Based on the nucleotide sequence of Japanese encephalitis virus(JEV) SA_(14)-14-2 strain,a pair of primers was designed.The prM/E gene of JEV was cloned by RT-PCR.The sequence analysis showed that the prM genes of SA_(14) and SA_(14)-14-2 strains had 100% homology,while there are 4 mutants in the E gene,and it cause 3 amino acids change.The prM/E gene was digested with BamHⅠ and EcoRⅠ,and the fragment was inserted into the same sites of the universal vector pPgG-uni.A recombinant virus transfer vector pPgG-prM/E was constructed,the pPgG-prM/E digested with KpnⅠ was co-transfected PK-15 cells with PRV Ea TK~(-)/gG~(-)/LacZ~(+) mutant strain genomic DNA digested with EocRⅠ using liposome method.The recombinant virus TK~(-)/gG~(-)/prM/E~(+) was gained by the plaque purification and PCR amplification.This recombinant virus strain can be used for the research of genetic engineering vaccine against PRV and JEV.
A one-step fluorogenic RT-PCR(5′-nuclease probe-based) assay using a primer/TaqMan probe set designed against a highly conserved region of open reading frame 7(ORF7) of EAV genome was developed for the specific detection of EAV.The assay had an analytical sensitivity of 5 molecules of EAV RNA allowing the detection of EAV in clinical samples or tissue culture fluid(TCF) containing about 5 PFU/μL.Following intramuscular injection of two ponies with vaccine virus Bucyrus strain,the excretion of virus in respiratory passages can be detected more sensitively by the flunorogenic RT-PCR than by virus isolation test.The one-step RT-PCR method specifically detected EAV in samples from Customs and quarantine departments with rapider,more accurate,more convenient and higher sample throughout than our routine diagnostic procedures of ELISA and virus isolation in cell culture.Therefore it may be considered as a valuable tool to complement the routine diagnostic procedures for EAV diagnosis.
The purified N protein was used to be coated on the well of 96-well plate, each following step was optimized. As a result, an indirect ELISA was constructed to detect antibody against PRRSV. The various factors and conditions of ELISA were explored, and the optimal reaction conditions of ELISA were determined. It was shown that the optimal concentration of recombinant N protein for coating of plate was1 mg/L, the optimal coating condition of recombinant N protein for ELISA was 4℃ overnight, the dilution of serum sample was 1∶40, serum sample for detecting and HRP-labeled rabbit anti-porcine IgG should be incubated at 37℃ for 30 min and 40 min respectively, the substrate for ELISA was incubated at 37℃ for 10 min before terminated with the stopping solution.The blocking test for the reaction between the PRRSV positive serum and the recombinant N protein was carried out by using the PRRSV antigen.The results showed that PRRS virus could block the reaction completely. Meanwhile, it was confirmed that there was no cross reaction between the antibodies against other porcine infectious diseases.The results revealed that the indirect ELISA by the purified recombinant nucleoprotein had good specificity for the detection of PRRSV antibody in serum. The difference value among wells in a plate and among plates for ELISA was both less than 7%, which showed the assay had a good retrievality.
Seven strains Escherichia coli isolated from provinces having edema were identified by gram dyeing,biochemical reaction and serotying.These strains had pathogenicity and were able to produce Vero toxin,which could be confirmed by following examinations.Their broth culture could lead Kunming mice to death.Axenic filtrate of the broth culture could induce Kunming mice and Vero cell death.These strains were tested by PCR to detect genes of SLT-Ile,F18,LT1 and ST1.Seven strains were positive for SLT-Ile,five strains were positive for F18,but all were negative for LT1 and ST1.Which accorded with the literature previously reported.Antibiotics susceptibility test showed their resistance patterns were different.All of the seven strains could reproduce the edema disease of weaning piglets.
The envelope glycoprotein E2 of classical swine fever virus(CSFV) was a protective antigen inducing neutral antibody.In order to get soluble recombinant E2 glycoprotein and inspect the protein expression dynamics convinently,the enhanced green fluorescence protein(EGFP) and hog cholera lapinised virus(HCLV) E2 gene were cloned into baculovirus transfer vector pBlueBacHis2A and the recombinant plasmid pBGFPTE2 was constructed.After co-transfection pBGFPTE2 and linear baculovirus Bac-N-Blue into sf9 cell with cationic lipid reagent Cellfectin,homological recombination and plaque purification,the pure recombinant baculovirus clone named rBACTE2-339 were harvested and proliferated in sf9 cell to generate P2 stock.After infection with P2 stock,brilliant green fluorescence was detected under the fluorescence microscopy confirming the heterogenous protein expression of recombinant baculovirus in sf9 cell.
The VP2 gene of 9 IBDV field isolates that isolated from egg chicken(5 isolates) and dark-born chicken(4 isolates) from several parts of Luoyang area in 2000,2001 and 2002 years,were amplified by reverse transcription-polymerase chain reaction(RT-PCR) and sequenced.The results showed that there were 4 amino acids changed at the sites of 394(Val/Ile),381(Lys/Arg),375(Pro/Ser),and 320(Gln/His).The variations of nucleotide mainly occurred the replace of synonymous codons.Inoculated egg chicken embryo with 3 IBDV isolates isolated from dark-born chicken and inoculated dark-born chicken embryo with 3 IBDV isolates from egg chicken,respectively.Isolating IBDV after propagated blindly 7 generation from embryo chorin.Sequence analysis showed that there were preference of synonymous codons of IBDV-VP2 gene in egg chicken and in darkborn chicken.It appeared mainly at the sites of 220 Tyr(TAC/TAT),260 and 420 Thy(ACC/ACT,ACA/ACT),271 Val(GTA/GTG),279 Asp(GAC/GAT),305 Ile(ATC/ATA),328 Ser(TCA/TCG) as well as 224,360,393 Gly(GGA/GGG).The usage of synonymous codons in different strains chicken is not only one probable reason of susceptible of IBDV to egg chicken and dark-born chicken,cut also the main way of virus variation and new strains production possibly.
The gene encoding P15 of Cryptosporidium parvum (C. parvum) was amplified by PCR, and then inserted into the BamH Ⅰ site of pUC19 vector. Nucleotide sequencing showed that the gene was a 479 bp fragment and encoded a protein composed of 148 amino acid. The P15 gene was ligated into the BamH Ⅰ site of the expression vector pGEMEX-2, and then expressed in E. coli JM109 (DE3) and mammal cells. SDS-PAGE and Western blot analyses showed that the P15 gene of C. parvum was expressed in E. coli and the molecular weight of the fusion protein was estimated as 50000; the protein expressed in the mammal cells had a similar molecular weight (18000-22000) with the native P15 of C. parvum.
A pair of primers were designed and synthesized according to fedA of fimbriae F18ab (fedA/ab),and the coordinate subunit genes were amplificated from ten different Escherichia coli strains,F107/86,YCED1,YCED2,C,D,E,12F,2134P,8199 and 8813.Thereinto,F107/86 is display fimbriae F18ab,whereas 2134P,8199 and 8813 are all display fimbriae F18ac.All the PCR products were cloned into pGEM-T vector respectively.Color screening,PCR and restriction endonucleases analysis were used to identify the recombinant plasmids,and ten recombinant plasmids,TF107A,TYCED1A,TYCED2A,TCA,TDA,TEA T12FA,T8813A,T8199A and T2134PA were obtained.The ten recombinant plasmids sequences were analyzed,and compared with the published sequences of fedA/ab.The data of the sequences of the ten genes were showed that they belong to two different genic group,fedA/ab (513 bp) and fedA/ac (516 bp).F107/86,YCED1,YCED2,C,D,12F are fall into fedA/ab group,whereas E,8813,8199 and 2134P are fall into fedA/ac group,although they are possessed of high identity at nucleoide and amio acid level with fedA/ab group respectively.
The Interleukin-2 gene cDNA was cloned into the Pichia pastoris expression vector pPICZB,which is under the control of the alcohol oxidase promoter AOX1. The linearized recombinant plasmid of BoIL2-pPICZB,digested by Sac I ,was transformed into X-33 strains by electroporation. The multi-copy insert transformants were screened by Zeocin-resistance and induced by 1% methanol. The intracellular expression products were tested by SDS-PAGE analysis and Western blotting. Purified recombinant BoIL2 was gained by metal-chelating affinity chromatographic (MCAC). Assay with murine CTLL-2 cells showed that the recombinant BoIL2 exhibited the biological activity.
In this paper,30 microsatellite markers with medium or high polymorphisms were selected to detect the genetic diversity of 7 indigenous chicken breeds in Jiangxi.According to allele frequencies of 30 microsatellites,the polymorphism information content(PIC),mean heterozygosity(h),Nei′s standard genetic distance(D_S) and D_A genetic distance were calculated for each breed.The results showed that 24 of 30 microsatellite loci were highly polymorphic,so the 24 microsatellite loci were effective markers for analysis of genetic relationship among the chicken breeds.The mean heterozygosity(h) in the Yugan Silky was the lowest(0.615 5),whereas in the Chongren spotted chicken was the highest(0.652 5),and in other breeds was also more than 0.5,indicative of their wide diversity.D_A genetic distances between 7 chicken breeds were consistent with D_S,but the values of D_A were bigger than those of D_S. Two same UPGMA trees were obtained through the analysis of D_A genetic distance and Nei′s standard genetic distance(D_S).In both trees,Yugan Silky,Wahui chicken,Kangle chicken and Ningdu Sanhuang chicken were first grouped together,Chongren spotted chicken and Dongxiang Green-shell chicken were second grouped together,but Silky had its own branch.
The VP2 gene of infectious bursal disease virus HK46 strain was amplified by polymerase chain reaction with special primers containing BglⅡ and NheⅠ. Then the digested and purified VP2 gene of infectious bursal disease virus was cloned into plant expression vector pCAMBIA3301. The transformation of Agrobacterium tumefaciens LBA4404 was performed by electroporation with the binary vector containing VP2 gene and CAMV 35 s promoter. Using PCR technique, it is confirmed that the recombinant plasmid pCAMBIA3301 with VP2 gene was successfully transferred into Agrobacterium tumefaciens LBA4404.
In order to explore the possibility of developing a HBV/HAV combined gene vaccine,a multi-epitope antigen gene VPX of HAV was fused with HBsAg gene and SVPX gene was constructed.SVPX was sub-cloned into an eukaryotic expression plasmid pVAX1 to construct the eukaryotic recombinant expression plasmid pVAX1/SVPX.The identified recombinant was then transfected into CHO cells with liposome mediated method.The transfected cells was tested by RT-PCR,Western-blot,ELISA method 48 hours after culture.The results showed that the multi-eptitope gene SVPX can be transcripted and expressed,and expressed fused protein could react with HAV and HBV antisera.
A high-performance liquid chromatographic method(HPLC) was reported for the quantitative determination of residues of ciprofloxacin in eggs of laying hen.Ciprofloxacin was extracted from eggs with acetonitrile solution.The extract solution was dried in vacuum condition(45℃) and residues were dissolved in mobile phase.Ciprofloxacin was separated on a Symmetry C_ 18 Column(5 μm,3.9 mm×150 mm) by 0.015 mol/L tetrabutyl ammonium bromide/acetonitrile(94/6) and determinated at λ_ EX 280 nm,λ_ EM 455 nm by fluorescence detector.Recoveries of ciprofloxacin added to blank eggs at levels of 0.05,0.50,1.00 mg/kg were(82.2±6.8)%,(84.5±5.9)% and (87.9±4.7)%,respectively.This method permitted the minimum detection limit of ciprofloxacin residues in eggs at 0.01 mg/kg.After the laying hens orally administered successively ciprofloxacin hydrochloride solution of 10 mg/kg and 20 mg/kg of body weight one time every day for 5 days,respectively,residues of ciprofloxacin in eggs were eliminated slowly.Residues of ciprofloxacin in eggs were lower than 0.03 mg/kg at 8 th-9 th withdrawal day.Residues of ciprofloxacin in eggs were all lower than the minimum detection limit(0.01 mg/kg) at 9 th-10 th withdrawal day.With the increase of ciprofloxacin orally adminietered doses,residues of ciprofloxacin in eggs increased accordingly.
Cerebellar grannule neuron is one of crucial reactive cells in the occurrence and development in animal transmissible spongiform encephalitis(TSE).Culture in vitro,purified and identification of cerebellar grannule neurons is an important procedure in establishing the cell model for transmissible spongiform encephalitis.Based on the different properties of cellular growth pattern,using the measure of controllability filtration,cerebellar grannule neurons were cultured and purified.Immunostaining by symbolic enzyme NSE of neurons,cerebellar grannule neuron were identified and the purification of cell culture is high.Cultured astrocytes can be used for establishing the cell model.
The positive clone pBK-CMV-WN1 from the cDNA library of newborn larvae of Trichinella spiralis was isolated by immunoscreening with T.spiralis infected swine serum.Sequence analysis showed that this clone contained cDNA transcript of 474 bp in length with a full open reading frame(ORF) of 354 bp which encoded 117 amino acids with molecular weight of ~13 200 and isoeletric point of 4.25.The signal peptide sequence indicated the putative peptide a secretory protein.The WN1 cDNA was amplified by PCR and cloned into pET28 expression vector.The recombined transformant was induced and the recombinant protein of ~15 000 was successfully expressed.
One hundred and fifty Landrace×Large-White geld piglets were randomly allotted to six groups of 25 each.Pigs in group 1 and group 4 were given basal diet without any medicine,pigs in group 3 and 6 being fed basal diet supplemented with 100 mg/kg cyadox,and pigs in group 2 and 5 being fed basal diet supplemented with 100 mg/kg olaquindox.Pigs in group 4,5 and 6 were orally given 10~(10) CFU of Escherichia coli(E.coli,O_(139)∶K_(88)) while pigs in group 1,2 and 3 were given equal volume of alkaline broth as a placebo.There were no differences(P0.05) in the fecal shedding of total E.coli or the inoculated E.coli between olaquindox-supplemented pigs and cyadox-supplemented pigs.However,the fecal shedding of the inoculated E.coli in the olaquindox-or cyadox-supplemented was lower(P0.05) than that in the inoculated control.The incidences of Escherichia coli infection in group 6 and 5 were both 16%,lower(P0.01) than that in group 4(68%).Average daily gain(ADG) was improved by the additives(P0.05).ADG in the cyadox-supplemented was higher than that in the olaquindox-supplemented.ADG and feed conversion ratio(FCR) in group 6 were improved by 11.9% and 7.9% respectively,higher than those in group 5(4.2% and 1.5%),whereas ADG and FCR in group 4 were decreased by 9.6% and 8.4%.These results indicated that cyadox can not only prevent Escherichia coli infection efficiently,but also improve ADG and FCR significantly.
This study was done to examine immunohistochemically the presence of LIF,LIFR and gp130 in the monkey oviduct during the menstrual cycle.The results showed that the oviduct epithelial cells were the primary site of immunoreactive LIF,LIFR and gp130,which were present to a lesser extent in the stromal cells,smooth muscle cell layers and serosal tissue.The immunostaining for LIF,LIFR and gp130 was cycle-dependent,and considerably higher during mid-to-late proliferative to early-to-mid secretory phases than during early proliferative and late secretory phases of the menstrual cycle.These results demonstrate that rhesus monkdy oviduct contains immunoreactive proteins for LIF,LIFR and gp130 during the menstrual cycle.The cycle dependency suggests a potential regulation of their expression by ovarian steroids.The results imply the importance of LIF in a variety of oviduct biochemical and physiological functions and possibly in early embryonic development.
