High temperature induces heat stress, adversely affecting the growth and lactation performance of cows. Research has shown the protective effect of taurine against hepatotoxicity both in vivo and in vitro. This study aimed to investigate the effect of taurine on the metabolomics of mammary epithelial cells of dairy cows under high-temperature conditions. Mammary epithelial cells were exposed to 0 mmol/L (HS, control), 8 mmol/L (HT-8), and 32 mmol/L (HT-32) of taurine, then incubated at 42°C for 6 h. Metabolomics analysis was conducted using Liquid Chromatograph Mass Spectrometer (LC–MS). Compared with the HS group, 2,873 and 3,243 metabolites were detected in the HT-8 group in positive and negative ion modes. Among these, 108 and 97 metabolites were significantly upregulated in positive and negative ion modes, while 60 and 166 metabolites were downregulated. Notably, 15 different metabolites such as palmitic acid, adenine and hypoxanthine were screened out in the HT-8 group. Compared with the HS group, 2,873 and 3,243 metabolites were, respectively, detected in the HT-32 group in the positive and negative ion modes. Among those metabolites, 206 metabolites were significantly up-regulated, while 206 metabolites were significantly downregulated in the positive mode. On the other hand, 497 metabolites were significantly upregulated in the negative mode, while 517 metabolites were reported to be downregulated. Noteworthy, 30 distinct metabolites, such as palmitic acid, phytosphingosine, hypoxanthine, nonanoic acid, and octanoic acid, were screened out in the HT-32 group. KEGG enrichment analysis showed that these metabolites were mainly involved in lipid metabolism, purine metabolism and other biological processes. Overall, our study indicates that taurine supplementation alters the metabolites primarily associated with purine metabolism, lipid metabolism and other pathways to alleviate heat stress in bovine mammary epithelial cells.
To construct a lentiviral vector overexpressing JSRV-env,screen human bronchial epithe-lial cells-2b(BEAS-2B)stably expressing Env,and detect the effect of Env on cell proliferation and migration.The target gene env was amplified by PCR and inserted into the pMT194 plasmid to construct the pMT194-JSRV-env lentiviral overexpression plasmid.The recombinant plasmid was mixed with the packaging auxiliary plasmid and co-transfected with the transfection reagent com-plex into 293T cells.After collection,purification and concentration of the recombinant lentivirus,the virus was used to infect BEAS-2B cells,the infection conditions were optimized.The stable cell strain was screened by puromycin(Puro)according to the resistance gene puro carried by the lentiviral vector.The expression of the target gene was detected by qPCR and Western blot at the transcriptional and translational levels.The cell proliferation ability was detected by CCK-8 and the cell migration ability was detected by scratch test.The target gene env was successfully amplified and inserted into the PMT194 vector.The enzyme digestion identification was consistent with the expectation,and the PMT194-JSRV-env expression vector was successfully constructed.The ex-pression of env in 293T cells was significantly higher than that in blank control group and negative control group(P＜0.001).The average titer of JSRV Env recombinant lentivirus was 1.03 × 109 IU/mL.The optimalmultiplicity of infection(MOI)of the recombinant lentivirus on BEAS-2B cells was 40,the optimal Puro concentration for screening cell strain was 0.3 mg/L,and fluores-cence microscopy showed that BEAS-2B cells expressed red fluorescence.The results of qPCR and Western blot showed that the expression of env in the experimental group was significantly higher than that in the blank control group and the negative control group(P＜0.001).The Env-FLAG fu-sion protein was successfully expressed in the host cells.Compared with the blank control group and the negative control group,the proliferation and migration ability of the cells in the experimen-tal group were significantly enhanced(P＜0.05).In summary,the JSRV-env lentiviral overexpres-sion vector was successfully constructed,the BEAS-2B cell strain stably expressing Env was screened,and Env can significantly increase the proliferation and migration of BEAS-2B cells.
To isolate the porcine reproductive and respiratory syndrome virus(PRRSV)in Tianjin and analyze its molecular genetic evolution,RT-PCR tests were performed on samples suspected of PRRSV infection from a pig farm in the area.Positive samples were used to infect porcine alveolar macrophages(PAMs),and PAMs with cytopathic effect(CPE)were identified by indirect immuno-fluorescence(IFA)after limited dilution.RT-PCR was used to isolate and purify the whole genome sequence of the strain and homology and genetic evolution analysis were performed.Recombinant analysis and cellular tropism tests were performed on the isolate.The results showed that the isola-ted PRRSV strain(named TJbc2021)did not infect Marc-145 cells.The whole genome length of this strain was 15 111 bp,which has the highest nucleotide homology with NADC34 with a nucleo-tide homology of 95.2％.Analysis revealed the strain belongs to the NADC34-like PRRSV that is currently mainstream in China,and its Nsp2 has the molecular characteristics of 100 amino acid deletions.Recombinant analysis showed that the strain had genetic recombination at 14 082-14 635 nt(ORF6-ORF7)with lineage 1.5 strain(NADC34-like)as the main parent and lineage 1.8 strain(NADC30-like)as the secondary parent.The results of this study provide reference for the preven-tion and control of reproductive and respiratory syndrome in pigs.
To clarify the genetic variation and antigenic characteristics of the GAstV Liaoning strain,diseased materials such as goose liver and kidney were aseptically collected from a goose farm in Liaoning Province.The pathogen was isolated and identified by goose embryo inoculation,nucleic acid detection,and electron microscopy,then genetic variation analysis and antigenic epitope prediction were carried out for the isolated strain.By observing the pathological characteristics of inoculated goose embryos,PCR nucleic acid amplification and sequence analysis of the virus,and morphological identification of transmission electron microscope,the results showed that one strain of GASTV was successfully isolated,it was tentatively named GAstV/LN/202101(abbrevia-ted as LN202101).The genetic evolution tree and homology analysis were conducted for the conserved domains N and P2 capsid domain encoding capsid protein,the results showed that the strain was closely related to Jiangxi strain JX01(MZ576222.1)and other strains found in recent two years,branched in the same cluster and belonged to GAstV-Ⅱ type.However,its homology has shown differences.Taking the verified epitopes of HAstV and the antigen-antibody binding model as a reference,combined with the bioinformatics methods,the antigen epitopes of GAstV can be speculated as the following four sequences:453-467aa PQADSRSRYNANITF,508-513aa VCNTLA,525-553aa TAVLRVNTSTTSTGGQITELRNRLNIADG,and 585-597aa DSNP-GETFQSFKM.The above results showed that the epidemic strain of GAstV in Liaoning and the strains from other regions in China were homologous,but it appeared mutations;Liaoning strain could be considered as a representative of the epidemic strains in recent two years because it had many dominant epitopes of capsid protein.The above results laid a foundation for further clarifying the epidemic and immune characteristics of the virus and developing corresponding vaccines,anti-bodies,and other biological prevention and control products.
This study aimed to investigate mechanisms of resistance to β-lactams and quinolones for 28 cephalosporin-resistant Salmonella isolates from pig-borne food chain of Henan Province.Sero-typing,antimicrobial susceptibility testing and extended-spectrum β-lactamases(ESBLs)were per-formed;the presence of β-lactamase genes,plasmid-mediated quinolone resistance genes and amino acid mutations in the quinolone resistance-determining region(QRDR)was detected by PCR am-plification and sequencing.The results showed that the prevalence rate of cephalosporin-resistant Salmonella isolates was 0.89％(28/3 137)in all samples from pig-borne food chain of Henan Province,with the highest prevalence in liver samples(4.98％,16/321).All Salmonella isolates were classified into 7 serotypes,and the main serotypes were Indiana(46.43％,n=13)and monophasic Salmonella typhimurium(25％,n=7).Except for colistin,the resistance rate of all strains to other 11 drugs was higher than 60％,and the multi-drug resistance rate was 100％.All the isolates tested carried at least one β-lactamase gene,and the bla CTX-M gene had the highest carrying rate(85.71％,n=24).There were six combinations of p-lactamase gene profiles.Four quin-olone resistance genes(AAC(6)-Ib-cr,oqxAB,qnrS and qnrC)were detected,and no amino acid mutation was found in GyrB and ParE.Fifteen isolates with both gyrA(Ser83Phe and Asp87Asn/Gly)and parC(Thr57Ser and Ser80Ile/Arg)mutations were resistant to ciprofloxacin at high lev-els(MIC≥16 mL/L)regardless of the presence of quinolone resistance genes.This study showed that the prevalence of cephalosporin-resistant Salmonella strains in the pig industry chain in Henan Province was relatively low,and these Salmonella strains had serious resistance to a variety of drugs,of which the phenotype of resistance to β-lactams and fluoroquinolones was consistent with the genotype of resistance.
The objective of this study is to investigate the effects of cadmium induced cytotoxicity,immune adjustment on avian macrophage,the protective effects of zinc and NAC on cadmium-ex-posed macrophage.In this experiment,cadmium poisoning(20,50 μmol/L)cell model was estab-lished.Cadmium toxicity damage to HD-11 cells was investigated at different time periods(6,12 h).Additionally,the effects of Zn2+and NAC on cytoskeleton,apoptosis rate,reactive oxygen species(ROS)content,level of mitochondrial membrane potential(MMP)of cadmium induced macrophages by supplementing the cell culture medium with Zn2+(20 μmol/L),NAC(500 μmol/L)was investigated.The regulatory mechanisms of Zn2+and NAC on cadmium-stained macropha-ges were revealed by detecting the transcript levels of cellular metal-element binding protomer(MTF-1)and metallothionein(MT-1)genes.The results showed that cadmium poisoning caused the degradation of HD-11 cytoskeletal proteins,ROS production,decrease of MMP level,increase of apoptosis,and decrease of IL-1β,IL-8,and IL-10 genes expression,and the addition of Zn2+and NAC reduced the structural damage of cadmium-poisoned macrophages to different degrees,de-creased the production of ROS and the depolarization level of MMP,and strengthened the cellular antioxidant capacity and immune function,as well as promoted the transcription levels of MTF-1 and MT-1,and the regulation of MTF-1 and MT-1 genes.and promoted MTF-1 and MT-1 gene ex-pression.The results showed that cadmium contamination could cause concentration-time-depend-ent structural damage and immune dysfunction in chicken macrophages,and the addition of Zn2+and NAC alone or in combination could effectively inhibit the toxicity damage of cadmium-contaminated chicken macrophages and improve the cellular immune function by enhancing the cellular antioxidant level.
In order to establish a rapid serological diagnostic method for the detection of infectious bovine rhinotracheitis virus(IBRV)antibody,the gD protein was labeled with colloidal gold and coated on the binding pad as the detection probe,the unlabeled gD protein was coated with NC membrane as the detectionline,the gold labeled mouse IgG and goat anti-mouse IgG were used as independent quality control systems.The colloidal gold immunochromatographic test paper for the detection of IBRV antibody was established by the double antigen sandwich method,and the per-formance of the test paper was evaluated.The results showed that the test paper was negative when detecting the standard positive sera of MAP,AKAV,BVDV,M.bovis,PPRV,Brucella and FMDV.The sensitivity of the test paper in detecting IBRV standard positive serum was 1∶16.The test paper and the imported commercial ELISA kit were used to detect 60 bovine sera to be tested.The positive coincidence rate was 93.3％,the negative coincidence rate was 100％,and the total co-incidence rate was 96.7％.The colloidal gold immunochromatographic test paper established in this study can complete the detection in 10-15 min,which is rapid,accurate and simple,and provides an effective tool for clinical qualitative detection and field diagnosis of IBR.
In order to understand the pathogenic bacteria species causing subcutaneous abscess,and its distribution characteristics in goats in Yunnan Province,bacterial isolation and identificationand PCR were used to detect 128 subcutaneous abscess samples collected from goatsin goat farms in Xishan District of Kunming City,Shilin County,Yiliang County and Mile County,and the results of the two methods were compared.Among the 128 subcutaneous abscess samples,the PCR detec-tion rates of Arcanobacterium pyogenes,Corynebacterium pseudotuberculosis and S.aureus were 17.19％(22/128),20.31％(26/128)and 67.19％(86/128),respectively.The isolation rates of A.pyogenes,C.pseudotuberculosis,S.aureus and Streptococcus ovis were 9.38％(12/128),20.31％(26/128),65.63％(84/128)and 1.56％(2/128),respectively.Among the 128 subcutaneous abscess samples,the detection rates of single bacterial species in the bacterial isolation and identification method and the PCR method were 95.31％(122/128)and 92.19％(118/128),respectively.The total coincidence rate of the bacterial isolation and identification method and PCR method was 90.63％(116/128).The results showed that S.aureus was the most common pathogenic bacteria species causing subcutaneous abscess in goats in Yunnan Province.Moreover,S.ovis strains were i-solated from goat subcutaneous abscess samples for the first time,suggesting that S.ovis may be a potential new pathogen causing subcutaneous abscess in goats.This study provides basis for the prevention and control of subcutaneous abscess in goats in Yunnan Province.
Novel goose parvovirus(NGPV)is a variant of goose parvovirus(GPV),which can in-fect young ducks and cause an infectious disease characterized by duck short-beaks and dwarf syn-drome.In 2015,the disease broke out in duck herd in China,which seriously affected the emergence rate of ducks and brought huge economic losses to breeding industry of China.To establish an indi-rect ELISA method for the detection of antibodies against NGPV for the serological detection of parvovirus infection in ducks and the monitoring of antibody levels in ducks immunized with parvovirus vaccine,the antigenic epitope of the structural protein VP3 of NGPV YICH strain was predicted by bioinformatics,and the antigen rich region(250-525 aa)was selected for prokaryotic expression.After protein purification,the soluble truncated protein VP3-tr was obtained.Then,u-sing VP3-tr as coating antigen,a new indirect ELISA method for detection of NGPV antibody was established by optimizing the reaction conditions through checkerboard titration method.The opti-mized indirect ELISA method was used to detect 30 duck serum samples negative for GPV,and the cut-off value of negative and positive was 0.219.The sensitivity,specificity and repeatability of ELISA showed that the method had high sensitivity and good specificity,and did not cross react with the positive serums of duck Tambusu virus,Newcastle disease virus,avian influenza virus,Riemerella anatipestifer and E.coil.The coefficients of variation of intra-and inter-assay repeated tests were less than 6％,which showed good reproducibility.The results showed that the positive rate of ELISA was 85.0％,and that of Western blot was 69.4％.The coincidence rate of ELISA and Western blot was 84.4％.In conclusion,this study successfully established an indirect ELISA method for the detection of antibodies to the NGPV from duck,which provides a simple and relia-ble method for the surveillance of ducks infected with NGPV and the evaluation of vaccine immu-nity.
Senecavirus A(SVA),a newly emerged virus in recent years,it can cause swine vesicular disease,the virus has become more and more widespread in China,causing huge economic losses to the pig industry.Conventional inactivated SVA vaccine developed in some laboratories can effec-tively prevent and control the disease,but there are some defects such as low antibody level and short immune period.In order to improve the immune effect of inactivated vaccine,this study based on SVA infectious clone,and the porcine GM-CSF-T2A gene was inserted between 2A and 2B of SVA by fusion PCR,and the recombinant plasmid SVA-GM-CSF was successfully constructed,then it was transfected into BHK-21 cells for viral rescue and passage.The results showed that the recombinant virus in BHK-21 cells could cause obvious cytopathic effect.RT-PCR amplification and gene sequencing showed that the target gene was inserted correctly.Indirect immunofluores-cence showed that the foreign gene GM-CSF was successfully expressed in this virus.Biological characterization analysis showed that the recombinant virus and the parent virus had similar growth characteristics in BHK-21 cells,and the recombinant virus had good genetic stability during the passage,the construction of the recombinant virus provides theoretical support and technical guidance for further clarifying the pathogenesis of SVA and developing a new vaccine.
In order to understand the genetic variation and pathogenicity for porcine epidemic diar-rhea virus(PEDV)epidemic strains,PEDV were isolated from PEDV-positive samples using Vero cells,and identified by indirect immunofluorescence assay(IFA).Whole genome sequencing,ho-mology comparison and genetic evolution analysis of the isolates were conducted based on RT-PCR and the second-generation sequencing technology.The virulence of the isolates was determined by pathogenic trial in suckling piglets.IFA results showed that the isolates reacted specifically with PEDV S protein antibody,which was identified as PEDV.The nucleotide homology between the two strains and the reference strain was 95.6％-98.7％,which belonged to the G Ⅱ b group.The two isolates were named as CH-HNKF-03 and CH-HNDS-01 respectively.Disease onset occurred at 18 h after challenge with CH-HNKF-03 and CH-HNPDS-01 to suckling piglets,and typical wa-tery diarrhea,vomiting,emaciation,dehydration and other symptoms appeared 24-56 h,and all died within 96 h.In conclusion,two highly virulent PEDV G Ⅱ b variants were isolated in this stud-y,which provides candidate strains for the development of novel PEDV vaccine.
This study aims to investigate the effects of aflatoxin B1(AFB1)on the antioxidant func-tion of bovine mammary epithelial cells(MAC-T).The MAC-T cells in logarithmic growth phase were cultured with AFB1 at different concentrations for 24 h.Reactive oxygen species(ROS),su-peroxide dismutase activity(SOD),glutathione peroxidase activity(GSH-Px),malondialdehyde(MDA),total antioxidation capability(T-AOC)were tested using the relative detection kit.The relative expression level of Nuclear factor erythroid-2-related factor 2(NrF-2),Heme oxygenase-1(Oxygenase-1),Heme oxygenase-1(HO-1)and NAD(P)H quinone oxidoreductase 1(NQO-1)mRNA were detected by real-time quantitative polymerase chain reaction(RT-qPCR)method.The relative expression levels of NrF-2,HO-1 and NQO-1 protein were detected by Western blot as-say.The results showed that compared to the 0 μmol/L group,the contents of ROS and MDA in MAC-T cells were significantly increased in 10,20 μmol/L AFB1 treatment groups,while the ac-tivities of SOD,T-AOC and GSH-px were decreased.The results of RT-qPCR assay indicated that compared with the 0 µmol/L group,the expression levels of NrF-2,HO-1 and NQO-1 mRNA in MAC-T cells were significantly down-regulated in AFB1 with different concentrations.Western blot results showed that 10 µmol/L AFB1 significantly decreased the protein expression levels of NrF2,HO-1 and NQO-1 in MAC-T cells compared with the 0 μmol/L group.These results indi-cate that AFB1 could induce oxidative stress injury of bovine mammary epithelial cells(MAC-T)by inhibiting NrF2 signaling pathway.
The optimal mass concentration and time of action of lipopolysaccharide(LPS),a compo-nent of the bacterial cell wall,and poly(i:c),a viral mimetic,on the nucleoplasmic shuttle of high mobility group protein B1(HMGB1)in BV-2 microglia were determined by fluorescence quantita-tive PCR.IBA-1,a specific marker of BV-2 microglia activation,was used to determine whether LPS and poly(i:c)could activate BV-2 microglia;the protein expression of pro-inflammatory cyto-kines(IL-1β,TNF-α,NLRP3 and iNOS)in BV-2 microglia induced by LPS and poly(i:c)was de-tected by Western blot.The results showed that 10 μg/L LPS,50 ng/L poly(i:c)and 6 h of stimu-lation significantly increased the protein expression of pro-inflammatory factors;immunofluores-cenceresults showed that BV-2 microglia were activated by LPS and poly(i:c)stimulation;nucleo-plasmic Isolation results showed increased cytoplasmic translocation of HMGB1 in BV-2 microglia stimulated by LPS,poly(i:c).The results suggest that either bacterial or viral infection can cause HMGB1 translocation from the nucleus to the cytoplasm in BV-2 microglia.
qseC and luxS gene are belong to quorum-sensing related genes in Pasteurella multoci-da,and the effects of qseC and luxS dual-deletion on virulence and cross-protection in P.multoci-da are still not well understood.In present study,the mutant of dual-deletion of qseC and luxS gene(ΔqseC ΔluxS)was constructed based on qseC mutant(ΔqseC)derived from bovine P.mul-tocida type A CQ2(PmCQ2).Compared with wild type PmCQ2,dual deleting of qseC and luxS gene promotes the mutant production of biofilm,and downregulates the mutant in capsule produc-tion,serum sensitivity and virulence.Compared with single qseC gene deletion,the dual deletion of qseC and luxS gene has positive or negative superimposition effects in some biological properties such as the production of biofilm and capsule.In terms of vaccine cross-protection,compared withΔqseC and wild-type PmCQ2,the deletion of luxS gene in ΔqseCΔluxS weakened the cross-pro-tection effect brought by qseC gene deletion,but increased the cross-protection effect with qseC gene deletion against bovine P.multocida serotype F.Compared with wild type PmCQ2 vaccine,the dual deletion of qseC and luxS gene still endowed the mutant good cross-protection against P.multocida infection.The results indicated that both qseC and luxS could regulate the virulence and cross-protection of P.multocida,and qseC and luxS gene mutant can be used as a candidate vac-cine against P.multocida strains of multiple serotypes infection.
The collected porcine COCs were placed in the in vitro maturation medium of porcine o-ocytes supplemented with different concentrations(0.0,0.3,0.6,1.2 μmol/L)of monoammonium glycyrrhizinate(MAG)for 46 h,the maturity rate was calculated,and the expression level of ROS in mature oocytes was detected by immunofluorescence staining.In vitro fertilization of mature o-ocytes,cleavage rate and blastocyst rate of IVF embryos were measured at 48 h and 120 h,respec-tively.Hochest fluorescence staining was used to detect the total number of blastocyst cells.The optimal concentration of MAG was added into the in vitro maturation medium,and 0.0 μmol/L MAG was used as the control group.After 46 h in vitro culture,the mitochondrial membrane po-tential and apoptosis of porcine mature oocytes were detected by immunofluorescence staining.Compared with the control group,the in vitro maturation rates of porcine oocytes in different MAG treatment groups were increased,but the differences were not significant(P＞0.05).The levels of ROS in porcine oocytes were decreased by MAG supplementation at different concentrations,and there were significant differences in 0.3,0.6 μmol/L groups(P＜0.05)and ex-tremely significant differences in 1.2 μmol/L groups(P＜0.01).The 0.3 μmol/L supplementation group significantly increased the blastocyst rate in vitro fertilization(P＜0.05),but there were no significant effects on the embryo cleavage rate and total blastocyst cell number in different MAG supplementation groups(P＞0.05).Compared with the control group,the mitochondrial mem-brane potential level of oocytes in 0.3 μmol/L MAG supplemental group was significantly in-creased(P＜0.05),and the apoptosis level was significantly decreased(P＜0.05).0.3 pmol/L MAG could increase mitochondrial activity,reduce the oxidative stress and apoptosis level of por-cine oocytes,improve the quality of in vitro maturation of porcine oocytes,and significantly im-prove the developmental ability of IVF embryos.
It has been shown that lysosomes are closely associated with β-coronavirus replication.To investigate the effect of the key protein particle protein progranulin(PGRN),which regulates lysosome function,on β-coronavirus porcine hemagglutinating encephalomyelitis virus(PHEV)replication,the role of PGRN in the process of PHEV intracerebral infection in mice with PGRN deficiency and its control mice was investigated.It was found that PGRN-deficient mice survived longer after intracerebral infection with the virus compared to PHEV-infected wild-type mice,while intracerebral viral levels were significantly lower and cytokine IFN and TNF-a levels were higher;in addition,the expression of the neurodegenerative disease-associated protein TDP-43 was significantly higher,indicating that PGRN deficiency slowed viral replication,enhanced the immune response,and affected viral pathogenicity.The results of the study provide a research basis to fur-ther reveal the role of the lysosomal protein PGRN in the replication process of PHEV and provide a reference for the study of the pathogenesis of β-coronavirus.
In order to establish a rapid visualization method for detecting peste des petits ruminants virus(PPRV),the RT-RAA amplified primers and crRNA synthesized primers were designed,synthesized and screened according to the conservative sequence of PPRV N gene.Based on RT-RAA,Cas12a trans-cleavage reaction and lateral flow dipstick,a rapid visualization method for PPRV detection was developed and validated.Specificity assay showed this method only detected PPRV with no detection for BTV,EHDV,FMDV,CaPV,SVDV and PRV.Sensitivity test results showed that the detection limit of this method for PPRV RNA reached 4.30 X101 copies/μL.Paral-lel detection of the simulated samples by this method and the standard RT-qPCR demonstrated a high consistency with the Kappa value of 0.918.In conclusion,the established visualized method was specific,sensitive,simple,and fast,which provide a reliable tool for rapid screening and detec-tion of PPRV in field or basic farm.
Highly pathogenic porcine reproductive and respiratory syndrome(HP-PRRS)has been raging in China for a long time,domestic vaccines are mainly live attenuated vaccines,which can ef-fectively protect pigs from viruses,but are easy to recombine with wild strains,resulting in the e-mergence of new variants.Inactivated vaccines are safe but low vaccine titer,so this study is to de-velop a safe and effective vaccine that will not recombinant.Reverse genetic technology was used to insert HP-PRRSV antigenically active GP5 gene sequence into Newcastle disease virus vector.Re-combinant viruses that stably express GP5 was rescued,identified by PCR,enzyme digestion,Western blot,indirect immunofluorescence(IFA)and virulence detection experiments.The results showed that the D value of specific antibody at 35 d was 0.47(P＜0.01),and neutralizing antibody reached 1:8(P＜0.01),which could induce efficient antibody expression in mice.The proliferation of splenic lymphocytes in rL-GP5(LQ)group was significantly increased(P＜0.01),indicating that rL-GP5(LQ)could induce the immune response in vivo,providing new vaccine candidates and ideas for the prevention and control of HP-PRRS.
According to the reference sequence of chicken transmembrane 9 superfamily 2(chTM9SF2),the coding sequence amplification primers were designed,and cDNAs of primary chicken embryo fibroblasts(CEF),chicken fibroblast line DF1 and chicken liver cancer cell(LMH)were used as templates,and chicken TM9SF2 coding sequence was obtained from the above three templates by PCR method,and bioinformatics tools such as MEGA 7.0,Clustal W,SWISS-MOD-EL were used to obtain the encoding sequence of chicken,human,duck,murine TM9SF2,then the sequence was analyzed for genetic evolution,homology,domain and structural simulation.Western blot and laser confocal microscopy were used to analyze the expression and subcellular distribution of chTM9SF2.The results showed that the chTM9SF2 gene with consistent coding sequence was successfully obtained in three types of cells,and the homology with bird was the highest,and the extracellular region domain was found to be highly conserved.This gene could be expressed in DF-1 cells and the protein was mainly localized in the late endosomal or lysosomal membranes.The chicken-derived TM9SF2 gene was successfully expressed in DF-1 cells,and the protein localized in the late endosomal or lysosomal membranes,which laid a foundation for further study of the phys-iological function of chTM9SF2,and its role in pathogenic infection.
Pseudorabies virus(PRV)is the causative agent of pseudorabies(PR)that infects most mammals.Since its discovery,it has become popular all over the world and has caused huge eco-nomic losses to the pig industry.In recent years,to prevent and treat PR,the pathogenic mecha-nism of PRV has been deeply studied,and some progress has been made in the changes of host in-nate immune signal transduction after PRV infection,as well as the impact on host immune re-sponse,but there are still many signaling pathways and virus mechanisms to be revealed.There-fore,this paper reviews the research progress of virus-host interaction by binding to different re-ceptors after PRV infection,in order to provide reference for the subsequent research on the patho-genic mechanism of PRV and antiviral drugs.
