon-gamma expressed in CHO cells and its antiviral activity

The aim of this study was to produce recombinant porcine interferon gamma (rPoIFN-γ) by Chinese hamster ovarian (CHO) cells expression system and to analyze its antiviral activity. Firstly, we constructed the recombinant eukaryotic expression plasmid pcDNA3.1-PoIFN-γ and transfected into suspension cultured CHO cells for secretory expression of rPoIFN-γ. The rPoIFN-γ was purified by affinity chromatography and identified with SDS-PAGE and Western blotting. Subsequently, the cytotoxicity of rPoIFN-γ was analyzed by CCK-8 test, and the antiviral activity of rPoIFN-γ was evaluated using standard procedures in VSV/PK-15 (virus/cell) test system. Finally the anti-Seneca virus A (SVA) of rPoIFN-γ activity and the induction of interferon-stimulated genes (ISGs) and cytokines were also analyzed. The results showed that rPoIFN-γ could successfully expressed in the supernatant of CHO cells. CCK-8 assays indicated that rPoIFN-γ did not show cytotoxicity on IBRS-2 cells. The biological activity of rPoIFN-γ was 5.59×107 U/mg in VSV/PK-15 system. Moreover, rPoIFN-γ could induced the expression of ISGs and cytokines, and significantly inhibited the replication of SVA. In conclusion, the high activity of rPoIFN-γ was successfully prepared by CHO cells expression system, which showed strong antiviral activity on SVA. This study may facilitate the investigation of rPoIFN-γ function and the development of novel genetically engineered antiviral drugs.

Characterization of a D-mannitol oxidase from Paenibacillus sp. and its application in the preparation of D-mannose

D-mannose has many functional activities and is widely used in food, medicine, agriculture and other industries. D-mannitol oxidase that can efficiently convert D-mannitol into D-mannose has potential application in the enzymatic preparation of D-mannose. A D-mannitol oxidase (PsOX) was found from Paenibacillus sp. HGF5. The similarity between PsOX and the D-mannitol oxidase (AldO) from Streptomyces coelicolor was 50.94%. The molecular weight of PsOX was about 47.4 kDa. A recombinant expression plasmid pET-28a-PsOX was constructed and expressed in Escherichia coli BL21(DE3). The Km and kcat/Km values of PsOX for D-mannitol were 5.6 mmol/L and 0.68 L/(s·mmol). Further characterization of PsOX showed its optimal pH and temperature were 7.0 and 35 ℃, respectively, while its enzyme activity could be stably remained below 60 ℃. The molar conversion rate of 400 mmol/L D-mannitol by PsOX was 95.2%. The whole cells of PsOX and AldO were used to catalyze 73 g/L D-mannitol respectively. The reaction catalyzed by PsOX completed in 9 h and 70 g/L D-mannose was produced. PsOX showed a higher catalytic efficiency compared to that of AldO. PsOX may facilitate the enzymatic preparation of D-mannose as a novel D-mannose oxidase.

Role of Cherry Valley duck IRF1 mediated signal pathway in host anti-duck Tembusu virus

China is the country with the largest amount of duck breeding as well as duck meat and egg production. In recent years, the emergence and spread of duck Tembusu virus (DTMUV) has become one of the important factors in reducing the amount of duck slaughter, which seriously endangers the duck breeding industry in our country. In-depth research on the mechanism of duck innate immunity facilitates the exploration of new models for the treatment of DTMUV infection. IRF1 can induce the expression of many antiviral immune factors in the animal organism and play an important role in the innate immune response. In this study, we used interfering RNA to knock down the IRF1 gene in DEF cells and then the cells were infected with DTMUV. We found that knockdown of IRF1 promoted DTMUV replication at an early stage and caused downregulation of the expression of several major pattern recognition receptors (PRRs), interleukins (IL), interferons (IFN), antiviral proteins, and MHC molecules by assay, showing that the duIRF1-mediated signaling pathway plays an extremely important role in DTMUV-induced host innate immunity. In addition, we constructed the recombinant expression plasmid pET32a(+)-duIRF1-His, and finally prepared the polyclonal antibody of duIRF1 with good specificity, hoping to provide a detection means for research on the mechanism of IRF1 in innate immunity in our laboratory and in this field.

Preparation of polyclonal antibodies against the Drosophila deacetylases SIRT 6 and SIRT 7

SIRT6 and SIRT7, as members of the Sirtuins family, are indispensable for the growth and development of Drosophila. They play crucial roles in maintaining genome stability, regulating metabolic senescence, and controlling tumorigenesis. To investigate their involvement in the Drosophila life cycle, we focused on describing the expression and purification of recombinant Drosophila SIRT6 and SIRT7 proteins. Subsequently, these proteins were utilized for generating polyclonal antibodies against Drosophila SIRT6 and SIRT7. The recombinant expression plasmid was introduced into E. coli cells to enable the production of SIRT6 and SIRT7 proteins. Following immunizations of New Zealand white rabbits and guinea pigs with the recombinant proteins as antigens, specific polyclonal antisera against both proteins were obtained. After purification, the specificity of SIRT6 and SIRT7 was confirmed using ELISA and western blot analyses, demonstrating strong specificity. These antibodies hold promise for the development of detection assays required for further research.

Minimized antibiotic-free plasmid vector for gene therapy utilizing a new toxin-antitoxin system

Approaches to improve plasmid-mediated transgene expression are needed for gene therapy and genetic immunization applications. The backbone sequences needed for the production of plasmids in bacterial hosts and the use of antibiotic resistance genes as selection markers represent biological safety risks. Here, we report the development of an antibiotic-free expression plasmid vector with a minimized backbone utilizing a new toxin-antitoxin (TA) system. The Rs_0636/Rs_0637 TA pair was derived from the coral-associated bacterium Roseivirga sp. The toxin gene is integrated into the chromosome of Escherichia coli host cells, and a recombinant mammalian expression plasmid is constructed by replacing the antibiotic resistance gene with the antitoxin gene Rs_0637 (here named Tiniplasmid). The Tiniplasmid system affords high selection efficiency (∼80%) for target gene insertion into the plasmid and has high plasmid stability in E. coli (at least 9 days) in antibiotic-free conditions. Furthermore, with the aim of reducing the size of the backbone sequence, we found that the antitoxin gene can be reduced to 153 bp without a significant reduction in selection efficiency. To develop its applications in gene therapy and DNA vaccines, the biosafety and efficiency of the Tiniplasmid-based eukaryotic gene delivery and expression were further evaluated in CHO–K1 cells. The results showed that Rs_0636/Rs_0637 has no cell toxicity and that the Tiniplasmid vector has a higher gene expression efficiency than the commercial vectors pCpGfree and pSTD in the eukaryotic cells. Altogether, the results demonstrate the potential of the Rs_0636/Rs_0637-based antibiotic-free plasmid vector for the development and production of safe and efficacious DNA vaccines.

Hp0521 inhibited the virulence of H. pylori 26,695 strain via regulating CagA expression

Hp0521 is the number of cag pathogenicity island (cagPAI) family in Helicobacter pylori (H. pylori, Hp), which encoded Cag2 protein. The aim of this study was to investigate the role of hp0521 on the H. pylori 26,695 strain. We constructed the recombinant prokaryotic expression plasmid pET-32a-hp0521 and pET-32a-hpc0521. Then, we co-cultured the H. pylori wild strain 26,695 and Δhp0521 strain with GES-1 cells to detect CagA protein transport and IL-8 secretion. We found that Δhp0521 mutation increased the expression of cagA, rpoB and promoted the transportation of CagA protein in GES-1 cells. In addition, we also observed that Δhp0521 mutation had no effect on other cagPAI protein stability and the expression of IL-8. Our findings suggested that hp0521 may down-regulated the expression of cagA, rpoB and inhibited the transportation of CagA protein in GES-1 cells and had no effect on growth.

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.

Biological activity of recombinant bovine IFN-α and inhibitory effect on BVDV in vitro

Type I interferon has great broad-spectrum antiviral ability and immunomodulatory function, and its receptors are expressed in almost all types of cells. Bovine viral diarrhea virus (BVDV) is an important pathogen causing significant economic losses in cattle. In this study, a recombinant expression plasmid carrying bovine interferon-α(BoIFN-α)gene was constructed and transformed into E. coli BL21 (DE3) competent cells. SDS-PAGE and Westernblotting analysis showed that the recombinant BoIFN-α protein (rBoIFN-α) was successfully expressed. It is about 36KD and exists in the form of inclusion body. When denatured, purified and renatured rBoIFN-α protein stimulated MDBK cells, the expression of interferon stimulating genes (ISGs) such as ISG15, OAS1, IFIT1, Mx1 and IFITM1 were significantly up-regulated, and reached the peak at 12 h (P< 0.001). MDBK cells were infected with BVDV with moi of 0.1 and 1.0, respectively. The virus proliferation was observed after pretreatment with rBoIFN-α protein and post-infection treatment. The results showed that the denatured, purified and renatured BoIFN-α protein had good biological activity and could inhibit the replication of BVDV in MDBK cells in vitro, which provided a basis for BoIFN-α as an antiviral drug, immune enhancer and clinical application of BVDV.

Fatty acid binding protein 1 and fatty acid synthetase over-expression have differential effects on collagen III synthesis and cross-linking in Zongdihua pig primary adipocytes.

The purpose of this study was to determine whether FABP1 and FAS regulate expression of collagen and its crosslinking via lysyl oxidase in isolated adipocytes from Zongdihua pigs. We aimed to identify biochemical processes affecting meat quality using molecular tools to provide a basis for breeding improvement of these animals. We measured expression levels of FABP1 and related genes using qRT-PCR in longissimus dorsi muscle and subcutaneous adipose tissues. Primary adipocytes from fat tissues were isolated and FABP1 and FAS were over-expressed from recombinant plasmids. Sequence analysis of the cloned genes indicated that FABP1 encodes a hydrophobic protein of 128 amino acids and contained 12 predicted phosphorylation sites and no transmembrane regions. The basal levels of FABP1 and FAS expression in pig tissues were 3-3.5-fold higher in subcutaneous fat compared with muscle (P < 0.01). Recombinant expression plasmids were successfully transfected into the cloned preadipocytes and (a) over-expression of FAS resulted in significantly increased expression of COL3A1 (P < 0.05) and significantly inhibited lysyl oxidase LOX expression (P < 0.01); (b) over-expression of FABP1 significantly increased COL3A1 expression (P < 0.01) and significantly inhibited LOX expression (P< 0.05) and significantly reduced lysyl oxidase activity (P < 0.01). Therefore, FAS enhanced FABP1 expression resulting in increased collagen accumulation and this preliminarily suggested that FAS and FABP1 can serve as fat-related candidate genes and provide a theoretical basis for the study of fat deposition in Zongdihua pigs.

Establishment of a neutralization assay for Nipah virus using a high-titer pseudovirus system.

To construct a high-titer Nipah pseudovirus packaging system using the HIV lentivirus backbone vector and establish a safe neutralization assay for Nipah pseudovirus in biosafety level 2 facilities. Nipah virus (NiV) fusion protein (F) and glycoprotein (G) recombinant expression plasmids, psPAX2, and pLenti CMV Puro LUC (w168-1) were transiently transfected into 293T cells for 72h for the generation of a NiV pseudovirus. The neutralization ability of Nipah virus F and G protein antibodies was assessed using the pseudovirus. A NiV pseudovirus was constructed using 293T cells. The ideal mass ratio of plasmid psPAX2: w168-1: F: G for transfection was determined to be 4:4:1:1. The specificity of recombinant F and G protein expression was indicated by indirect immunofluorescence and western blotting. The pseudovirus particles showed obvious spikes under a transmission electron microscope. The NiV pseudovirus titer was 4.73 × 105 median tissue culture infective dose per mL, and the pseudovirus could be effectively neutralized by polyclonal antibodies specifically targeting the F and G proteins respectively. A NiV pseudovirus was successfully generated using HIV vector systems, and was used as a platform for a safe and reliable pseudovirus-based neutralizing assay that can be performed in biosafety level 2 facilities.