Acta Veterinaria et Zootechnica Sinica

Molecular Cloning, Characterization and Polymorphism of the Porcine (Sus scrofa) Cathepsin B Gene

CHEN Lei;LI Xue-wei;ZHU Li;LI Qiang;LI Ming-zhou

2008, 39(4): 385-392. doi:

Abstract ( 1488 )

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The porcine cathepsin B (CTSB) nucleotide and amino acid sequence were obtained by RT-PCR and sequencing. The open reading frames (ORFs) were 1 008 bp long (encoding for 335 amino acids). The nucleotide sequence identities of porcine cathepsin B ORFs were 85%, 81% and 90% comparing to that of the human, rat and bovine, respectively. The deduced amino acid sequence identities of the porcine cathepsin B protein were 81%, 79%, and 91% comparing to that of the rat, human and bovine, respectively. The structure of cathepsin B protein was predicted by homologous modelling which was similar to other papains. The substratebinding cleft and the catalytic triad were found in the structure model. The polymorphisms of porcine cathepsin B gene was detected in 147 pigs by PCR-SSCP. There were six genotypes be detected on the SSCP locus. The genotype FF on CTSB gene SSCP locus had the highest tenderness traits, it had significantly higher shear force (6.56 kg) and toughness (23.55 kg·s) than genotype EE and EF (P<0.01), and it had significantly higher mean shear force (4.85 kg) than genotype EF (P<0.05).

Porcine Lung Surfactant Protein A: Complete DNA Cloning and Bioinformatics Analysis

QIAO Li-juan;WANG Li-xian;SU Zhen-huan;YAN Hua

2008, 39(4): 393-398. doi:

Abstract ( 931 )

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Porcine surfactant protein A (SP-A) is the most in content and the first found in porcine surfactant protein which has the function of regulating inflammation and immune. The complete DNA sequence of porcine SP-A was attained by designing six primers during PCR cloning in this study. The length of the complete DNA sequence of porcine SP-A was 4 037 bp (gi: DQ985806) including the promoter regions , a 747 bp open reading frame coding 248 amino acid(aa). The molecular weight and the isoelectric point of porcine surfactant protein A was 26.37 ku and 5.20, respectively. It’s predicted the 1-20 aa was signal peptide sequence, and the 1-20 aa，120-135 aa, 145-160 aa was hydrophobicity domain; porcine surfactant protein A was in the surface of membrane by transmembrane helices analysis. Meanwhile, as human SP-A, the porcine SP-A has conservative collagen-like region, neck region, and carbohydrate recognition domain . The present results would contribute to study on the association between SP-A and porcine respiration disease .

Molecular Cloning and Tissue Specific Expression Profile of Porcine Ubiquitin B Gene

XIE Shui-hua;LI Jia-qi;CHEN Yao-sheng;ZHANG Hao;WANG Chong;MEI Ying-jie;MO De-lin;LI Ze-yue

2008, 39(4): 399-404. doi:

Abstract ( 2087 )

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Blast (http://www.ncbi.nlm.nih.gov/blast) searches was employed to obtain homologous porcine ESTs in the porcineEST databases with the mRNA sequence of human ubiquitin B (UbB) gene (GenBank accession No NM018955). And a 894 bp cDNA fragment (GenBank accession No. EF688558) was amplified and subsequently sequenced using total RNA of Landrace pig longissimus dorsi muscle as the template after in silico cloning. The cDNA fragment of porcine UbB gene contains a complete ORF encoding a deduced protein of 229 amino acid residue. The porcine UbB gene contained two exons and one intron spanning approximately 1.5 kb, and the exon/intron boundaries confirmed the GT-AG rule. Analysis of the porcine UbB gene genomic DNA sequences showed that it was highly homologous to that of other species such as Homo sapiens (92%), Pan troglodytes (91%), Mus musculus (91%), Rattus norvegicus (91%) and Gallus gallus (89%), and all these species shared the same amino acid sequence. Result of semiquantitative RT-PCR revealed that UbB gene was highly expressed in heart, liver, kidney and cerebellum, followed by spleen, lung, stomach, bladder, longissimus dorsi muscle, cerebrum, and the lowest in fat and hypothalamus. At the same time, we find that the UbB gene was differentially expressed between Lantang pig and Landrace pig in liver, spleen, lung, kidney, stomach, bladder and longissimus dorsi muscle.

Association of Polymorphisms in the Porcine CACNA1S Gene with Carcass and Meat Quality Traits

FANG Xiao-min;GUO Xiao-ling;XU Ning-ying;REN Shou-wen

2008, 39(4): 405-409. doi:

Abstract ( 954 )

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The Jinhua pigs (40), Pietrain pigs (30) and the second generation of crossbred of Jinhua and Pietrain (126) were used in the study, their genotype of SNPs at the position 187 of exon 44 and at the position 37 and 179 of intron 37 for CACNA1S were investigated by PCR-RFLP. The association analysis of the SNPs genotype with the meat quality traits was also conducted in the study, results showed that the genotype AA of Eco 72 I had significant effects on M longissimus dorsi area and meat lightness (L) (P<0.01), the genotype BB of Eco 72 I had significant effects on lean meat weight (P<0.01) and pH value for ham (0.01＜P＜0.05). The site Xba I had significant effect on backfat thickness at the thorax and waist joint position (0.01<P<0.05), but no significant difference was found among the genotype AA, AB and BB. The site Cfr 42 I showed polymorphisms only in Jinhua and Pietrain pigs and with the only genotype BB in the second generation of crossbred of Jinhua and Pietrain.

Polymorphisms Scanning in Exons of the PON2 Gene and Analysis of Exon 9 with SSCP in Seven Bovine Breeds

JI Ai-guo;XU Shang-zhong;HUAI Ya-hong;GAO Xue;ZHOU Zheng-kui;REN Hong-yan;CHEN Jin-bao

2008, 39(4): 410-416. doi:

Abstract ( 944 )

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Paraoxonase-2 (PON2) gene production is an antioxygen for the metabolism of circulating triacylglycerols. It is considered to be a major candidate gene affecting fat deposition in blood vessel and human lifespan, respectively. In this study, direct sequencing method was used to detect the single nucleotide polymorphisms in the exons of PON2 gene; polymerase chain reaction-singlestrand composition polymorphism (PCR-SSCP) was applied to analyze the polymorphisms of exon 9 of the PON2 gene in 478 cattle that comprised of seven breeds, namely Luxi, Nanyang, Jinnan, Chinese Simmental, Holstein, Murrah buffalo and Nili-Ravi buffalo. Four single nucleotide polymorphism sites were discovered in exons, which had not caused the change of amino acids. A 167 bp long PCR product of exon 9 was detected with SSCP. BB genotype had one substitution mutant T98C. Except Luxi and Holstein, other five breeds were all at Hardy-Weinberg equilibrium (P > 0.05). In Luxi, Nanyang, Jinnan and Holstein, the polymorphisms information contents were moderate polymorphisms (0.25 <PIC< 0.50). Others were in low polymorphisms. AA genotype was not detected in Murrah buffalo and Nili-Ravi buffalo, while AA, AB and BB genotypes present in other five breeds. The predominated frequency of the genotypes were difference in these seven breeds, AA>AB>BB was in Luxi and Nanyang; AA/AB/BB was near to 1∶1∶1 in Jinnan; there was serious skewness of BB in Chinese Simmental, which was the predominated genotype and the allele B was the dominance allele; and in two Buffalo breeds, B allele was the superiority. According to statistical analyze with SAS 9.1 GLM procedure, there were significant difference between breeds and genotypes (P < 0.); and no significant difference were found among several age groups in Luxi cattle (n=238) between genotypes (P > 0.05), but significant difference among different genotypes was found (P < 0.01). Combination with the tendency of the development of the genotype of animals, the AA type will be the fitted one for longer lifespan.

Origin and Phylogenetics of Seven Chinese Sheep Breeds Based on D-loop Sequence

ZHAO Qian-jun;GUAN Wei-jun;GUO Jun;QIAO Hai-yun;HE Xiao-hong;PU Ya-bin;FU Bao-ling;AO Hong;LI Kui;MA Yue-hui

2008, 39(4): 417-422. doi:

Abstract ( 1509 )

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To investigate the origin, evolution and genetic diversity of Chinese domestic sheep (ovis), complete D-loop sequences of 59 individuals from 7 local breeds from Xinjiang, Inner Mongolia, Tibet, Yunnan, Ningxia and Shandong and 1 imported breed were sequenced. Haplotype diversity (Hd) and nucleotide diversity (π) among 7 Chinese sheep population were 0.996 0 and 0.030 6 averagely, which indicate genetic variance within control region of Chinese sheep is abundant. Neibour-joining tree was constructed based on 59 D-loop sequences from 8 domestic sheep populations together with previously published wild sheep sequences. It supports there are three distinct lineages (A, B and C) in Chinese sheep. Mouflon was clustered with lineage B, which showed that it had close relationship with Chinese sheep. Further medianjoining network analysis also showed three lineages were determined in Chinese sheep. The result suggests that Chinese sheep derived from three independent maternal ancestors. Analysis of mismatch distribution showed unimodal distribution of three lineages. And Fu’s test of selective neutrality revealed that three lineages were in departure from the neutrality, which the difference were significant (P<0.001). It implies three lineages of Chinese sheep ever experienced population expansion possibly.

Association of PCR-RFLP at the Growth Hormone Gene with Early Growth and Developmental Traits in the Nanjiang Brown Goats

ZHANG Hong-ping;ZHANG Guo-jun;XIANG De;LIU Cheng-jian;WANG Bo;CHEN Yu;LI Li

2008, 39(4): 423-428. doi:

Abstract ( 1111 )

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The partial fragments of GH gene polymorphisms of 90 Nanjiang Brown goats were detected by PCR-RFLP.Association of PCR- RFLP at the GH gene with early growth and developmental traits in the Nanjiang Brown goats was also analyzed. The result showed that the sequence of PCRamplified products was 985 bp which included goat GH gene sequences of exon 3, exon 4, exon 5 and intron 3, intron 4. The PCR products were digested by two kinds of restriction endonuclease named FokⅠ and AciⅠ, respectively. Both of them had polymorphisms.The FokⅠ produced GG, GA and AA genotypes which frequencies were 0.522, 0.289 and 0.189, but the allele frequency of G (0.666) was higher than that of A (0.334).The AciⅠ produced CC, TC and TT genotypes which frequencies were 0.467，0.322 and 0.211， respectively, but the allele frequency of C (0.628) was higher than that of T (0.372).Chi-square test revealed that the genotype frequencies produced by restriction endonucleases were non-equilibrium Hardy-Weinberg. Association analysis of PCR-RFLP at the GH gene with early growth and developmental traits indicated that the least square means of weight, body height and body length with GA and TC genotypes were both significantly higher than that with other genotypes at different month ages (0.05<P<0.01 or P<0.01)

Variation of Goat MSHR Gene G676A in Different Populations with Different Colors

LI Lan-hui;LI Xiang-long;ZHOU Rong-yan;WANG Li-ze

2008, 39(4): 429-436. doi:

Abstract ( 1338 )

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Variation of goat MSHR gene G676A mutation in 22 populations containing 634 individuals was analyzed. The results indicated that the dominate genotype in the goat populations with white hair were homozygote GG，and the allele G was the superiority gene with a frequency of 0.99. The allele A was the superiority gene with a frequency of 0.33 in red and brown hair goat populations containing 197 individuals, especially up to 0.89 in those of abroad breeds. The frequency of A was 0.21 in 271 goat individuals with black coat color mostly, which was higher than that in the white (0.01) but lower than that in the red and brown (0.33). It could be inferred that genotype AA might be related with red and brown coat color, and GG with white coat color. The results also indicated that the G676A mutation was with higher genetic diversity, but poor in Jianchang Black goat, Leizhou goat, Wuan goat, Duan goat and Jining Grey goat. The genetic differentiation among imported goat populations (0.449) was higher than that among Chinese indigenous goat breeds (0.237), and it was lower between indigenous southern and northern goat populations. The higher gene flow of G676A mutation existed between black hair populations and red and brown hair populations. The clustering of populations based on G676A mutation was basically consistent with the variation of coat and hair color.

Studies of Single Nucleotide Polymorphism of CAST Gene and Its Association with Muscle Fiber Traits in Chicken

LIU An-fang;LIU Yi-ping;JIANG Xiao-song;LI Liang;DU Hua-rui;ZHU Qing

2008, 39(4): 437-442. doi:

Abstract ( 1479 )

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Calpastatin(CAST) has profound effects on the muscle growth and tenderness It was indicated that calpastatin could affect the protein metabolism of poultry In order to investigate the effect of calpastatin gene on muscle tenderness traits of chicken, the adequate quality broilers new strains bred by Sichuan Daheng pourty breed limited company were used．The single nucleotide polymorphisms (SNPs) were detected by PCR-SSCP and DNA sequencing．A SNP site（G→T） was found in coding region of CAST gene. The different genotypes detected by PCR-SSCP were defined as AA, BB and AB. Correlation between genetype and chicken muscle fiber traits was analyzed with SAS 8.01. The statistics analysis results showed that the CAST polymorphism was significant related to the muscle fiber density (MFN) and diameter (MFD)（P＜0.05）. And the chicken in identical day with the genotype of AA has a higher MFN and lower MFD than those with the genotype of AB（P＜0.05）, while there was no significant difference between the genetypes of BB and AA/AB（P＞0.05）. The chicken in identical body weight with the genotype of BB has a higher MFN and a lower MFD than those with the genotype of AB（P＜0.05）, while there was no significant difference between the genetypes of AA and AB/BB（P＞0.05） The results indicated that the CAST gene might play an important role in chicken muscle fiber formation. The significant correlation between CAST and development of muscle fiber suggested that this gene is a good marker in marker assisted selection program for muscle tenderness traits.

Polymorphism of Intron 3 of Growth Hormone Gene and Its Relationship with Body Weight and Carcass Traits in Zi Geese

ZHAO Wen-ming;CHEN Qing;CHENG Jin-hua;WU Xin-sheng;CHEN Guo-hong

2008, 39(4): 443-448. doi:

Abstract ( 896 )

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The single nucleotide polymorphism (SNP) of GH gene was investigated in Zi geese. The primers for intron 3 in GH gene were designed according to the homologous sequence of duck and the SNPs were detected by PCR-SSCP method.The results showed that the length of intron 3 of goose GH gene was about 364 bp, seven SNPs were detected, which were A160G, C167G and T264C, in addition, deletions were found at the base position 176, 177, 231 and 232, respectively, in the two kinds of homozygote. Three kinds of genotypes (AA, AB and BB) were detected and BB genotype was dominant in population, the frequency of genotypes were BB>AB>AA. The results of ChiSquare test indicated that the frequencies of GH gene fit with Hardy-Weinberg equilibrium in Zi geese. The correlation between the genotypes and body weight and carcass traits of Zi geese showed that 7 weeks body weight, 9 weeks body weight, 10 weeks body weight, breast muscle weight, breast muscle rate and heart weight of Zi geese with BB genotype were significantly higher than those with AA genotype (P<0.05), 8 weeks body weight with BB genotype were significantly higher than those with AB genotype (P<0.05). As the whole, the average values of these traits of the individuals with BB genotype were the highest, while those with AA genotype showed the lowest.

Effects of Soybean Oil on Concentrations of Conjugated Linoleic Acid and Accumulation of Intermediates of Ruminal Biohydrogenation in vitro

HOU Jun-cai;LIU Yan-ping;GUI Shi-lin;HUO Gui-cheng;JIA Zhi-yuan;JIANG Ning;ZHANG Yong-zhong

2008, 39(4): 449-454. doi:

Abstract ( 926 )

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This study was conducted to investigate the effect of addition of soybean oil in dietary on concentrations of conjugated linoleic acid and intermediates of ruminal biohydrogenation of mature goat in vitro by batch culture. The addition level of these soybean oil were 0(control group ) and 100 mg/flask, respectively. Results indicated that the cis-9, trans-11 CLA content of soybean oil supplementation treatment was significantly（P＜0.05）higher than that of the control all the time, and which reached its maximum after 8 hours’ incubation (6.05 mg/g DM). And the trans-10, cis-12 CLA content of soybean oil supplementation treatment was also significantly（P＜0.05）higher than that of the control all the time, and which increased gradually, with the extension of incubating time. The total CLA content of soybean oil supplementation treatment reached its maximum after 16 hours’ incubation (11.61 mg/g DM). During in vitro study, that trans-11 C18∶1 and trans-10 C18∶1 were accumulated with treatment of soybean oil, and the treatment was abstractly higher than the control (P＜0.05). The total trans-C18∶1 content of soybean oil supplementation treatment increased gradually, with the extension of incubating time, while the total cis-C18∶1 content of soybean oil supplementation treatment decreased gradually.

Effects of Forage to Concentrate Ratio on Homeostasis of Rumen and Oxidative Stress in Cows

HOU Zhi-gao;WANG Zhen-yong;CHAI Tong-jie;JIA Yu-dong;GONG Qing-liang;MA Jian;WANG Yun-tian

2008, 39(4): 455-459. doi:

Abstract ( 1424 )

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To explore effects of forage on oxidative stress and homeostasis of rumen, pH and anti-oxidative indexes of rumen, anti-oxidative indexes of serum were studied. Five Holstein cows during the dry period were used with 30 days in the first 8 periods, the last period was 3 days. Forage to concentrate ratio were 30∶70，35∶65，40∶60，45∶55，50∶50，55∶45，60∶40，70∶30，100∶0, respectively. The result indicated that rumen pH was the lowest in 100∶0 (P<0.05).The activities of SOD in the rumen was the highest in 55∶45 (P<0.05); The contents of MDA and OH·in the rumen was the lowest in 55∶45 (P<0.05); The activities of GSHPx in the rumen was the lowest in 55∶45 (P<0.05); The activities of TAOC in 55∶45 was higher than that in 30∶70，35∶65，40∶60，45∶55，50∶50，100∶0 remarkably in the rumen (P<0.05). The activities of SOD，T-AOC in the serum was the highest in 55∶45(P<0.01); The activities of GSH-Px in the serum was the lowest in 55∶45 (P<0.01); Compared with 50∶50 and 60∶40,the contents of MDA in the serum in 55∶45 had no significant difference（P＞0.05）, but was lower than that in other concentrate ratio (P<0.05); Compared with 60∶40, the contents of OH· in 55∶45 was not significantly different in the serum（P＞0.05）, but was lower than that in other concentrate ratio (P<0.05).The results indicated that forage to concentrate ratio significantly influenced the homeostasis of rumen and oxidative stress of body.

The Immunologic Effect of Identified Avian Influenza Viruses T Cell Epitopes in vitro

LI Xin-sheng;CHEN Hong-ying;GAO Feng-shan;XIA Chun;CUI Bao-an

2008, 39(4): 460-465. doi:

Abstract ( 1438 )

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To identify whether avian influenza viruses (AIV) derived peptides KILTIYSTV and LLLAIVSLV are authentic T cell epitopes, 6 weeks old BALB/c mice were immunized with KILTIYSTV, LLLAIVSLV and KILTIYSTV respectively. The blood samples were collected at the 1st, 2nd, 3rd, 4th and 5th week after reimmunization. CD8⁺ T cell was detected by fluorescence activated cell sorting (FACS). MTT test was carried out at 14 days after boost immunization; Peptide-specific delayed-type of hypersensitivity assay was done on third weeks after the first immunization. Proliferation of secreted IFN-γ in serum of mice was detected by ELISA. The results showed that the peptides KILTIYSTV, LLLAIVSLV and TIGECPKYV stimulated 4.2%, 4.0% and 0.2% growth of peptide-specific CD8⁺ T lymphocyte; Secreted IFN-γ level, specific delayed-type of hypersensitivity (DTH) response and MTT test in KILTIYSTV and LLLAIVSLV group were all significantly higher than that of control groups. In a word, animal immunity experiment revealed that two T cell epitopes KILTIYSTV and LLLAIVSLV could lead to specified CTL reaction, but the peptide TIGECPKYV that don′t bind with reconstructed MHC Ⅰ couldn′t.

Study on Immune Efficacy of Trivalent Combinations and Fusion DNA Vaccine from Mycobacterium bovis

GONG Qiang;LIU Si-guo;WANG Chun-lai;WANG Yong;LIU Jian-dong;LIU Hui-fang;SHI Yuan-xiang;A Man-gu-li;LI Fa-bin;KONG Xian-gang

2008, 39(4): 466-471. doi:

Abstract ( 1556 )

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Mycobacterium bovis is the etiological agent of bovine tuberculosis (BT). The secretory proteins Ag85B, MPB64 and ESAT-6 are the major immunogenic antigens of M. bovis, and they play important roles in inducing immune responses that confer resistance against infection. In the present study, using pcDNA3.1(+) as vector, various DNA vaccines were constructed with the genes encoding the three antigens as follows: fusion of three genes (pcDNAMPB64/Ag85B/ESAT-6, pcDNA-MAE) and trivalent combinations (pcDNA-Ag85B + pcDNA-MPB64 + pcDNA-ESAT-6). The immune efficacy of the DNA vaccines were evaluated based on serum antibody titers, lymphocyte proliferation assay and content of cytokine (interferon-γ and interleukin-2)titers. Protective efficacy following challenge with M. bovis Bacille Calmette-Guérin (BCG) was evaluated based on lung tissue bacterial loads and histopathologic changes. The data demonstrated that immunization with fusion DNA vaccines (three genes) resulted in significantly higher serum antibody levels, lymphocyte proliferation (SI) values, IFN-γ and IL-2 levels than immunization with polyvalent DNA vaccines (P <0.05). Additionally, fusion DNA vaccines provided superior protection than polyvalent DNA vaccines following BCG challenge. The protective efficacy of the fusion DNA vaccines was equivalent to that of the BCG vaccine, suggesting that fusion DNA vaccines provide a promising approach for the prevention of BT.

Phenotypic and Aminoglycosides Genotypic Antimicrobial Resistance Characterization of Escherichia coli Isolated from 95 Pig Farms

TANG Jing-yuan;WANG Hong-ning;ZHANG Peng-ju;GANG Ting-ting;ZENG Bo

2008, 39(4): 472-477. doi:

Abstract ( 945 )

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The objective of this study was to investigate the prevalence of antimicrobial resistance and aminoglycosides resistance genes(aadA1,aaC2, aaC4 and aphA3) among 480 commensal Escherichia coli isolates, which were collected from weaning piglets of 95 large scale pig farms among 19 provinces in China from 2006 to 2007.The resistance of those E.coli strains toward 19 broadspectrum antibiotics were tested by K-B(Kirby-Bauer) method, and 4 aminoglycosides resistance genes were detected by multiplex PCR. The results showed that E.coli isolates were relatively sensitive to polymycin B (85.6%)，cefotaxime sodium (85.3%)，amikacin (84.8%) and spectinomycine (65.0%) Most of 480 isolates were multidrug resistant strains. The number of E-coli isolates, which were resistant to 13，14，15，16 or 17 antibiotics, was relatively more than others. Moreover, there were 26 isolates resistant to 18 antibiotics and 14 isolates resistant to 19 antibiotics. By WHONET5.4 analysis, the number of swine farms resistant to amikacin,cefotaxime sodium and polymycin B was relatively less than others, and they were 29,26 and 24, respectively. The mainly detected aminoglycosides resistance genes were aadA1(65.89%), aaC2(52.35%)，aaC4(12.91%) and aphA3(10.95%). The antimicrobial resistance genetype of aadA1/aaC2(29.61%) and aadA1(18.04%) were detected frequently among commensal E-coli isolates. The antimicrobial resistance genotype of aadA1/aaC2(43), aadA1/aaC2/aaC4(20) and aadA1/aaC2/aaC4/aphA3(20) were relatively prevalent among 95 pig farms by excel analysis. By comparing the coincidence rate of antimicrobial resistant phenotype and genes, the coincidence rate of amikacin(68.39%) was relatively higher than other antibiotics. The results indicated that the commensal E-coli could produce multidrug resistant to antibiotics, be potential reservoir to preserve and disseminate antimicrobial resistance genes between pathogenic and nonpathogenic bacteria, and could be used as indicator to survey antimicrobial resistance in swine farm environment.

Establishment of a Stable PK-15 Cell Line Expressing the P1 Gene of Swine Vesicular Disease Virus

TIAN Hong;WU Jin-yan;GONG Zhen-li;ZHENG Hai-xue;SUN Shi-qi;SHANG You-jun;LIU Xiang-tao;XIE Qing-ge

2008, 39(4): 478-482. doi:

Abstract ( 844 )

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P1 gene of swine vesicular disease virus (SVDV) was amplified by PCR using specific primers from SVDV HK/70 genome.The amplified fragment was cloned into pBABE puro vector for sequence analysis,and the recombinant plasmid was named pBABE puro-P1. Then pBABE puro-P1 and pVSV-G were cotransfected into GP2-293 packaging cells by liposomes, and recombinant retrovirus was acquired.The recombinant retroviruses was transfected into PK-15 cell by polybrene. The transfectants were selected by paromycin.Results of immunofluorescence, PCR analysis and Western blot showed that the foreign gene was integrated into the chromosome of transfected PK-15 cells and the expressed protein could react with positive serum against SVDV.

Effects of L-Arginine and L-Aminoguanidine During the Eimeria stiedai Infection in Rabbits

YIN Xun-he;QIU Jian-hua;WANG Shu-ying;WANG Cui-mei;SUN Yi-dong;CHEN Bing-feng;LI Wei

2008, 39(4): 483-487. doi:

Abstract ( 942 )

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To probe the activity of inducible nitric oxide synthase (iNOS) in liver , the concentration of nitric oxide (NO) in serum and its inhibition or killness on coccidia in Eimeria stiedai infected rabbits, the infected rabbits were injected with the substrate of nitric oxide synthase L-arginine (L-Arg) and the inhibitor of inducible nitric oxide synthase L-aminoguanidine (L-AG) through the abdominal cavity. Then the activity of iNOS in liver , the concentration of NO in serum，the coccidian oosysts per gram feces (OPG), the anatomic structures and microscopic pathological changes of rabbits’ livers and their scores were studied. The results showed that : ① The activity of iNOS of the uniform slurry of the liver and the concentration of NO in serum increased gradually after infection, and that in control group, L-arginine group, and L-AG group reached the top at 12 days after infection, then decreased gradually and reached the level before infection at ~23 days; ② L-Arg can enhance the activity of iNOS and the release of large amount of nitric oxide, consequently it inhibit or kill Eimeria stiedai and ease the pathological changes caused by Eimeria stiedai; while L-AG decreased the activity of iNOS and reduce NO production in vivo, so that it aggravates the pathological changes caused by Eimeria stiedai. The results showed that NO and iNOS participate in the infection process of Eimeria stiedai in rabbit and NO plays an role of inhibition or killness on coccidia of E.stiedai.

Construction and Screening of ScFv Phage Display Library of the Bursin Antiidiotype Antibody from Chicken Immunized with Anti-Bursin McAb

WU Xiang-dong;QU Yue;CHEN Nan-hui;WANG Ping;HE Hou-jun

2008, 39(4): 488-493. doi:

Abstract ( 858 )

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By using RT-PCR, the chicken heavy chain variable region gene(V_H, 370 bp) and light chain variable region gene (V_L, 320 bp)were amplified from spleen cells of chicken immunized with McAb against bursin, subsequently the acquired segments were assembled together to get ScFv gene through SOE by using (Gly₄Ser)₃ as a linker. The acquired ScFv genes were transformed into E.coli TG1 and amplified with co-infection of helper phage M13K07 to obtain chicken ScFv library of Bursin anti-idiotype antibody. Results of ELISA，animal experiment confirmed that five positive clones screened from the library can combine with anti-bursin antibody.

Study on Adherence and Invasion of Holstein Cows Mammary Epithelial Cells by Klebsiella pneumoniae in vitro

WANG Heng;MENG Xia;QIU Chang-wei;MA Chong;WU Pei-fu;HAN Chao;QI Chang-ming

2008, 39(4): 494-498. doi:

Abstract ( 894 )

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Adherence and invasion of Klebsiella pneumoniae to bovine mammary epithelial cells can be the potentially pathogenic mechanisms. In the present study, the cultured bovine mamary epithelial cells was applied to detect the adherence and invasion of Klebsiella pneumoniae. The cells were determined by immunohistochemistry with anti-cytokeratin antibody and transmission electron microscopy. Positive immuno-reaction and desmosomes were observed respectively. Klebsiella pneumoniae, originating from clinical bovine mastitis cases, Salmonella typhimurium and Escherichia coli DH5α were tested for their ability of adherence and invasion to bovine mammary epithelial cells in vitro Results showed that Klebsiella pneumoniae adhered to bovine mammary epithelial cells in dose and time dependent until saturation, and invaded the cells without attenuation. In addition, no significant influence on the bacteria viability in cells was observed during the experimental time. The results of the present paper may partially contribute to an understanding of the pathogenesis of Klebsiella pneumoniae mastitis by surviving in the cells to protect themselves from antimicrobial drugs and host defense system.

Effects of Berberine, Chlorogenic Acid and Baicalin on the Expression of ICAM-1 in Rat Intestinal Mucosa Microvascular Endothelial Cells Injured by LPS

ZHANG Tao;HUANG Hui-ling;HU Ge;DUAN Hui-qin;TENG Ke-dao;MU Xiang

2008, 39(4): 499-502. doi:

Abstract ( 869 )

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To investigate their anti-inflammatory mechanism, the effects of berberine (Ber.), chlorogenic acid (Ch. A.) and baicalin (Bai) on the expression of intercellular adhesion molecule 1 (ICAM-1) in rat intestinal mucosa microvascular endothelial cells (RIMMVECs) challeged by lipopolysaccharide (LPS) were analyzed by immunocytochemical staining, and gray scale and statistical analysis were performed. The results showed that RIMMVECs constitutively express ICAM-1 in low level and LPS significantly increased it, while LPS-induced strong expression of ICAM-1 in RIMMVECs was suppressed by Ber, Ch- A and Bai. The results indicated that the anti-inflammatory role of Ber, Ch- A and Bai is related to their downregulating effect on LPS-mediated ICAM-1 expression, which provide data for clarifying their mechanism.

The Analysis for the Single Nucleotide Polymorphism of TGF-β1 and Its Association with Reproduction in Different Breeds Sheep

GAO Li-xia;LI Hong-bin;DU Li-xin;SONG Xue-mei;LI Zhen-zu;LI Shan-gang;WEI Cai-hong

2008, 39(4): 503-507. doi:

Abstract ( 842 )

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A single nucleotide polymorphism (SNP) of sheep 805 bp sequence in 6th intron region of transforming growth factor β1(TGF-β1) gene was identified in 196 sheep among Small-tailed Han，Tong,Tan and Oula sheep by PCR-SSCP，which declared one mutation point. Comparative sequencing analysis of cloned products revealed a AGAC deletion at upstream 294 of extron 7 of TGF-β1 gene (site 14 201 in gi76871756). The statistical results of genotype and allele frequency in different breeds showed that the genotype AB dominated in the Small-tailed Han sheep. The genotype BB was in majority in low-fecudity sheep. The results of χ2 test indicated that all the populations were in Hardy-Weinberg equilibrium.

Cloning and Characterization of the CDK2 Gene cDNA of Inner Mongolia Cashmere Goats

GUAN Ze-hong;XU Ri-gan;ZHAO Yan-fang;BI Li-ge

2008, 39(4): 508-512. doi:

Abstract ( 1276 )

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The complete CDK2 cDNA sequence from Inner Mongolia Cashmere goat testis was cloned in the first time by homology cloning approach combined with RTPCR and RACE technique ( GenBank Accession No. EF035041). The CDK2 cDNA sequence was 1 355 bp length, containing an open reading frame of 894 bp, flanked by 174 bp 5′UTR and 266 bp 3′ UTR, encoding 298 amino acids. The homology of the goat CDK2 cDNA sequence with bovine was 98%.The amino acids sequence of the CDK2 protein shared 100% identity with bovine CDK2 protein. The homology of the Goat CDK2 cDNA sequence and amino acids sequence with the other mammalian CDK2 genes was more than 92% and 93%, respectively, showing that the structure and function of CDK2 gene is highly conservative.

The Research of the Impact of the State of Donor Cell on the Growth of Reorganization Embryos

LI Xiang-chen;YUE Hua;LIU Peng;WU Yun Qi Qi Ge;GUAN Wei-jun;MA Yue-hui

2008, 39(4): 513-516. doi:

Abstract ( 880 )

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This study conducted in-depth research on the effect factors of the growth of reconstructed embryos, through comparing the different serum starvation days, the number of generations with the recovery of cryopreserved of Donor fibroblast and so text conditions. The research results show that the difference of reorganized embryo cleavage rate is not significant (P> 0.05) as donor serum were starved 0, 1-3, 4-6 and 7-9 days, but blastocysts rate was the highest as donor serum were starved 1-3 days, and the difference was significant (P＜0.05) compared with the 4-6 and 7-9 days groups; the difference of embryo cleavage rate wasnot significant(P>0.05) and morula rate of 1-3 generation was the highest as the cells of 0、1-3、4-6 and 7-9 generation as a source of donor cells; The cleavage rate of reorganized embryos from 2 generation frozenthawed cell was significantly higher compared with 4 and 8 generation. 2 and 6 generation thawed cells of after frozen as were donor cells, egg(embryo) cleavage rate did not differ significantly, and morula rate of 8 generation was lower compared with the other groups. This experiment provide important theoretical basis and technological support to improve donor cells re-program(procedure) in oocyte, the growth of follow-up reorganization embryo and the research of related fields.

Establishment and Application of Dulex PCR Assay for Differentiating Riemerella anatipestifer and Escherichia coli Infection in Ducks

QIN Zong-hua;CAI Jian-ping;LV Min-na;YU Jin-shu;WU Cai-yan;WEN Lie-na;XIE Ming-quan

2008, 39(4): 517-521. doi:

Abstract ( 1002 )

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In this study, partial sequence of Riemerella anatipestifer 16S rRNA gene was amplified by polymerase chain reaction(PCR) using the bacteria 16S rRNA gene universal primers, which its length was 1 512 basepair (bp) and it has been recorded in GenBank and named as DQ078779. The BLAST analysis indicated that DQ078779 was the most similarity with reported 16S rRNA gene of R. anatipestifer. Moreover, speciesspecific primers were designed according to BLAST results of different 16S rRNA gene sequences of R. anatipestifer and E. coli, which 354 and 718 bp specific DNA fragment could be amplified by the two pairs of primers from R. anatipestifer and E. coli, respectively. Finally, dulex PCR with the specific primer for differentiating R. anatipestifer and E. coli infection in ducks was established, and genomic DNA or cultured bacteria or boiled liver tissue of ducks infected by R. anatipestifer and E. coli could be used as template.

Development of Serotype Specific LUX Real-time RT-PCR Assay for Rapid Detection of Vesicular Stomatitis Virus

CHEN Ru;LIU Zhong-yong;ZENG Bi-jian;CAO Yong-chang;CHEN Jun-wei;ZHAO Yin;LIN Zhi-xiong;BI Ying-zuo

2008, 39(4): 522-528. doi:

Abstract ( 1462 )

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Serotype specific LUX (Light Upon Extension) real-time RT-PCR assays were developed for rapid detection of vesicular stomatitis virus (VSV). The LUX primers were designed based on the NS gene sequence of the NJ and IND serotype respectively. The real-time PCR assay could differentiate VSV from FMDV and other related RNA viruses. The detection limit of the assay could reach 1 TCID50 as showed by tests on serial ten fold dilution of cell propagated virus samples. Comparison tests indicated that the LUX real-time RT-PCR assay had at least ten-fold increase of sensitivity than the gel-based RT-PCR method. The assay could detect VSV infection and identify the serotype of the virus from import bovine clinical samples and artificial infected guineapig tissues. The whole procedure, including RNA extraction, realtime amplification and dissociation analysis, could complete within 3 hours.

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