Porcine circovirus type 2 (PCV2) has been recognized as the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), which results in tremendous economic losses in swine industry worldwide. PCV2 infection leads to impaired immune system in animals, severe immunosuppression, as well as co-infection and secondary infection among various bacteria and viruses, which will bring about tremendous obstacles in diagnosis and therapy. It is an effective way to control PCV2 infection by vaccination. The study on PCV2 vaccines is one of the hot research areas. To date, the inactivated vaccines of PCV2, subunit vaccines and chimeric virus vaccines are commercially available in North America and Europe, and the inactivated vaccines of PCV2 have also been developed in China. Development and application of the commercial PCV2 vaccines play a significant role in the prevention and control of PCV2 associated diseases effectively. However, the commercial PCV2 vaccines could not totally block the transmission of PCV2 among pigs and eliminate the virus in pigs. Therefore, it is the main research trend to develop efficient and inexpensive genetically engineered vaccines such as live vector vaccines, DNA vaccines and marker vaccines in the future. This article summarizes the characteristics and efficacy of currently commercial PCV2 vaccines, and reviews the recent achievements on PCV2 vaccines including live vector vaccines, DNA vaccines and marker vaccines. It may become a useful contributor to the next generation vaccines for control of PCV2-associated diseases.

The study aimed to establish the primary culture system of Laiwu Black pig preadipocyte for better studying the properties of hyperplasia of Laiwu Black pig adipose tissue. Preadipocytes were isolated from subcutaneous adipose tissue of 1 day piglet by the combination of collagenase and trypsin digestion and were successfully cultured. The induction and differentiation of preadipocytes were studied, the expression of the important transcription factor PPARγ in the process of preadipocytes differentiation were detected. The result showed that the isolated cells started adherence at 8 h after initiation of primary culture and displayed short spindle or irregular triangle. On 3rd day, the cells entered logarithmic growth phase, displayed fibroblast-like, and the cells growth showed ‘S’ shape; the induction result showed that fat droplets occurred in small amount of cells on 2nd day, in large amount on 3rd day, those small fat droplets gradually converged into big ones from 7th to 8th day, and on the 12th day become to one big fat droplets, the adipocytes showed jacinth by oil red O staining; the PPARγ mRNA levels gradually increased during the process of the preadipocytes differentiation, and reached the top at 48 h after induced differentiation culture, then the PPARγ mRNA levels gradually decreased. The Laiwu Black pig primary culture system and the model of the induced differentiation in vitro were established successfully. This study laid the basis for further researching Laiwu Black pig adipocyte proliferation, differentiation and porcine fat deposition.

The study aimed to elucidate the structures of SLA Class Ⅱ genes of Guangxi Bama Minipigs and the potential in pigs to humans xenotransplantation. 4 specimens of ear tissues were sampled from four Guangxi Bama Minipigs, and the classical swine leukocyte antigens class Ⅱ genes (SLA-DRA, SLA-DRB1, SLA-DQA, SLA-DQB1) were amplified by PCR after reverse transcription. Purified products were cloned into pMD18-T vectors and bi-directional sequenced. Analyzing obtained sequences by BLAST in NCBI, a total of eight alleles were found, and five alleles have been found in previous reports, the other three were novel. From the homology analysis between alleles of SLA and HLA in GenBank and phylogenetic trees analysis, the results showed that the Guangxi Bama Minipigs possessed high homologous in MHC information with human, and indicated that Guangxi Bama minipigs would have potential utilization values in the study of pig to human xenotransplantation.

In the study, the full-length cDNA of the liver receptor homolog-1 (Lrh-1) was detected from liver of Hu sheep by reverse transcription PCR and RACE methods. Lrh-1 cDNA sequence, protein physicochemical properties, membrane structure, signal peptide and secondary structural characterisitics were analyzed and predicted with bioinformatics softwares and online tools ,such as DNAman, Tmpred, SignalP 3.0, TargetP 1.1, Expasy, and PSORT II prediction. The expression of Lrh-1 mRNA in different tissues of Hu sheep was also examined by quantitative real-time PCR. The results showed that the cDNA of Hu sheep Lrh-1 contained 1 488 nucleotides, and the nucleotide sequence had 98% homology with that of Bos taurus. It encoded 495 amino acid residues, which shared 98%, 97%, 96%, 97%, 97%, 86% and 86% identity with those of Bos taurus, Homo sapiens, Equus caballus, Macaca mulatta,Canis familiaris, Mus musculus and Rattus norvegicus, respectively. The relative expression level of Lrh-1 mRNA was detected in brain, digestive system and reproductive system, and the expression levels were showed differently and had tissue-specific. Relatively, Lrh-1 mRNA expression level was the highest in hypothalamus of Hu sheep.

The objectives of this study were to investigate the developmental expression characteristics of PPARα and PPARγ mRNA in two sheep breeds of distinct tail types and to discuss the probable relationship between the mRNA expression and metabolism of adipose tissue. Guangling Large Tailed (GLT) sheep and Small Tailed Han sheep (STH) at 2, 4, 6, 8, 10 and 12 month old were used to detect the mRNA expression in seven adipose tissues (great omental, small omental, tail, subcutaneous, mesenteric, perirenal and retroperitoneal fat) by real-time PCR. The results showed that PPARα and PPARγ expressed in all tissues studied. Generally, the mRNA expressed lower in superficial fats than that in deep fat deposits. As the main effects, months of age and breed did not basically influence the mRNA expression of PPARα and PPARγ. Though sex did not influence PPARα expression in both breeds, low expression of PPARγ in females of GLT resulted in the significant sex effect and interaction between sex and breed. Based on the expression differences between tissues and ages, respectively, the expression of the two genes was of significant spatiality but not of temporality. The two genes had a synergistic effect on the metabolism of adipose tissues despite their opposite functions.

In order to clone the DNA sequence of Gfrα1 gene in sheep and deeply research the sheep spermatogonial stem cells (SSCs), in the present study, the partial sheep Gfrα1 cDNA were cloned using RT-PCR and molecular cloning technique. The cloned partial Gfrα1 gene was 1 312 bp in length，including an ORF of 1 311 bp encoding 437 amino acids. The nucleotide sequences of sheep Gfrα1 gene were compared with the counterpart sequences of Bos Taurus，homo sapiens, Pongo abelii, Rattus norvegicus and Mus musculus, and the nucleotide homology was 98.5%, 91.3 %, 91.0%, 88.9% and 88.0%, respectively. According to the cloned Gfrα1 gene sequence, the epitope was forecasted and then the cDNA was inserted into pET-44a（+） vector．Subsequently, the recombinant was induced by IPTG. The recombinant epitope polypeptide was purified according to Affinity column chromatography. Western blot analysis indicated that the molecular weight of the expressed epitope polypeptide was the same as predicted size of approximate 18.2 ku. The data collected provided the important information for further making Gfrα1 gene polyclonal antibody and authenticating on molecular level for spermatogonial stem cells in sheep.

In order to elucidate the roles of DHRS7 gene in formation of goose fatty liver, the goose DHRS7 gene full-length cDNA sequence was cloned by RACE method, and then was analyzed by bioinformatics method. Meanwhile, expression of DHRS7 in goose different tissues and organs was analyzed using qRT-PCR method, as well as the mRNA expression influenced by forcing-feed was analyzed in goose liver. Results showed that the full sequence of goose DHRS7 gene was 1 297 bp, including an coding domain sequence (CDS) encoding for 336 AA, and a 25 bp 5′ UTR and a 243 bp 3′UTR. Results of bioinformatics analysis showed that the geese DHRS7 posses a transmembrane region and a NADP binding region, on which functional region there is few variants. The DHRS7 had a higher expression level in geese liver and adipose tissues. And the expression of DHRS7 up-regulated significantly in geese liver tissues after a force feeding (P<0.05). The results indicate that the goose DHRS7 gene may play an important role during formation of geese fatty liver.

This experiment was conducted to study the effects of feeding different linseed level diets on performance, carcass characteristics and meat quality of lamb, and its mechanism. Thirty-two F1 lambs (two months old) of Suffolk sheep(♂)×Small-Tailed Han sheep(♀) were allocated into four groups with eight replicates in each group. Lambs were fed the diet with 0% (control group), 5%, 10% or 15% linseed (DM basis). The experiment lasted 60 days. All the diets were designed to contain similar contents of crude protein and digestible energy. The results showed that feeding diet containing 5% and 10% linseed could improve the performance, ADG（P<0.05） and carcass weight（P<0.05）of lambs, and decrease the ratio of Feed/Gain（P<0.01）. With the increasing of linseed content in the diet, water content of muscle was decreased （P<0.01） and the fat content in the muscle was increased（P<0.01）. The contents of total cholesterol, TG and free fatty acid in the blood tended to increase（P>0.05）. The activity of lipase decreased. The diet containing 5% to 15% linseed significantly modified meat quality of lamb. Linseed improved cooking percentage, intramuscular fat and MFI （P<0.01）of muscle, so it improved the muscle tenderness. Linseed in diet declined the oxidation resistance, but had no effect on the meat color(P>0.05). Linseed supplement improved the fatty acid profile, especially increased the amounts of PUFA（P<0.01）, CLA（P<0.01）, linolenic acid（P<0.01） and the ratio of n-6 to n-3 PUFA（P<0.01）. Diet containing 5% to 10% linseed could increase growth performance and improve the meat quality through optimizing fatty acid profile and increasing PUFA content, especially CLA content of meat.

This study was conducted to investigate the difference of intestinal fluid composition along with the intestine of Peking duck fed with wheat-soybean meal diet and corn-soybean meal diet, respectively, which would provide biological basis for simulating intestinal fluid in duck feedstuff evaluation using in vitro digestion. The completely random design was adapted. Thirty adult ducks with similar weight were divided into 2 groups consisting of 5 replicates of 3 birds each and fed ad libitum with wheat-soybean meal and corn-soybean meal diets, respectively. The digesta of duodenum, anterior jejunum, posterior jejunum and ileum were sampled after slaughter at the 19^th day of the test period. Intestinal fluid was made by centrifuging digesta samples at 4 ℃. The activites of digestive enzymes and electrolyte concentration were determined. The results showed that there were higher amylase, lipase activities in duodenal and anterior jejunal fluid than that in posterior jejunal and ileal fluid (P<0.05). The activity of trypsin was higher in anterior and posterior jejunal fluid than that in duodenal and ileal fluid (P<0.05). The anterior jejunal fluid had higher activity of chymotrypsin than that in duodenal, posterior jejunal and ileal fluid (P<0.05). There was significant difference in chymotrypsin activity between in any two of duodenal, posterior jejunal and ileal fluid. Along with intestine of Peking duck, the pH was increased from 6.22 to 7.94. The Na⁺ and Ca²⁺ concentration were increased and then decreased. The K⁺ concentration was slowly decline. The Mg²⁺ concentration in duodenal fluid was lower than that in anterior jejunal, posterior jejunal and ileal fluid. Diet treatments significantly affected amylase, lipase, trypsin, and chymotrypsin activities and K⁺，Mg²⁺ concentration of intestinal fluid. In conclusion, along the duck’s intestine, jejunal fluid had higher 4 digestive enzyme activities than that in duodenal or ileal fluid, and there were significant difference in the ion concentration of intestinal fluid from duodenum, anterior jejunum, posterior jejunum and ileum.

 This study was conducted to evaluate the effect of adding wet hullessbarley distillers- grains (WHDG) on the fermentation quality of mixed silage of common vetch and tall fescue during ensiling. Mixture of common vetch and tall fescue were ensiled by adding wet hullessbarley distillers'grains(WHDG), there were four adding levels (0, 10%, 20% and 30% of fresh weight). The silos were opened on 7, 14, 30 and 60 days after ensiling in triplicate and the fermentation quality was analyzed. The results showed that WHDG addition markedly improved the fermentation quality of mixed silage, which was well indicated by significantly (P<0.05) higher lactic acid content and lactic/acetic acid and significantly (P<0.05) lower pH and ammonia-N/total N in WHDG addition silages than these in control. WHDG addition greatly enhanced lactic acid production and pH decline during the early stage of ensiling, effectively inhibited the activity of aerobic bacteria, and resulting in lower loss of dry matter and water soluble carbohydrate. Based on the results, it can be concluded that the fermentation quality of mixed silage was considerably improved by 10% or more WHDG addition.

The objective of the study was to establish a judging standard of colloidal gold-based immunochromatographic assay strip (GICA) for detecting antibodies of serotype O of foot-and-mouth disease (FMD) rapidly. The GICA of 3 batches were tested. There were a hundred and eighty FMD negative serum samples (containing pig, cattle and sheep), a hundred and ninety-seven FMD weak positive serum samples (containing pig, cattle), three hundred and seventeen FMD serotype O positive serum samples (containing pig, cattle and sheep), two hundred and twenty-three FMD serotype A and Asia 1 positive cattle sera, six hundred filed samples. To compare three methods of GICA, liquid-phase competition ELISA (LB-ELISA) of FMDV serotype O and Indirect Hemagglutination Test (IHA) of FMDV serotype O for detecting antibodies of FMD serotype O, all the serum samples were detected, the judging standard was established. According to the color of test line and control line, compared with the titers of LB-ELISA, the colorimetric card was drawn. The judging standard of GICA was as follows: Using a 1:2 dilution of the sample, the result was negative, and the anti-FMDV serum was negative; Using a 1:8 dilution of the sample, the result was positive, which implied that the serum had serotype O FMDV positive antibodies. Compared with GICA and LB-ELISA, the colorimetric card was drawn which was helpful for reading results. The correlation of the GICA and LPBE was y = 1.142x + 0.742 1; the correlation coefficient (r) was 0.922 1; the correlation of the GICA and IHA was y = 1.184 5x + 0.862 3; the correlation coefficient (r) was 0.903 3. These results indicated that the judging standard of GICA was prior to detecting antibodies of FMD serotype O; the colorimetric card was easy to read, which was just monitoring the anti-FMD serotype O antibodies of immunized animals fast and accurately. This judging standard could promote the qualitative analysis to semi-quantitative analysis, and it had provided that it was more suitable for using in laboratory and the field.

To identify the Torque teno sus virus (TTSuV), a PCR-DHPLC assay was developed in this study. Primers specific for the partial region of the TTSuV1 and TTSuV2 were selected to conduct the PCR-DHPLC assays. The specificity test was performed with PPV, PCV-Ⅱ and PRV and no cross reaction was found. The PCR-DHPLC for TTSuV had good specificity and nice repeatability. Sensitivity analysis showed that the developed PCR-DHPLC could detect 1.0×10¹ copy·μL^－1. The established method and the Real-time PCR were used to detect 80 serum samples, the results showed that 63 samples were TTSuV1 positive by PCR-DHPLC, and it was consistent with real-time PCR test; 69 samples were TTSuV2 positive by PCR-DHPLC, and it was consistent with real-time PCR test, 56 samples were positive by normal RT-PCR. The method showed nice specification, sensitivity, repeatability, and quickness, and it could be used in epidemiological investigation of TTSuV.

 According to the gene sequences in GenBank, six pairs of specific primers were designed to amplify part fragments of the specific genes of PRV gE, PPV NS1, PCV ORF2, PRRSV ORF7, CSFV E2 and JEV E, respectively. The results of sensitivity and specificity tests showed that the minimum amounts of nucleic acid detection by the mPCR were as follows: PRV 6.6 pg, PPV 96 pg, PCV2 12.9 pg, PRRSV 10.5 pg, CSFV 51 pg, and JEV 46 pg respectively, and the amplification results of Swine Influenza Virus (SIV) and Escherichia coli (E. coli) were both negative. The results of 103 clinical samples from Guizhou province revealed the six viral diseases exist universally in pig farms of Guizhou province, and the mixed infection was much serious. The Six-plex PCR method could detect the infection of PRV, PPV, PCV, PRRSV, CSFV and JEV simultaneously, could detect DNA and RNA virus synchronously, and could differentiate and diagnose the clinical samples of single infection or mixed infection infected by the six viruses quickly.

The study explored the role of glycosyltransferase in inflammation response of embryo trophoblast cells infection by Brucella. WboA gene was amplified from Brucella melitensis M5-90 strain by PCR and cloned into vector pET-28a(+). The constructed recombinant plasmid pET-WboA was transformed to competent E. coli BL21. The protein WboA was identified by SDS-PAGE, Western blot, and purified. The suicide plasmid pGEM-7zf-ΔWboA-SacB was constructed. The WboA gene was knocked out of the genomic DNA of Brucella melitensis vaccine M5-90 strain by homologous recombination. Then purified the WboA protein and ΔWboA affects in the embryo trophoblast cells separately, the inflammatory cytokines of IL-6, IL-10, TNF-α and the lactic dehydrogenase (LDH) were ELISA examination. The protein WboA was successfully purified and the mutant ΔWboA was constructed. Compared with the M5-90 infected group, the HPT-8 cells infected by ΔWboA released much less inflammatory cytokine IL-6, IL-10 and TNF-α (P<0.05). The inflammatory cytokine IL-6, TNF-α  and LDH released by HPT-8 effected with WboA protein were significantly higher than the PBS control group(P<0.01). These results indicate that the protein WboA has the function of cytotoxin and the inflammation response of Brucella is related to the Lipopolysaccharides O chain.

In order to identify the linear B-cell epitope of structural protein BP26 of Brucella abortus, the BP26 protein was dissected into three overlapping fragments，and expressed in Escherichia coli, respectively. Western blot analysis of the three fused proteins was carried out with sera from Brucella infected cattle. The results showed that the linear B-cell epitope of BP26 protein was located in the 51-165 aa. Based on these, the short peptide protein BP26 2 was dissected into five overlapping fragments further. Finally, we identified that the linear B cell epitope on BP26 is located in the 109-141 amino acid. It was concluded that the MAbs against the protein BP26 could be useful tools for further studying on the structure and function of BP26, as well as for development of diagnostic methods.

To construct human adenovirus type 5 vectored recombinant virus expressing S1 glycoprotein gene of avian infectious bronchitis virus (IBV), S1 gene of IBV QS10 strain obtained through RT-PCR was inserted into shuttle plasmid of the adenoviral expression system, forming pacAd5 CMV-S1, as well as the backbone plasmid, which was linearized with PacⅠand transfected into 293AD cells. Detection with PCR revealed that the recombinant virus was successfully packaged with a titer up to 10⁷ TCID₅₀·mL^－1. Furthermore, The results of Western blotting and indirect immunofluorescence assay (IFA) indicated that the S1 gene was significantly expressed. Sera antibodies against IBV of the SPF chicken immunized with the recombinant virus were detected by enzyme-linked immunosorbent assay (ELISA). The recombinant adenovirus expressing S1 gene of IBV was successfully constructed with potent capacity to induce the specific antibodies in chickens and had the good immunogenicity, which laid the foundation for further development of recombinant avian infectious bronchitis vaccine.

Our previous studies demonstrated that anti-idiotypic antibody against porcine reproductive and respiratory syndrome virus (PRRSV) GP5 protein specifically binds nonmuscle myosin heavy chain II-A (NMHC II-A) on Marc-145 cells and the binding site is in the NMHC II-A C-terminus region (named PRA). In this study, PRA protein was expressed in eukaryotic system and examined for its roles in PRRSV infection of Marc-145 cells. PRA protein containing 310 amino acids was expressed in Bac-to-Bac baculovirus expression system and identified by Western blot. Its binding to Marc-145 cells was detected by indirect immunofluorescence assay. The ability of PRA protein to block PRRSV infection of Marc-145 cells was examined by virus neutralization test, fluorescent focus neutralization test and quantitative RT-PCR. PRA protein was expressed in eukaryotic system with the highest expression level at MOI 1 after 96 hours of infection. The results showed that PRA protein specifically bound with Marc-145 cells and inhibited the PRRSV infectivity up to 60%. Our results indicated that NMHC Ⅱ-A C-terminus protein was successfully expressed in baculovirus expression system and could inhibite the PRRSV infectivity of Marc-145 cells up to 60%. These results provided additional information on the function of NMHC II-A during PRRSV infection.

The present experiment was conducted to investigate effects of conjugated linoleic acid (CLA) on growth performance, oocyst shedding, duodenumal mucosal immune response and lipid peroxidation of broiler chicks infected with E. aceruvlina. After 21 days of dietary supplementation with either basal diets or 1.5% CLA diets, half of replicates in each treatment were inoculated with 10 000 E. aceruvlina sporulated oocysts. Compared with basal diets, 1.5% CLA diets ameliorated growth inhibition (P<0.01), and decreased oocyst shedding between 6-9 d post-inoculation resulted from E.aceruvlina infection (P<0.01). Whether infection or not, duodenumal intraepithelial lymphocyte （IEL） CD8⁺T lymphocyte subpopulations were significantly higher in chicks fed CLA diet than the controls (P<0.05), but interferon γ （IFNγ） gene expression was not (P>0.05). Dietary CLA notably suppressed the increase of MDA in duodenumal mucosa (P<0.05), and inhibited the decrease of glutathione (GSH) concentration （P<0.01） resulted from E.aceruvlina infection, but didn’t influence the activity of total superoxide dismutase （TSOD） and catalase （CAT）（P>0.05）. Dietary CLA decreased oocyst shedding and ameliorated growth ihibition of broiler chicks resulted from E.aceruvlina infection at least in part through increasing duodenumal IEL CD8⁺T lymphocyte subpopulations and inhibiting the decrease of duodenumal mucosal GSH in broiler chicks.

The objective of this study was to ascertain the species, distribution and genetic development of the endophytic fungus from the major locoweed in the west meadow of China and detect the antibacterial activity of their secondary metabolites. The study collected Oxytropis flabra, Oxytropis Ochrocephala Bunge, Oxytropis Kansuensis, Oxytropis Deflexa, Oxytropis Coerulea and Astragalus variabilis samples to isolate and purify the endophytic fungi in the different tissues, then used the morphology techniques and ITS sequence analysis to identify their species, and built the phylogenetic tree by Neighbor-Joining method. Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Salmonella were selected as indicators to do the antibacterial tests of the fungi secondary metabolites by paper disc diffusion method. 265 fungal colonies were isolated from the collected locoweed, which were combined to 38 kinds of different endophytic fungus. The distribution of the endophytic fungus in the tissues of locoweed was different; Fusarium sp. Alternaria sp. and Paecilomyces sp. were the dominant strains, which occupied 16.98%, 11.33% and 9.81% of the total isolated colonies respectively. Phylogenetic analysis showed that 38 kinds of endophytic fungus could be divided into two populations based on the evolutionary kinship. The endophyte species and the distance of genetic relationship belonged to locoweed species, tissues, ecological environment and other factors. The antibacterial tests results showed that the secondary metabolites of 29 strains at least (76.32% of the total strains isolated) presented the antibacterial activity against one of the tested bacteria, of which the secondary metabolites of two strains were found to have stronger antibacterial activity against the four tested bacteria. There are various species of endophytic fungus in the major locoweed in the west meadow of China, the endophytes in different ecological environment have a certain kinship but also with some differences. Diverse antibacterial natural products are existed in the secondary metabolites of endophytic fungus from locoweed, which could be a new resource to screen active substance with antibacterial activity.

 The experiment was conducted to study the effects of different Ca levels on functional injury of kidneys induced by fluoride in rat and provide the theory basis for the selection of calcium dose for treating fluorosis patients. Fifty health Wistar rats at the age of 21 days were chose and randomly divided into 5 groups: control group, high F group (including 150 mg·kg^-1 F^-), high F and low Ca group (including 150 mg·kg^-1 F^-and 2％ CaCO₃ ), high F and middle Ca group (including 150 mg·kg^-1 F^-and 3％ CaCO₃), high F and high Ca group (including 150 mg·kg^-1 F^- and 4％ CaCO₃). After 60 days, blood was taken to measure the content of calcium (Ca), phosphate (P), blood urea nitrogen (BUN), serum creatinine (SCr) in the serum. The kidneys were weighted to calculate viscera index. And the enzymatic changes including lactate dehydrogenase (LDH), succinic dehydrogenase (SDH) and alkaline phosphatase (ALP) were detected. Immunohistochemistry methods were employed to determine the expression of CD₁₁ and TNF-α protein in kidney of male rats. The result showed that, compared to control group, the weight of high F group was decreased significantly (P<0.01); its P in serum was much higher than normal group (P<0.05); the content of LDH, SDH and ALP of high F group were successively increased (P<0.01); CD₁₁ and TNF-α protein expression was markedly higher (P<0.01). Compared with the high fluoride group, high F and low Ca group and high F and middle Ca group increased body weight obviously (P<0.05); the levels of Ca and P in serum and LDH, SDH and ALP in kidney were significantly decreased; expression levels of CD₁₁ and TNF-α in high F and low Ca group were significantly inhibited (P<0.01). Between results of high F and high Ca group and high F group, there was no significant difference (P>0.05) in viscera index and the amounts of LDH, ALP and SDH, the BUN and SCr levels was impermissibly high (P<0.05), and the expression levels of CD₁₁ and TNF-α didn’t show significant changes (P>0.05). The results suggested that fluorine in high doses may cause the functional injury of rat kidney. Calcium in both low and middle doses could reduce the toxic effects of kidney caused by fluorine and have the protective role in kidney tissue. The excessive calcium, instead of alleviating the toxic effects of fluorine on kidney, may make the kidney damage more serious.

Fibroblast cells were isolated from adult Banna Miniature Inbred pig with cold trypsin digestion method and subcultured. Through study of cell biology characteristics, effects of fibroblast cells as the donor cells on somatic cell nuclear transfer was investigated. The results showed that isolated fibroblast cells were morphologically consistent with typical fibroblast, cell viability was 97.2% before freezing and 92.6% after thawing (P>0.05). The cell growth curve was ‘S’ shape and population doubling time was approximately 36 h. The karyotype was normal after 14 passages of subculture in vitro. When isolated fibroblast cells were used as donor cells for somatic cell nuclear transfer, the cleavage rate, the blastocyst rate and cell number of blastocyst were 61.9%, 13.0% and 37, respectively. One of three surrogate pigs after somatic cell nuclear transfer with fibroblast cells as donor cells was pregnant and gave birth to one clone pig. In brief, fibroblast cell line could be established from adult Banna Miniature Inbred pig. The isolated fibroblast cells could be used as donor cells for somatic cell nuclear transfer and gave birth to the clone pig of Banna Miniature Inbred pig.

To screen and analyze the genes associated with growth of goats’ wool and cashmere, differential display reverse transcription polymerase chain reaction (DDRT-PCR) technology was used to study the differentially expressed sequence tags ( ESTs ) at different embryo ages in skin primary and secondary follicles of Inner Mongolia Cashmere goats. 15 ESTs were identified (Acc_N: JK711022-JK711036) , of which 3 ESTs might play a role in the development of hair follicle, and 5 ESTs might be related to secondary follicles and sebaceous glands. The results of bioinformatics analysis showed that JK711036 shared homology with other species’ TRIP12 gene, but the function of other ESTs were unknown. All these were showed that many differential expressed genes was associated with primary and secondary wool follicles on metabolism pathways, which would establish molecular foundation for the formation of wool and cashmere in goats.

 Based on multiplex PCR combined with denaturing high-performance liquid chromatography technique, this study was carried out for simultaneous detection of Mycobacterium tuberculosis complex and identification of major pathogenic species for human and animal tuberculosis. A novel multiplex PCR-DHPLC method was established which identified four gene targets in an reaction，including IS6110 and IS1081 insertion sequence specific for Mycobacterium tuberculosis complex and specific structure gene for M. tuberculosis and M. bovis respectively. The specification of the quadruplex PCR-DHPLC assay was verified by tests on 13 species of mycobacteria reference and isolated strains and 23 species of other microorganism. The limit of detection was measured by testing on purified mycobacterium DNA and cloned plasmid DNA. Clinical performance of the assay was demonstrated by detection on bovine tissue samples from infected herds and compared with culture. Results were as follows: The assay specifically detected strains of Mycobacterium tuberculosis complex and simultaneously differentiated M. tuberculosis and M. bovis strains. The limit of detection on cloned plasmid DNA was 10² to 10³ gene copy or 3.6 to 6.8 fg DNA per reaction. For the detection on 39 suspected tissue samples, the assay detected 33 samples positive for Mycobacterium tuberculosis complex and identified 28 as M. bovis positive, while 21 samples were showed positive by culture method. The whole detection procedure of the multiplex PCR-DHPLC assay on clinical samples could finish within 1 day, and the detection on purified DNA sample took around 2 hours. This study provided a novel rapid molecular method for differential identification of pathogenic mycobacteria for human and animal tuberculosis.