In order to screen antigenic genes of Clonorchis sinensis (C.sinensis).Firstly,a cDNA expression library from adult worms of C.sinensis was successfully constructed with lambda ZAP express vector.Total RNA of C.sinensis adult worms were extracted by the Trizol reagent and mRNA were further purified through oligo-dT cellulose.The first strand eDNA was synthesized by using MMLV reverse transcriptase.After the synthesis of the second strand,the cDNA were purified by CHROMA SPIN-400 kit and then ligated with lambda ZAP express vector,then packaged in vitro and amplified.The original library contained 1.5 × 106 pfu cDNA clones,the titer of amplified library reached 1.5 × 1010 pfu/mL,in which about 99% clones were recombinants and most of insert DNA fragments were 0.4-2.0 kb.Secondly,immunoscreened using the naturely infected man serum from C.sinensis.The positive clones were sequenced and analyzed.From 2.0 × 105 recombinant clones of the eDNA library,41 positive clones were obtained.Sequence analysis indicated that the cDNAs encoded proteins including glycine rich antigen 2,proline rich antigen 2 and antigen Cs44 from C.sinensis,anothers were lower similarity to predicted protein from Nematostella vectensis,transcription elongation factor GreA from Bartonella quintana str.Toulouse and NM_132090 CG3446 gene product in Drosophila melanogaster from Schistosoma japonicum.These data may form a foundation for identifying recombinant antigens that can be used in the diagnosis or vaccination against clonorchiasis.

The σC gene of ARV S1133 was designed to amplify by reverse transcription chain reaction(RTPCR).The σC gene was inserted into the vector pMD19-T,identified by PCR method and restriction enzyme,and sequenced.It showed that the insert cloned gene fragment was the σC gene of ARV.Then the gene was inserted intc the pET32a(+) and indicated that fusion expression vector pET32a-σC was constructed.The recombinant fusion protein was highly expressed in E.coli BL21 induced by 1.0 mmol/L IPTG for 5 hours in the form of inclusion bodies.The weight of recombinant fusion protein molecular is 54 000.Western-blot with ARV antibodies against the fusion protein showed the recombinant protein has a favourable reactivity.

The levels of the antibody response against,classic swine fever (CSF) vaccine were measured by blocking ELISA at different time for pigs vaccinated with CSF vaccine (CSFV group,n= 3),pigs inoculated with porcine eircovirus type 2 (PCV2) and with CSF vaccine two weeks later (when PCV2 viremia was detected by PCR,PCV2/CSFV group,n=3),or inoculated with both CSF vaccine and PCV2 at the same time (CSFV/PCV2 group,n=3),respectively.And distribution of genome or antibodies specific to PCV2 of the PCV2 infection group,PCV2/CSFV group and CSFV/PCV2 group were also detected by indirect ELISA or PCR,respectively.The results showed that experimental infection of PCV2 was successful.In pigs inoculated with both PCV2 and CSF vaccine,the antibody response to CSF vaccine was significantly lower than that of animals treated with CSF vaccine vaccination alone at 52 days postinoculation (DPI).The average titers of antibodies against CSFV in animals of CSFV group were obviously higher than those of the PCV2/CSFV or CSFV/PCV2 group at 49 and 52DPI,respectively.The positive rate of antibodies against CSFV in pigs of CSFV group was 100% in comparison with that of animals inoculated with PCV2 and the vaccine (only 67%).The results suggested that the infection of PCV2 could suppress the antibody response to classical swine fever vaccine.

The amplified VP2 gene (PPV strain SC-1) and PCV20RF2 gene were inserted into the eukaryotic expression vector pPI-2.EGFP.Then the recombinant plasmid named pPI-2.EGFP.VP2.ORF2 was obtained.Mediated by liposome,the recombinant plasmid was transfected into Veto cells and expressed.Using immunofluorescence assay,the fluorescence of expression products were first detected at 20 h after transfection and peaked 36 h.Under electronmicroscope,virus-like particles (VLPs) can be observed in the transfected cells.To confirm the obtained VLPs to be recombinant particles,piglets were immunized using purified VLPs.The dynamic variation of blood T lymphocytes and serum antibody level of PPV and PCV2 were measured.The results showed that the ratio of CD3+,CD4+ T lymphocyte in peripheral blood lymphocyte of immunized piglets raised in a certain degree,the number of CD8+ T lymphocytes fell at 7-14 d after immunization,and then raised.Relatively high level of PP-V,PCV2 specific antibody could be detected.This indicated that the expression of recombinant plasmid pPI-2.EGFP.VP2.ORF2 was successful,the virus-like particles were formed and showed favourable immunogenicity.

Animal health systems initially established in Australia,Canada and other countries,played a very important role in prevention and control of animal diseases.Based on comparing the animal health systems of Australia with the veterinary medical system in China,some strategies was obtained for establishing a more reasonable official veterinary system and animal health system in China,thus speeding up the integration process of veterinary and human medical public health system.

The study was conducted to investigate the effect of different forms of trivalent chromium on growth hormone (GH) secretion and pituitary mRNA expression in finishing pigs.A total of 96 finishing pigs with an initial average body weight 65 kg were blocked by body weight and randomly assigned to four treatments with three replicates.Pigs were offered one of four diets including a control diet or the control diet supplemented with 200 μg/kg chromium from either chromium chloride (CrCl3),chromium picolinate (CrPic) or Chromium nanocomposite (CrNano) for 40 days.During the trial,all pigs were given free access to feed and water.At the end of the feeding trial,blood samples were taken via auriculares at 15 min intervals for 3 hours.Eight pigs from each treatment were slaughtered for backfat thickness and longissimus muscle area (LMA),and three pituitaries were collected to determine GH mRNA level.The results showed that average daily gain of pigs fed supplemental CrNano was increased by 6.31 (P<0.05),feed gain ratio was decreased by 4.61% (P<0.05),backfat thickness was decreased by 24.32 % (P<0.05),and LMA was increased by 20.22% (P<0.05).The results of GH dynamic secretion showed that supplemental CrNano increased the mean level,lowest value,peak value and peak duration of GH by 42.62% (P<0.05),87.94%(P<0.05),26.60% (P<0.05),and 17.19%(P<0.05),respectively.The mean level and peak value of GH in pigs fed Supplemental CrPic was increased by 36.58% (P<0.05),27.18%(P<0.05),respectively.Pituitary mRNA expression of GH in pigs fed diet containing chromium from CrNano was increased by 27.63%(P<0.05).These results indicated that chromium nanocomposite increased GH mRNA expression and secretion in finishing pigs,through which growth was promoted and carcass characteristic was improved.

Sperm-mediated gene transfer (SMGT) is one of the most methods in the transgenic animal research and the efficiency of spermatozoa binding and internalization exogenous DNA after sperm/DNA co-culture is important to a successful SMGT.In this study,the influencing factors of exogenous DNA uptake by spermatozoa were detected using DIG labeled EGFP as exogenous gene.The results demonstrated that porcine spermatozoa could spontaneously take up exogenous DNA which mainly binding occurs on the sub-acrosomal and nuclei region of the sperm head.The rate of spermatozoa binding exogenous DNA increased with the extending action of time.At 37℃ and 39℃,the rate of spermatozoa uptake exogenous DNA would not increase after 60 min incubation,and the similar result was observed on 90 min at 17℃.Binding rates and internalization rates of washed ejaculated sperm cells from the 15 boars varied between 6.57%-35.81% and 2.990%-24.66%,respectively.The binding rate and intemalization rate were mostly inhibited by seminal plasma.The binding rates were significantly increased by liposome and DMSO,respectively.Dead-spermatozoa could bind exogenous DNA,the intermalization process could not be completed.Furthermore,the highest binding rate was found in membrane broken spermatozoa as a result of freeze-thawing and this was independent of the sperm donors.

A single factor duplicate test was designed to investigate whether bovine recombinded resistin impacts the expression of pyruvate carboxylase (PC) mRNA and the activity of PC in vitro culture bovine hepatocyte.Bovine recombinded resistin was added to the media with 0,25,50,100,200 and 400 ng/L.Abundance of PC mRNA in bovine hepatocyte,which was cultured with bovine recombinded resistin for 12 hours,was determined by real-time fluorescence quantitative RT-PCR,and activity of PC was determined by colourimetry.The results showed that bovine recombinded resistin could downregulate the expression of PC mRNA and the activity of PC in vitro culture bovine hepatocyte.

A model of infectious bronchit was developed in SPF chickens by repeated intranasal infectious routes,and then the influence of compound Chinese traditional medicine on cellular immunity and humoral immunity during preventing and curing infectious bronchitis was studied by MTT,flow cytometry and serum neutralization test in tracheal organ culture.The results showed that compared with the infected group,the compound Chinese traditional medicine group could significantly increase the weight gain of chickens(P<0.05),promote the growth of immunity apparatus,enhance the T lymphocytes proliferate response of chickens and increase serum neutralization antibody titers of chickens significantly(P<0.05),and the ratio of CD4+/CD8+T lymphocytes was improved significantly(P<0.01).The aforementioned results indicated that the compound Chinese traditional medicine could reinforce immune function via preventing both cellular and humoral immunity from depression in the chickens with IBV.

To evaluate the effects of supplemental probiotics and xylo-oligosaccharide on performance,digestive enzyme activities,blood index and intestinal microflora.Two hundred and forty crossbred early weaned PIC piglets with an average initial weight of (6.83 + 0.9) kg,weaned at (21 ± 2) d of age,were divided into four groups.Control Ⅰ (positive diet,with no probiotics and xylo-oligosaccharide but more fish meal,whey powder and little soybean meal),Control Ⅱ (negative diet,with no probiotics and xylo-oligosaccharide but less fish meal,whey powder and more soybean meal),Negative diet + 0.035 % probiotics,negative diet + 0.002% xylo-oligosaccharide.The results of theses studies suggested that whey powder and fish meal from 5.50% to 2.50%,soybean meal from 22.50% to 26.50% could result in decreasing of alimentary canal enzyme activity,blood sugar,total serum protein and the quantities of Bacillus in appendix(P<0.05),and raising of serum urea nitrogen,the quantities of Escherichia coli in appendix,diarrhea indexes and Feed/Gain(P<0.05).Addition of probiotics and xylo-oligosaccharide could significantly improve the activities of alimentary canal protein,lipase and amylase(P<0.05),decrease Feed/Gain and the levels of blood sugar and decrease the levels of serum urea nitrogen and improve protein equilibrium in blood and energy metabolism.The quantities of bacillus were improved,Escherichia coli were decreased in appendix(P<0.05).Diarrhea indexes were decreased(P<0.05).Whether adding probiotics and xylo-oligosaecharide or not could not affect ADG and ADFI (P>0.05).Addition of probiotics and xylo-oligosaccharide could improve digestive function,beneficial microbial population of post-alimentary canal,Feed/Gain and prevent the diarrhea of piglets.