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.
Swine ; Animals ; Cricetinae ; Interferon-gamma/pharmacology* ; Cricetulus ; CHO Cells ; Sincalide ; Recombinant Proteins/pharmacology* ; Antiviral Agents/pharmacology*

The aim of this study was to produce E^rns protein of bovine viral diarrhea virus (BVDV) by using suspensively cultured CHO cells expression system and to analyze the immunogenicity of the purified E^rns protein. In this study, the recombinant eukaryotic expression plasmid pcDNA3.1-BVDV-E^rns was constructed based on the gene sequence of BVDV-1 NADL strain. The E^rns protein was secreted and expressed in cells supernatant after transfecting the recombinant expression plasmid pcDNA3.1-BVDV-E^rns into CHO cells. The expression and purification of the E^rns 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 E^rns 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 E^rns protein was up to 0.886 mg/mL by BCA protein quantification kit. The results showed that the E^rns 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 E^rns 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 E^rns 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.
Rabbits ; Animals ; Cricetinae ; Cricetulus ; CHO Cells ; Antibodies, Viral ; Diarrhea Viruses, Bovine Viral/genetics* ; Antibodies, Monoclonal/genetics* ; Diarrhea ; Viral Vaccines/genetics*

We researched the mechanism of African swine fever virus (ASFV) protein E248R in regulating the cGAS-STING pathway. First, we verified via the dual-luciferase reporter assay system that E248R protein inhibited the secretion of IFN-β induced by cGAS-STING or HT-DNA in a dose-dependent manner. The relative quantitative PCR analysis indicated that the overexpression of E248R inhibited HT-DNA-induced transcription of IFN-b1, RANTES, IL-6, and TNF-α in PK-15 cells. Next, we found that E248R interacted with STING by co-immunoprecipitation assay and laser confocal microscopy. Finally, we demonstrated that E248R inhibited the expression of STING protein by using Western blotting. We demonstrated for the first time that the E248R protein of ASFV suppressed the host innate immune response via inhibiting STING expression. The results are pivotal in extending the understanding of the ASFV immune escape and can guide the design of vaccines against ASFV.
African Swine Fever Virus/genetics* ; Animals ; DNA ; Immunity, Innate ; Nucleotidyltransferases/metabolism* ; Signal Transduction ; Swine

To develop Senecavirus A (SVA) virus-like particles (VLPs), a recombinant prokaryotic expression plasmid pET28a-SVA-VP031 was constructed to co-express SVA structural proteins VP0, VP3 and VP1, according to the genomic sequence of the field isolate CH-FJ-2017 after the recombinant proteins were expressed in E .coli system, and purified by Ni+ ion chromatographic method. The SVA VLPs self-assemble with a high yield in vitro buffer. A typical VLPs with an average diameter of 25-30 nm which is similar to native virions by using TEM detection. Animals immunized by SVA VLPs shown that the VLPs induced high titers neutralizing antibodies in Guinea pigs. This study indicated that the VLPs produced with co-expressing SVA structural proteins VP0, VP3 and VP1 in prokaryotic system is a promising candidate and laid an important foundation for the development of a novel SVA VLPs vaccine.
Animals ; Antibodies, Neutralizing ; Escherichia coli/genetics* ; Genomics ; Guinea Pigs ; Picornaviridae/genetics*

AIM To investigate the effects of frost-like powder,steaming,stir-frying with wine,stir-frying with salt-water and stir-frying with vinegar on compositions and contents of fatty oils in Descurainiae Semen.METHODS Descurainiae Semen was processed by five methods,respectively.The fatty oils were extracted from various processed products by petroleum ether,which were then derivatized.GC-MS was adopted in the qualitative identification and quantitative determination.RESULTS Except for frost-like powder,various processing methods could increase the extraction rate of fatty oils.Compared with raw product,the quantities of fatty oils in various processed products were decreased,together with the increased contents.The main compositions of obtained fatty oils were unsaturated fatty acids,whose contents in various processed products (except stir-frying with vinegar product) were higher than those in the raw product.CONCLUSION The effects of different processing methods on compositions and contents of fatty oils in Descurainiae Semen show obvious differences,among which the processing effect of stir-frying with vinegar is not satisfactory.

Integrin alphavbeta3 plays a major role in various signaling pathways, cell apoptosis, and tumor angiogenesis. To examine the functions and roles of alphavbeta3 integrin, a stable CHO-677 cell line expressing the murine alphavbeta3 heterodimer (designated as "CHO-677-malphavbeta3" cells) was established using a highly efficient lentiviral-mediated gene transfer technique. Integrin subunits alphav and beta3 were detected at the gene and protein levels by polymerase chain reaction (PCR) and indirect immunofluorescent assay (IFA), respectively, in the CHO-677-malphavbeta3 cell line at the 20th passage, implying that these genes were successfully introduced into the CHO-677 cells and expressed stably. A plaque-forming assay, 50% tissue culture infective dose (TCID50), real-time quantitative reverse transcription-PCR, and IFA were used to detect the replication levels of Foot-and-mouth disease virus (FMDV) in the CHO-677-malphavbeta3 cell line. After infection with FMDV/O/ZK/93, the cell line showed a significant increase in viral RNA and protein compared with CHO-677 cells. These findings suggest that we successfully established a stable alphavbeta3-receptor-expressing cell line with increased susceptibility to FMDV. This cell line will be very useful for further investigation of alphavbeta3 integrin, and as a cell model for FMDV research.
Animals ; Animals, Suckling ; CHO Cells ; Cloning, Molecular ; Cricetulus ; DNA, Complementary/genetics/metabolism ; Disease Susceptibility/virology ; Foot-and-Mouth Disease/*genetics/virology ; Foot-and-Mouth Disease Virus/*physiology ; Integrin alphaVbeta3/*genetics/metabolism ; Mice

This study was aimed to clone the GGPS (geranylgeranyl pyrophosphate synthase) gene from Lepidium apetalum, to analyze its sequence, and to express the protein in E.coli expression system. Specific PCR cloning primers were designed for GGPS gene from Lepidium apetalum according to the full-length sequence from a previous transcriptome sequencing project. PCR amplification was performed with this primer pair on a leaf cDNA template. TA cloning, sequencing and sequence analysis were performed.GGPS gene from Lepidium apetalum was expressed in the E.coli expression system. The results showed that the full-lengthGGPS cDNA from Lepidium apetalum was 1 146 bp coding a protein of 381 amino acids. The LaGGPS protein had an isoprenoid synthase domain. According to a phylogenetic tree constructed with multiple alignment of GGPS protein sequences from various plant species, GGPS protein from Lepidium apetalum was the closest to Arabidopsis thaliana and Sinapis alba. The prokaryotic expression vectorpET-32a-LaGGPS was also constructed successfully. The protein was expressed in E.coli BL21 strain. It was concluded that the cloning and prokaryotic expression of LaGGPS gene provided a foundation for a follow-up research of its function with protein purification and activity analysis.

This study was aimed to establish the method for fraction-splitting of seeds of Descurainia sophia, in order to study the stability and unoverlapping property of fractions of seeds of D. sophia. The fraction-splitting of seeds of D. sophia were obtained through the combination of various methods including decoction, distillation, extraction, ethanol precipitation and gradient elution of macroporous adsorption resin. HPLC and chemometrics software were used to analyze the stability and unoverlapping property of the fractions of D. sophia. The results showed that the chemical components from seeds of D. sophia was divided into six fractions. HPLC data and chemometrics analysis showed good stability of technology. The fraction-splitting of seeds of D. sophia was unoverlapping. It complied with the research model of chemical constituents of seeds of D. sophia which can be split and combinatorial. It was concluded that the established method for splitting fractions of seeds of D. sophia had good stability and repeatability. Each splitting fraction uncrossed others.

This article was aimed to study the immunomodulatory effect of ethanol sediments of the seeds of Descurainia sophia(L.) Webb. ex Prantl. both in vitro and in vivo. The lymphocyte proliferation test in vitro was carried out to explore the effect of the ethanol sediments on the proliferation of T cell and B cell in the spleen of normal mice. And, the carbon clearance test, serum hemolysin test, and delayed-type hypersensitivity test were used to investigate the influence of fraction on non-specific immunity, humoral immunity and cellular immunity in the immunosuppressive mice induced by cyclophosphamide. Besides, the immunosuppressive model was used to evaluate the effect of fraction on immune organs and content of cellular factors in blood serum. The results showed that the ethanol sediments promoted Concanavalin A (Con A) induced T cell and Lipopolysaccharides (LPS) induced B cell (P < 0.01). It increased the carbon clearance index K, phagocytic index α, half value hemolysis (HC50), and swelling degree of auricula (P < 0.05 or P < 0.01). It reduced the body weight and atrophy of thymus and spleen index (P < 0.05 or P < 0.01). It increased the contents of IL-2, IFN-γ and IL-4 in serum in immunosuppressive mice (P < 0.05 or P < 0.01). It was concluded that ethanol sediments of the seeds of D. sophia(L.) Webb. ex Prantl. can boost the lymphocyte proliferation, protect the immune organs, and enhance the non-specific and specific immunity in immunosuppressive mice, which indicated that it had immune-promotion effect.

This article was aimed to study the chemical constituents from the chemical split fractions of Mori Cortex. The compounds were isolated with Diaion HP-20, Toyopearl HW-40, Sephadex LH-20, MCI Gel CHP-20, Silica gel column chromatography and preparative HPLC. Structures of compounds were identified by physicochemical properties and spectral analysis. The results showed that 23 compounds were obtained. And their structures were identified. The 16 compounds were obtained from the 30% ethanol fraction as vanillic acid (1), 3,4-dimethoxyphenol (2), benzoic acid (3), syringic acid (4), kelampayoside A (5), p-hydroxyphenylpropionic acid (6), caffeic acid (7), hydroferulic acid (8), 6,7-dihydroxycoumarin (9), 5,7-dihydroxycoumarin (10), morin-7-O-β-D-glucopyranoside (11), liriodendrin (12), 2,3-trans-dihydromorin (13), 2,3-cis-dihydromorin (14), 2,3-trans-dihydroquercetin (15), 2,3-cis-dihydroquercetin (16). The 4 compounds were obtained from the 50% ethanol fraction as scopoletin (17), morin (18), kaempferol-7-O-β-D-glucopyranoside (19), umbelliferone (20). The 3 compounds were obtained from the 80% ethanol fraction as sanggenon R (21), cis-mulberroside A (22), resveratrol (23). It was concluded that compounds 2, 4-6, 11, 16, 19 were isolated from this plant for the first time.