Cystic echinococcosis (CE) is a worldwide zoonotic parasitic disease caused by infection with the larvae (metacestodes) of Echinococcus granulosus,it is an important public health problem to many countries and regions.Over the past 20 years，scholars around the world have done a lot of work on the development of vaccine of hydatidosis (including various vaccines applied in intermediate and definitive hosts)，and some useful results have been acquired.Among them，the most successful vaccine of hydatid disease is EG95 vaccine；this paper mainly summarizes the research status and progress of EG95 vaccine.

 To gain further insight into the biological functions of IRF7 gene，the open reading frame of the target gene was amplified from the cDNA of porcine duodenum，and cloned into the prokaryotic expression vector pET-21b(+) to generate a pET-21b-IRF7 recombinant plasmid.After verification by sequencing，the pET-21b-IRF7 was constructed，and then transformed into E.coli BL21(DE3).The His-IRF7 fusion protein was induced by IPTG and then identified by Western blot.The results shown that：(1) The open reading frame of 1 461 bp was successfully cloned.(2)His-IRF7 fusion protein was efficiently expressed in the lysate in the form of both soluble cysteine and inclusion bodies,with a molecular weight of about 60.5 ku.These results indicated that IRF7 fusion protein was successfully expressed in E.coli BL21(DE3)，which lays the foundation for the related function studies of porcine IRF7.

The experiment was conducted to study the composition and diversity of the mucosa-associated microbiota (MAM) in ileum and cecum between high quality chicken line A (pure line ♂，group A) and Huiyang Beard chicken (pure line ♂，group B) at 28 days old.Two chicken intestinal tracts were collected in group A and B，and mucosa-associated microbiota DNA were extracted at 28 days old，respectively.The random clone library of 16S rDNA V3 region of the mucosa-associated microbiota (MAM) in ileum and cecum between group A and B was established by analyzing 16S rDNA gene sequences.Analysis of the library in ileum showed that 6 operational taxonomic units (OTUs) (form 86 gene sequences or clones) were detected in the group A，as compared with 2 OTUs (form 97 clones) in the group B.Sequence being related to Enterococcus，Lactobacillus，Coprococcus and Clostridiaceae were found in the clone library of the MAM in ileum in group A.Clostridiaceae was only detected in group B.Analysis of the library in cecum shown that 44 OTUs (form 93 clones) were detected in the group A，as well as 42 OTUs(from 97 clones) in the group B.Unclassified Ruminococcaceae (18.28%),Coprococcus (13.98%)，Bacteroides (10.75%)，Blautia (10.75%) and Unclassified Lachnospiraceae (7.53%) were predominant in the group A，and Faecalibacterium (17.53%)，Coprococcus (11.34%)，Bacteroides (9.28%) and Unclassified Ruminococcaceae (8.25%) were abundant in the group B.The amount of other species found in cecum in group A were significant different from that in group B.In addition，sequence known being related to Colibacillus were not detected in group A and B.These results indicate that the composition of the MAM in ileum and cecum at day 28 days old are different between the high quality chicken line A and Huiyang Beard chicken，especially the proportion of butyric acid bacteria and Ruminococcus，and the host genetic factors may plays key role in this difference.

 To screen the reference genes of stable expression in different tissues，skeletal muscle of different development periods and skeletal muscle satellite cells (SMSCs) in goat，the expression levels of 9 reference genes of ACTIN，GAPDH，TOP2β，YWHAZ，SDHA，PGK1，RP2，RPL4 and HPRT1 mRNA were detected in different tissues (heart，liver，spleen，lung，kidney，brain，small intestine，large intestine,longissimus dorsi，triceps brachii and semimembranosus ) of 3-day-old postnatal kids，and the longissimus dorsi，triceps brachii and semimembranosus of 3，30，60，90 and 120 days old goat and the SMSCs cultured for 1，3，4 and 7 d.The data of expression levels were analyzed using the geNorm software.The results showed that the expressive stability of nine reference genes from high to low were HPRT1，RP2，TOP2β，SDHA，GAPDH，ACTIN，YWHAZ，PGK1，RPL4，and the 2 genes (HPRT1 and RP2) were needed to normalize the qRT-PCR data in different tissues of neonatal goat kids.Meanwhile，the stability of 9 references genes decreased as following: YWHAZ，ACTIN，HPRT1，RPL4，TOP2β，SDHA，PGK1，GAPDH，RP2，and the first 3 genes (YWHAZ，ACTIN and HPRT1) were suggested to accurately measure the true expression levels of target genes during the skeletal muscle of different development stages.Additionally，their expressive stability rank were PGK1，SDHA，ACTIN，RPL4，GAPDH，TOP2β，YWHAZ，RP2，HPRT1 in SMSCs，and 3 genes (PGK1，SDHA and ACTIN) were recommend as standardized factors.Here，the expressive stability of 9 reference genes and suitable genes were confirmed in different tissues，the skeletal muscle of different development periods and the SMSCs in goat.This will be beneficial to the research about the expression and function of gene at mRNA level.

The aim of this study was to clone ovine ApoER2 (apolipoprotein E receptor-2)，construct the plasmid vector ApoER2-siRNA，and investigate the inhibitory effect of ApoER2 gene silencing on expression of Sel P and PHGPx genes in co-culture Sertoli cells of sheep.The ovine ApoER2 was cloned by RT-PCR.Four pGPU6/GFP/Neo-ApoER2 plasmid expression vectors were constructed，and transfected into the co-culture Sertoli cells of sheep by Lipofectamine 2000^TM.The inhibitory effect of ApoER2-shRNA was detected by RT-PCR and Western blotting.The effect of ApoER2-shRNA inhibitory on expression of Sel P and PHGPx genes was detected by RT-PCR.The complete CDS of ApoER2 was 2 490 bp encoding a 892-amino-acid protein.The BLAST analysis showed that the entire nucleotide sequence of ApoER2 gene had 97.7% homology with bovine.ApoER2 mRNA levels in co-culture Sertoli cells were decreased significantly after transfection by the recombinant plasmids located in ApoER2-siRNA-1565 (P<0.01)and ApoER2-siRNA-2567 (P<0.01).Expression of Sel P and PHGPx mRNA were significantly decreased in the ApoER2-siRNA-2567 recombinant plasmid transfection group (P<0.01).The complete CDS of ApoER2 was obtained.Recombinant plasmid pGPU6/GFP/Neo-ApoER2-1565 and pGPU6/GFP/Neo-ApoER2-2567 were successfully constructed with effective inhibitory.Interference of ApoER2 gene decreased the expression of of Sel P and PHGPx mRNA，which could provide the scientific basis for further study of ApoER2 roles in pathway of selenium transport.

Based on the sequence of mitochondrial DNA Cytochrome oxidase subunit，the origin，taxon of Tibetan Mastiff and the phylogenetic relationship between Tibetan Mastiff and other large breed dogs were studied.Primers were designed according to the mitochondrial sequence of domestic dog，to amplify COI (Cytochrome oxidase subunit 1)，COII (Cytochrome oxidase subunit 2) and COIII (Cytochrome oxidase subunit 3) of the Tibetan Mastiff.Phylogenetic trees among Tibetan Mastiff and other 19 breeds of Canidae were constructed using giant panda (Ailuropoda melanoleuca) as the outgroup.The results shown that the length of COI，COII and COIII were 1 545，684 and 784 bp，respectively，three genes coded 514，227 and 261 AA，respectively.Phylogenetic analysis displayed that the Tibetan Mastiff，12 domestic dog breeds and grey wolf clustered together，which indicated Tibetan Mastiff and other domestic dog breeds originated from grey wolf.In Canis，Tibetan Mastiff clustered together with Saint Bernard，Old English Sheepdog and Leonberger，but German Sheepdog，Swedish Elkhound and Black Russian Terrier clustered one group，middle and little breed dogs clustered together，which suggested that there were closer relationship between Tibetan Mastiff and Saint Bernard，Old English Sheepdog and Leonberger，it was confirmed that many large breed dogs in the world such as Saint Bernard were possible of having blood lineage of Tibetan Mastiff based on molecular data.The result indicate that Tibetan Mastiff originates from grey wolf，about the zoological taxon，Tibetan Mastiff belongs to Canis lupus familiaris，Canis lupus，Canis，Canidae，Carnivora，the large breed dogs such as Saint Bernard probably have the blood lineage of Tibetan Mastiff in the breed forming.

This experiment was conducted to transfect chicken embryonic stem cells with recombinant plasmid pEGFP-hTERT，optimize culture system of chicken embryonic stem cells，and establish embryonic stem cell lines.hTERT gene was obtained and then subcloned into pEGFP-N1 to construct eukaryotic expression vector pEGFP-hTERT.cESCs were transfected with pEGFP-hTERT after transfection DF-1 cells，which were cultured with 80% conditioned medium，20% ES decarboxylase basal medium and 10 μmol•L^-1 vitamin C.After detection with AKP staining and associated specific surface antigen SSEA-1，SOX2 and OCT4，cESCs were selected with G418 and detected by real-time fluorescent quantitative PCR.The results showed that vitamin C could promote cESCs proliferation，green fluorescence could be detected in DF-1 cells aftert transfected with pEGFP-hTERT，cESCs transfected with pEGFP-hTERT could be passaged to 10th generation and remained undifferentiated state，and stem cell surface specific antigen detection was positive，as well there were no significant differences in expression levels of related genes（P>0.05）.The results indicated that cESCs could be passaged to 10th generation consistently，and remained undifferentiated state，which were transfected with pEGFP-hTERT and cultured with 80% BRL-CM added vitamin C.This would be used in embryonic stem cells immortalization and establishing cell lines.

To establish a simple and feasible method for isolation of highly purified rats brain microvascular endothelial cells（RBMECs）from 7-10 days SD rats，the relatively pure cerebral microvessel fragments were obtained by careful dissection in dispase II and three times centrifugation with dextran.After digested with collagenase/dispase enzyme，cells were grown in a humidified atmosphere of 5% CO2 at 37 ℃,then were identificated by the factor Ⅷ-related antigen.The results showed that primary endothelium cells formed monolayers with the characteristic cobble-stone morphology and were strongly positive for Ⅷ-related antigen factor.It was indicated that relatively pure primary culture of RBMECs was successfully established using this method.

To investigate expression of proteins from buffalo testicular seminiferous tubules at different developmental stages (nepionic and senescence).Tandem mass tag (TMT) coupled to two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS).Identification was based on tandem mass tag (TMT)-coupled to 2D liquid chromatography tandem mass spectrometry (2D LC-MS/MS) using LC-ESI LTQ-Orbitrap Elite MS/MS equipment.Detected proteins were processed by bioinformatics analysis using the SEQUEST search engine.Eighty-nine different proteins were obtained with a more than two-fold change between developmental stages.Compared with the nepionic stage，51 proteins were up-regulated and 38 proteins were down-regulated.TMT-coupled 2D LC-MS/MS was an appropriate method for isolating and identifying protein groups of buffalo testicular seminiferous tubules at different developmental stages.The results provided an experimental basis for exploring changes in protein expression during spermatogenesis，and found biological markers of spermatogenesis，possibly Glutathione S-transferase P，Catalase，Heat shock protein 90-alpha and Heat shock 70 ku protein 1-like.

The inefficiency of cloned animal producing is mainly due to uncompleted epigenetic reprogramming. In this study, the effect of Debao pig ear fibroblasts treated with different concentrations (0.00（control）, 0.25, 0.50 and 1.00 μmol•L^-1) of 5-Aza-CdR (DNA methyltransferase inhibitor) on the developmental potency of handmade cloned pig embryos were investigated. The results showed that all treated cells have “S” shape growth curve, meantime cells treated with 0.25 μmol•L-1 5-Aza-CdR have a growth curve which was similar to that of control group’s. With the concentrations increasing of 5-Aza-CdR, cells showed distortion, growth inhibition in a certain degree, and even death. Debao pig fibroblasts treated with 5-Aza-CdR from 0.00 to 0.50 μmol•L^-1 didn’t show significantly alteration on the chromosome ploidy.5-Aza-CdR could down regulate the expression of Dnmt1, Tet1, Tet2 and Tet3 on Debao pig ear fibroblasts following a dose-related quantity pattern. The cleavage and morula rates of handmade cloned embryos with donor cell treated with 5-Aza-CdR had no significant difference with that of the control group (P>0.05). However, the blastocyst rates of 0.25 μmol•L^-1 and 0.50 μmol•L^-1 treatment groups were significantly higher than that of 1.00 μmol•L^-1 group (P<0.05), but had no significant difference with that of the control group (P>0.05). The total blastocyst cells number of 0.50 μmol•L^-1 treatment groups was significantly higher than that of control group and 1.00 μmol•L^-1 group (P<0.05), but had no significant difference with that of 0.25 μmol•L^-1 group (P>0.05). In conclusion,high concentration of 5-Aza-CdR have side effect on the proliferation and chromosome ploidy of Debao pig fibroblasts. Applying low concentration (≤0.25 μmol•L^-1) of 5-Aza-CdR on donor cells reduced the methylation level of cloned embryos and consequently enhanced the developmental potency of handmade cloned pig embryos.

This experiment was aimed at studying the effects of exogenous Wnt3a on bovine mammary epithelial cell (BMEC) number and the expression of Frizzled 1 receptor and β-catenin.BMEC was cultured by adherent tissue culture.β-casein of culture medium was detected by Western-blot.BMEC was cultured in medium with 0，10，25 and 50 ng•mL^-1 Wnt3a separately，and then the BMEC and culture medium at 10 days were collected.The expression of Frizzled 1 receptor and β-catenin mRNA was checked by real time RT-PCR.The results showed that cell numbers in 50 ng•mL^-1 Wnt3a group were significantly higher than those of control group.The expression of Frizzled 1 receptor significantly increased，and there was not much difference in β-catenin expression between control group and Wnt3a group.It was concluded that Wnt3a exerts effects on the expression of Frizzled 1 receptor，moreover，promotes the number of BMEC.Current evidence suggested that Wnt signal played roles in the regulation of mammary development.

The aim of this study was to investigate the effects of biotin on the performance and hoof health of Chinese Holsein lactating cows.Sixty healthy Holstein cows (45 cows with lactation days in (67.4±35.5) d and 15 with lactation days in (167.6±27.5) d) in similar weight and milk yield were assigned randomly to four groups with 15 cows per group according to the lactating period of block experimental design.The cows in 4 groups were fed the basal diet supplemented with 10 (group A),20 (group B),30 (group C) and 40 mg•d^-1(group D) biotin，respectively.The test sustained 300 d，the coffin hardness and limb hoof score were detected every 75 d.Milk yield and milk copmposition were recored，blood indexes were detected，and the effect of biotin supplementary levels on lactating of cows was investigated on the 60th day of the test.The results shown that：1) Milk protein percentage decreased by supplementing botin and milk protein percentage in group A，B，C and D were 3.64%，3.26%，3.14% and 3.27%，respectively.Milk protein percentage in group A was significantly higher than that in the other biotin supplement groups (P<0.05)，but there were not significant difference in milk protein yield among groups;2) The plasma biotin and glucose concentrations increased by supplementing biotin.The plasma biotin concentration in supplementing 20,30 and 40 mg•d^-1 biotin groups were significantly higher than that in supplementing 10 mg•d^-1 biotin group (P＜0.01)，and the plasma glucose concentration in group B and D were 3.40 and 3.28 mmol•L^-1，which increased significantly(P<0.05)，there were no significant difference among group B,C and D in plasma glucose concentration; 3) Supplementing biotin enhanced the coffin hardness.For the coffin hardness determined at 75th and 105th day，there were significant difference among different groups(P>0.05)，the coffin hardness determined at 225th and 300th day in group B,C and D were higher than that in group A，but there were no significant difference among group B,C，D.The coffin hardness at 225th day in group B and C were significant higher than that in group A(P<0.05)，at 300th day in group B was significant higher than that in group A(P<0.05); 4) Status of limb hoof was negatively closely related to the coffin hardness(P<0.05)，and the correlation coefficient decreased with the experiment，which indicated that the effect of biotin decreased with supplementing.These results indicate that dietary biotin supplement can enhance the coffin hardness and improve the limb hoof health，the optimum biotin supplement level is 20 mg•d^-1.

The experiment was conducted to study the metabolic rule of the dietary protein of Chinese Holstein heifer with 300-350 kg body weight，and quantify the dietary protein requirement of heifers at this stage.Twenty one 10-month-old healthy heifers with similar initial body weight ((307.67±2.99)kg) were divided into 3 groups (A，B and C) in a randomized experiment design.The heifers in group A，B and C were fed diets with the same ratio of concentrate to forage and different nutritional levels (DM basis).The DE and crude protein content of experimental diet were 10.98 MJ•kg^-1 and 10.06%，11.43 MJ•kg^-1 and 11.04%，11.90 MJ•kg^-1 and 12.04% (dry matter basis)，respectively.The ADG of heifers in experiment groups were 620.09，820.30 and 887.30 g•d^-1，respectively.The average apparent digestibility of dietary DM，OM，NDF and ADF were 70.70%，72.51%，68.70% and 68.29% for heifer at this stage.The digestibility and deposition rate of dietary CP were 69.77% and 41.37%.The estimated requirement model of crude protein (CP)，digestible crude protein (RDCP)，rumen degraded protein (RDP) and the small intestine digestible protein (IDCP) of heifers were:CP（g•d^-1）=5.62W^0.75+595.31△W；RDCP（g•d^-1）=3.90W^0.75+418.04△W；RDP（g•d^-1）=2.50 W^0.75+266.19△W；IDCP（g•d^-1）=3.60 W^0.75+384.94△W(W was BW of heifers，kg；△W was ADG，kg•d^-1).

The study was conducted to develop a rapid and quantitative colloid gold immuno-chromatography assay of Asia 1 type Foot-and-Mouth disease (FMD) virus.Aiming at detecting FMD virus of Asia 1 type quantitatively，the colloid gold-based immuno-chromatographic test strip was prepared and the standard curve was constructed.Testing the laboratory samples and field samples，results showed that this assay had good sensitivity and specificity.This method could accomplish qualitative and semi-quantitative detection in 15 minutes; the sensitivity was up to 0.78 μg•mL^－1 with a linear ranging from 0.78 to 7.84 μg•mL^－1.The coefficient of variation was 0.2% to 5%，and the recovery reported to be 91% to 102%.Good reproducibility and accuracy of this method was demonstrated.The colloidal gold immuno-chromatographic assay is rapid，specific，sensitive，accurate，worthy to recommend its clinical use.

To prepare antibody with diagnosis value to infectious laryngotracheitis and to study the distribution of ILTV gC protein in LMH cells and the role of gC protein in the spread of ILTV among cells，the major antigen coded sequence of gC gene was amplified from ILTV-LJS09 genome by PCR.The prokaryotic expression vector pET-32a-gC was constructed by inserting the gC gene into pET-32a(+).The His-gC recombinant protein was purified，and then was used to immunize rabbits.The Western blot results demonstrate that the obtained polyclonal antibodies were specific to ILTV-LJS09 gC protein.The IFA results showed that the anti ILTV-LJS09 gC protein antibody could specifically response to ILTV-LJS09，HLJ0507，MDJ0442，SD0203，Zhj1298 strain.Intracellular localization of the ILTV gC protein in LMH cells using the anti-gC antibody showed that gC protein mainly located in cytoplasm and membrane of the virus infected cells.Antibody neutralization test indicated that the gC protein may be directly involved in virus transmission between the cells，thus affecting the virus plaque formation.In this study，we successfully prepared rabbit anti ILTV-LJS09 gC protein polyclonal antibody with diagnosis application value，and proved that LJS09 gC protein had the conservatism of α-herpesvirus on the distribution and function，which provided important data to the research of gC protein biological characteristics and ILTV infection mechanism.

A breeder chicken flock demonstrated 5%-6% positive rates in tests of antibodies to subgroups A/B avian leukosis virus (ALV) and egg albumen p27 antigen，although chickens were clinically health and their egg productivity was normal.However，no exogenous ALV was isolated when serum and egg albumen samples of more than 200 birds were inoculated in DF1 cell cultures.Then 23 breeders with high S/P values in ELISA for p27 antigen in their egg albumen were selected and kept from the breeder farms.Blood plasma and egg albumen samples were collected twice from each birds for inoculation of DF1 cells and SPF embryo-originated fibroblast (CEF)cultures，again there was no exogenous ALV isolated，in order to identify whether the virus is endogenous viruses.Fertilized eggs from these 23 breeders were incubated for 9-11 d and their CEF were prepared for each embryos，p27 antigen was detected in CEF supernatant from only one of the 23 breeder eggs.Its supernatant was further inoculated into DF1 and SPF embryo CEF culture，p27 was detected from only SPF embryo CEF but not DF1 cell cultures，indicating that there was endogenous but not exogenous ALV.By use of genomic RNA abstracted from viral particles in the supernatant with p27 detected as the template，gp85 gene was amplified by RT-PCR.Sequence comparison confirmed that the detected ALV belonged to subgroup E.After immunization with its gp85 protein expressed in E.coli，some chickens demonstrated antibody positive by ALV-A/B antibody detection ELISA kit.Influence or interference of ALV-E on results in tests with ALV antibody or p27 detection ELISA kits was discussed.

 To enrich the genomic information，the merozoites of ovine Babesia motasi Lintan were embedded in low-melting-point (LMP) agarose and subsequently digested with proteinase K to get their high-quality megabase size genomic DNA.Then the large size DNA fragments isolated by pulse-field gel electrophoresis (PFGE) were ligated to CopyControl pCC1BAC Cloning-ready Vector.The present results showed that the bacterial artificial chromosome (BAC) library containing 34 560 clones was successfully constructed by transformation of the ligated DNA into EPI300^TM Escherichia coli.In B.motasi Lintan BAC library，the average length of the insert DNA fragment was 70 kb and covers ×137 B.motasi Lintan genome.The BAC library in which the empty clone was less than 3% and the clones were stable should contribute to fine physical mapping，genome sequencing，and evolution and virulence genes derived from comparative genomics of Babesia species and so on.

Polyvalent coliphage Bp7 was isolated and identified as T4-like phage，the codon usage in gene gp37 was analyzed by various bioinformatics softwares.The results were showed as follows：The patterns of codon usage in gene gp37 of phage Bp7 and other 5 T4-like phages were almost similar.In gene gp37 of phage Bp7，the codon bias was a little low，and gp37 seemed like using A or U ending codon；17 kinds of high-frequency codons were identified except AUG (Met) and UGG (Trp).The codon bias comparison of gp37 among phage Bp7 and other 5 phages showed that codon usage in Bp7 was most similar to JS98，a little far from T4 and T4T relatively.The codon usage patterns of gp37 may be influenced not only by selection pressure，but also by neutral mutation.The results of anlysis of gp37 codons will benefit to the modification of tail fiber protein of Bp7 and further broaden the host range of phage Bp7，and improve the level of gp37 expression as well.

Type Ⅱ heat-labile enterotoxin (LT-Ⅱ)-producing Escherichia coli is a common zoonotic pathogenic agent responsible for diarrhea.In order to improve the understanding of the epidemiological situation of LT-Ⅱ toxin in cattle，a total of 138 E.coli isolates from 2010 to 2012 were detected for LT-Ⅱ gene by PCR，and ten of these were sequenced.The polycistron ORF of LT-Ⅱc1 was cloned into the pET30a expression vector，translated into Rossatta competent cells，and induced for expressing the recombinant LT-Ⅱ toxin.The toxicity of the recombinant LT-Ⅱ toxin was preliminary tested by mouse Y1 adrenal cells.The results showed that eight samples were LT-Ⅱc1 producing E.coli，one was LT-Ⅱc4 producing E.coli and one was LT-Ⅱc6 producing E.coli from ten LT-Ⅱ positive.The results of SDS-PAGE and Western Blot showed that recombinant LT-Ⅱc1 toxin was prepared successfully，and the recombinant toxin had induced the apparent cytopathic effect for mouse Y1 adrenal cells，with the minimum lethal dose of 17.8 pg.The results presented here demonstrate that we isolated the LT-Ⅱ producing E.coli for the first time in China，and lay a preliminary foundation for further study of the LT-Ⅱ toxin.

The aim of this study was to express the COE protein of porcine epidemic diarrhea virus (PEDV) in Pichia pastoris，and evaluate the neutralizing ability of mices serum after immunization with COE protein.A pair of primers was designed to clone PEDV COE gene，and the PEDV strain was isolated from a clinical sample.The PEDV COE ORF was amplified by RT-PCR.Then the PEDV COE gene was inserted into pPIC9K，and acquared the recombinant plasmid，pPIC9K-COE.After the pPIC9K-COE was linearized by SacⅠand transformed into P.pastoris GS115 by electroporation，the high copy positive recombinant bacteria were screened for inducible expression，which was induced by addition of methanol.SDS-PAGE and Western blot were conducted to analyze the recombinant protein and its immunogenicity.The neutralizing ability of mice serum after immuned with the recombinant PEDV COE was analyzed by virus neutralization test.An 530 bp gene, PEDV COE was amplified by RT-PCR，and the PEDV COE protein were expressed in P.pastoris.The result of SDS-PAGE showed that the 27 kDa PEDV COE protein was expressed in P.pastoris GS115，and the concentration was 30 mg•L^－1.Western blot analysis of the PEDV COE protein proved that the immunogenicity of the recombinant protein.And the virus neutralization test result showed that the sera from mice immunized with the recombinant protein possess neutralizing activity，and the neutralization titer was 1:36，while the control was less than 1:2.The results presented here demonstrate that PEDV COE protein can be correctly expressed in P.pastoris，and it has better biological activity.

 The objective of this paper was to explore if Cold-inducible RNA binding protein(CIRP) involved in the response of mice to LPS.Total 48 BALB/c mice were randomly divided into 4 groups，LPS was injected into abdominal cavity of each mouse in dose of 2.5，5，7.5 mg•kg^－1 body weight for 1-3 experimental groups.The 4th group(control group) was injected with the same volume saline solution in the same way with the above groups.Three mice were selected in each group and euthanized at 3，6，12 and 18 h post injection.The heart，liver，spleen and kidney of each mouse were collected.Real-time RT-PCR established in this study and immunohistochemistry were used to detect the transcription of CIRP mRNA and expression of CIRP protein in each organ.The results showed that the Cirp mRNA significantly increased in liver，spleen and kidney post LPS injection，while Cirp mRNA transcription in heart was inhibited at all time points.CIRP protein was highly expressed in all detected organs after LPS stimulation.In conclusion，the results in this paper demonstrated that CIRP involves in response to LPS in mice and present evidence to identify new feature of CIRP.

The distributional characteristics of VIP in different ages bursa of Fabricious of Chinese yellow feather quails were observed by using the immunohistochemical SABC method and microscopic image analysis.The result showed that VIP widely exists at newborn，1，3，5，9，13，17，21，23，25，32，36-week-old of Chinese yellow feather quail bursa，formed a long fusiform，horseshoe-shaped，oval，lumps or fine granular，mainly distributed in the submucosa，muscularis，serosa，and the distribution is the most typical at 3 weeks of age.To 25 weeks of age，the average optical density of VIP positive reaction reached the maximum，then gradually weakened.The result proves that VIP exists at all stage of growth in Chinese yellow feather quails bursa，and with the development and function of changes in bursal characteristics showing a certain correlation.

The current study was undertaken to study the protective effects of lycium barbarum polysaccharides (LBP) on the expression of Bax and Bcl-2 in hamster testicular tissue treated by diethylstilbestrol.Sixty-six adult male hamsters were randomly divided into the control group (A)，DES group (B)，1，10，50 mg•kg^－1 LBP group (C，D，E) and V_C+V_E group (F).Except the control group，the other 5 groups were subcutaneous injected with DES for 7 consecutive days，while the control group was injected equivalent amount of olive.Meanwhile hamsters in C，D，and E groups were gavaged 1，10，50 mg•kg^－1 LBP respectively，and the group of F were gavaged 100 mg•kg^－1 V_C+200 IU•kg^－1 V_E，while the groups of A and B were given equivalent amount of normal saline.After 24 hours of the last treatment，the expressions of Bcl-2 and Bax in testicular tissue were tested through RT-PCR and Western blot.Results showed that DES dramatically decreased the expression of Bcl-2 (P<0.01)，but the expression of Bax significantly increased.After treating with different dosage of LBP，the expression of Bcl-2 increased in a dose dependent manner，while the expression of Bax decreased，with 50 mg•kg^－1 LBP being the best (P<0.01).There was no significant difference between vitamin group and 50 mg•kg^－1 LBP group.These results indicated that regulating the expression of Bax and Bcl-2 may be one of the pathways that LBP alleviating the spermatogenic damage induced by DES on adult male hamster.

In order to analyze the effect of Emeria tenella infection on the expression of Zyxin，CD4 and TNFRSF1A in Jinghai Yellow chickens，the expression of the three genes in 10 tissues were detected by fluorescent quantitative PCR.The results showed that the expression of the 3 genes were significantly different between the infected and control groups in most tissues.Zyxin expression in glandular stomach was the highest among detected tissues in infected group，except for in lung，liver，kidney and cardiac muscle，the expression of infected group was significantly higher than that of control group(P<0.01)；CD4 expression of infected group in some immune organs，such as spleen，lung and thymus，was significantly higher than that of control group(P<0.01)；TNFRSF1A expressions in small intestine and caecum were at high level，and was significantly higher in infected group than that in control group(P<0.01) in small intestine，caecum，spleen，glandular stomach and thymus gland；Three gene expressions in bursa of Fabricius were at low level，and none significant difference was found between the infected and control groups except for Zyxin，which suggested that bursa of Fabricius probably did not play an important role in the infection of Emeria tenella.In caecum，the parasitic site of Emeria tenella，the expression of Zyxin and TNFRSF1A of infected group had significant difference with the control group(P＜0.01)，which indicate that the two genes had a close relationship with the infection of Emeria tenella.

The current study was undertaken to forecast the secondary structure and find out the localization of transfected cells of ORF125 from orf virus (ORFV).ORF125 gene was acquired by PCR and cloned into pEGFP-N1 eukaryotic expression vector directionally.The recombinant expression plasmid was identified by restriction enzyme digestion analysis，sequencing and named pEGFP-ORF125.Then the phylogenetic analysis of the gene was made by DNAStar and MEGA4.0.Also the secondary structure of ORF125 was predicted and compared with cellular Bcl-2 proteins by Expert Protein Analysis System (EXPASY).The recombinant plasmid was transfect into goat skin fibroblasts (GSFs)，subcellular localization of ORF125 was examined with laser con-focal microscopy after DAPI staining.The results of sequencing alignment showed that the homology of China Luoyang 2013 shared 97.1%-98.1% with other ORFV isolates and 84.1%（GQ329669）and 85.6%（GQ329670）with another two Pseudocowpox isolates from different regions at nucleotides level respectively.The sequence identity of ORF125 and cellular Bcl-2 proteins is low，but the secondary and tertiary structures are conserved.Laser con-focal microscopy results showed that ORF125 distributed dispersively in the cytoplasm of GSFs.This study helped to further understanding the function of ORF125.Results verified the ORFV ORF125 gene is conserved and have similar function with Bcl-2 protein，in addition，the ORF125 gene could be expressed successfully in GSFs，and the ORF125 protein localizes in the cytoplasm.

The purpose of this study was to optimize the manipulation of interspecies somatic cell nuclear transfer (ISCNT) between Sika deer and bovine.It was used the Sika deer ear skin fibroblast as nuclear donor cell，and bovine MII oocyte as recipient.McGrath-Solter，extrusion and DEME chemically assisted methods were used in enucleation.perivitelline microinjection (PM)，break-membrane-cell intracytoplasmic microinjection (BMCIM) and whole-cell intracytoplasmic microinjection (WCIM) were used in nuclear injection.Oocytes were electrofusion after enucleation of PM before chemical activated and others were chemically activated without electrofusion.Reconstructed embryos were cultured in media of CR1aa.The results showed that：(1) The rate of enucleation with the DEME method was the highest，and the rate of extrusion method was the lowest.There was significantly difference (P<0.05) in the rate of enucleation among all the three enucleate methods，but not significantly difference (P>0.05) in duration of enucleation.(2) The rate of nuclear injection by PM and WCIM was 100.0% and 94.8% separately，and the duration of nuclear injection by PM and WCIM was 31.0 and 35.1 s separately.There was no difference between them (P>0.05)，their rate of nuclear injection were higher than which in BMCIM group (P<0.05)，but duration of nuclear injection was converse (P>0.05).(3) The rate of activation by BMCIM and WCIM group were higher than it in PM group (P<0.05)，and which in BMCIM group of one-step method was the highest.There was no difference among the rate of activation，cleavage and blastocyst between one-step and two-step method (P>0.05).The results suggested that one-step manipulative method of DEME assisted enucleation and pizeo assisted BMCIM was efficient in Sika deer-bovine ISCNT.