Acta Veterinaria et Zootechnica Sinica

Cloning and Sequence Analysis of Mature Interferon-τ Gene from Chinese Hostein and A Classification for Bovine Interferon-τ

ZHANG Ming;GUAN Xiao-bin;ZHU Qing;LIU Xue-fang;FENG Yu-mei;LAI Song-jia

2007, 38(5): 417-424. doi:

Abstract ( 1261 )

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Complete sequence of mature interferon-τ gene from Chinese Hostein was cloned and sequenced for the first time(GenBank Accession Number: DQ680846、DQ680847). Its CDS sequence is 519bp, encoded 172 amino acid residues of bovine mature interferon-τ protein. All bovine interferon-τ sequences is divided into four groups by cluster analysis. A novel group(τ4) and six novel variant forms(τ1e、τ3f、τ3g、τ3h、τ4a、τ4b)were found. DQ680846 is a variant forms in τ4, signed τ4a; DQ680847 is a variant forms in τ3, signed τ3f. The nucleotide sequence homology of all bovine interferon-τ genes are 92.7%~99.8%, and the amino acid sequence homology of all bovine interferon-τ protein are 88.5%~99.4%. The nucleotide sequence homology between interferon-τ4a and other bovine interferon-τ is 94.0%~97.1%. The nucleotide sequence homology between interferon-τ3f and other bovine interferon-τ is 92.9%~99.6%. The amino acid sequence homology between interferon-τ4a and other bovine interferon-τ, interferon-τ3f and other bovine interferon-τ is 91.4%~96.0% and 88.5%~99.4% respectively. The results suggest bovine interferon-τ gene is high-degree conservation in the evolution. The secondary structure of bovine interferon-τ mature protein was predicted by MLRC and the results show that five α-helices(A-E) is substructure of interferon-τ mature protein, and Asn78、Cys1 、Cys29、Cys99、Cys139、Ile143、Lys160 and the 11 amino acid residues from carboxyl terminus are high-degree conservation region, which are essential for structure and function of bovine interferon-τ.

Cloning, expression and bioinformatics analysis of miR-206 precursor in the pig

CHEN Jian-hai;WEI Wen-juan;XIAO Xiao;XIE Sheng-song;FENG Yang;ZHAO Shu-hong

2007, 38(5): 425-431. doi:

Abstract ( 1687 )

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MiRNAs play important roles in skeletal muscle development. In this study, the porcine miR-206 precursor is cloned and sequenced from skeletal muscle through comparative genomics approach. The secondary structure was predicted. RT-PCR results indicated that the miR-206 precursor is expressed in most of the tissues and exhibited the highest expression level in skeletal muscle. Homology analysis revealed that pre-miR-206 is highly conserved among different species.

Cloning and Expression of Porcine Cathepsin D Gene Full-length cDNA

MEI Ying-Jie;LI Jia-Qi;CHEN Yao-Sheng;LI Xiao-Yun;ZHU Liang-rui;LING Fei;WANG Chong;ZHANG Hao;

2007, 38(5): 432-436. doi:

Abstract ( 1064 )

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Cathepsin D is a major, lysosomal aspartic proteinase, which has important function in proteins degradation. In this study through in silico cloning and RACE, the full-length cDNA of cathepsin D was isolated and sequenced. Sequence analysis revealed a 2032 bp cDNA containing a 93 bp 5’-untranslated region, a 706 bp 3’-untranslated region and a 1233 bp open reading frame coding 410 amino acid ( Genbank accession number：DQ018727). By means of bioinformatics and internet resources , The deduced 410 amino acids of cathepsin D was predicted to contain an Asn （Asparagine）domain, a number of transmembrane regions, a signal peptide with 20 amino acid. And 3D structure of pig cathepsin D was similar to mammalian cathepsin D（alignment score is 99.7%）. Northern hybridization result revealed this gene has only one transcript and expression analysis indicated that cathepsin D was expressed widely and there was no difference within the four pig breeds，and no difference among the tissues.

The Developmental Changes of IGF-I and IGF-IR Gene Expression in Testicular of Erhualian and Largewhite Pigs

LI Fa-di;XIA Dong;CHEN Jie;ZHAO Ru-qian

2007, 38(5): 437-441. doi:

Abstract ( 819 )

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To understand the relationship between the IGF-I and the porcine fertility development, testicular tissue samples were collected from male Erhualian and Large White pigs at 0, 3, 20, 30, 90, 120 and 180 days of age respectively. IGF-I and IGF-IR mRNA level were determined by relative quantitative RT-PCR with 18S rRNA as an internal standard. The developmental pattern of testicular IGF-I mRNA expression in Erhualian boars was similar with those in Large White boars from day 0 to 30, which increased with the age of boars. The levels of IGF-I mRNA in Erhualian boars had no significant changes from day 30 to 180, while those in Large White boars showed a temporal drop at day 90 and restored to the level of day 30 thereafter (P﹤0.05 ). Overall IGF-I mRNA level was higher in Erhualian boars than in Large White boars (P﹤0.05 ). The patterns of testicular IGF-IR mRNA expression in Erhualian and Large White boars were different. The levels of IGF-IR mRNA in Erhualian boars maintained consistent in general throughout the period of investigation, despite a tendency of decrease observed from day 90 to 120 (P﹤0.05 ). IGF-IR mRNA levels in Large White boars was high at day 0 and declined at day 3 (P﹤0.05 ) , and maintained invariable thereafter. Overall IGF-IR mRNA level was higher in Erhualian boars than in Large White boars (P﹤0.05 ). Testicular IGF-I mRNA level was found to be positively correlated with IGF-IR mRNA level (r=0.575, P<0.01). The results suggest that IGF-I and IGF-IR mRNA in porcine testis are coordinately expressed according to breed-specific developmental patterns, and it may be important in porcine testis development.

SNPs Analysis of 3′-Regulatory Region of Myostatin Gene in Three Greese Breeds

YANG Feng-ping;CHEN Yi-quan;LI Qi-song;ZHANG Liu;WANG Jin-yu

2007, 38(5): 442-446. doi:

Abstract ( 870 )

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5 pairs of primers were designed to identify SNPs in 3′-regulatory region of myostatin gene in 3 geese breeds, and the relativity of SNPs and slaughter characters was also analyzed. And the resulted two SNPs were detected, which were A88G and G353T. Population genetic analysis indicated that there were EE and EF genotypes in 88bp locus, and E allele was a preponderant one. The frequency of EF genotype was especially high in Yangzhou geese (0.55), but very low in Wanxi White geese (0.03). In 353bp locus, three genotypes, GG, GH and HH were detected .The frequency of GG was the highest and that of HH was the lowest in 3 geese groups. The relativity analysis showed, individuals with EF had higher slaughter performances than those with EE. For leg muscle weight(LMW) and leg muscle percent(LMP) , individuals with GG were significantly higher than those with HH in Yangzhou geese and Wanxi White geese (P﹤0.05).

Relationship between KAP Gene and Economic Traits in Inner Mongolia Cashmere Goats

ZHANG Ya-ni;ZHANG En-ping;WU Di;CHEN Yu-lin

2007, 38(5): 447-451. doi:

Abstract ( 877 )

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Choosing KAP gene as candidate gene, the genetic relationship between KAP gene and economic traits (cashmere yield, weight and cashmere fineness ) of 60 Inner Mongolia cashmere goats were analyzed by PCR-SSCP method, using GLM procedure of SPSS software. The result showed: BB genotype at S1 site and BB genotype at S2 site was favorable marker genotype for cashmere yield trait, BB genotype at S2 site and AA genotype at S3 site was favorable marker genotype for weight trait. BB genotype at S5 site was favorable marker for cashmere fineness trait. BB genotype at S2 site can be the marker genotype of multi-trait marker in assistance selection.

Molecular Cloning and Polymorphism Analysis of the Partial Coding Region of BMPR-IB Gene in Yunling Black Goat

LI Li-ping;CHENG Meiling;LIU Wei-quan;GAO Shi-zheng

2007, 38(5): 452-457. doi:

Abstract ( 1389 )

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The BMPR-IB gene, which is related to prolific traits of animal, was cloned by RT-PCR from the ovary tissue of Yunling black goat. The results showed that the amplified fragment sequence of the BMPR-IB gene was 467bp in length and it lied between the 497th base and the 963th base of the coding region of BMPR-IB. The homology of the amplified fragment sequence with the same coding region of the BMPR-IB gene in the wild type goat, sheep, pig, human and rat was 99%, 99%, 92%, 92% and 87%, respectively. Compared to the wild type goat, there were two polymorphic sites at the 498th and the 575th base of the nucleus acid sequence of the amplified region of BMPR-IB gene in Yunling black goat, which was that the T base mutated to C base at the 498th site and coding the same amino acid (aspartic acid, Asp), the C base mutated to T base at the 575th site and the coding amino acid changed from praline to leucine, and which might lead to the different secondary structure of BMPR-IB protein. These results would be the foundation for further researching the association between BMPR-IB gene and prolific traits of Yunling black goat.

Development of goat cloning embryos containing EGFP gene

ZHENG Yue-mao;LIU Feng-jun;AN Zhi-xing;LI Xiang-chen;ZHAO Xiao-e;QUAN Fu-sheng;ZHANG Yong

2007, 38(5): 458-463. doi:

Abstract ( 892 )

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Plasmid containing EGFP gene was transfected into the goat mammary gland epithelial cells by lipofection. Stable transfectants were selected by G418 and the positive cells were used as nucleus donors to create transgenic goat cloning embryos by nuclear transfer techniques. SOFaa culture medium supplied with 10 % normal goat serum (NGS) was the best combination for culturing the transgenic goat cloning embryos in vitro. Expression of the foreign gene in the embryos was detected by observation of green fluorescence. EGFP was expressed gradually after the 816 cell stages in most embryos, and the level of EGFP expression increased with development of the embryos. The result implied that EGFP gene could be used as reporter gene to check expression of foreign gene in the transgenic goat cloning embryos in their early stages.

Effect of Supplemental trans-10, cis-12 Conjugated Linoleic Acid on Growth Performance, Meat Quality and relative Enzyme Activities in Broiler chicks

Zhang Guang-min;Wen Jie;Chen Ji-lan;Zhao Gui-ping;Zheng Mai-qing

2007, 38(5): 464-470. doi:

Abstract ( 1331 )

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An experiment was conducted using a total of 288 day-old Arbor Acres male broilers to study the effect of supplemental t-10, c-12 CLA levels on growth performance, carcass traits, and abdominal fat of the broilers. Birds were randomly allotted by body weight to one of six treatments in a completely randomized design. They were fed either a CLA-unsupplemented corn-soybean meal basal diet or fed basal diets supplemented with 0.20, 0.40, 0.60, 0.80 or 1.00 % t-10, c-12 CLA for duration of 42 days. The result shows that the supplemental CLA had no effect (P＞0.05) on the ADFI, percentage of breast and leg muscles，but affected abdominal fat percentage (P< 0.001 ), ADG and F/G(P<0.05). The addition of 0.20, 0.40, 0.60, 0.80 or 1.00 % CLA resulted in a decrease in abdominal fat percentage (P<0.01), and broilers fed on the 0.60% t-10, c-12 CLA had a higher ADG and F/G (P<0.05). The addition of 0.20, 0.40, 0.60 or 1.00% CLA decreased b* value and shear force in the breast muscle (P<0.05), but not in the thigh muscle. The addition of CLA significantly increased taste estimate in breast muscle. The addition of 0.20, 0.40, 0.60 or 1.00% CLA increased MDH activity in abdominal fat, but decreased MDH activity in other tissues and serum leptin (P<0.01).Results from this study indicate that the addition of t-10, c-12 CLA to broiler diets might increase growth performance and feed conversion, decrease the percentage of abdominal fat by decreasing MDH activity in abdominal fat and leptin in serum, increasing MDH activity in liver, breast and thigh muscle.

Characteristics of Dipeptide Transport Kinetics in Small Intestinal Brush Border Membrane Vesicles of Piglets

DAI Jian-guo;LI De-fa;PIAO Xiang-shu;CHEN Hong-liang;PAN Bao-hai

2007, 38(5): 471-475. doi:

Abstract ( 2042 )

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In order to study the characteristics of dipeptide transport kinetics in small intestinal brush border membrane vesicles (BBMV) of piglets, radioactive isotope trace and invitro incubation technique were used to observe the transmembrane transport of Gly-Pro under different condition. The results showed that: (1) Transport of Gly-Pro was inhibited (P < 0.01) by Leu-Gly, Ala-His, Gly-Leu, and His-Leu both in the presence and absence of a Na+ gradient, while not by Glycine, L-proline, L-leucine , L-arginine, L-glutamine and L-methionine; (2) The transport of Gly-Pro was nonlinear but followed by Michaelis –Menten kinetics. The kinetic constants suggested that the apparent Kt (affinity constant) value was 0.64 mmol/L, while Vmax was 7.42 nmol/ (min•mg）membrane protein; (3) The transport of Gly-Pro across the small intestinal BBMV of piglets was due to transport directly into the intravesicular space of BBMV rather than binding to vesicular membrane itself. It was suggested that transport of Gly-Pro in small intestinal BBMV of piglet posses characteristics of carrier-mediated transport.

Development of TaqMan fluorescence quantitative RT-PCR assay for detection of transmissible gastroenteritis virus of swine

BAI Xing-hua;FENG Li;CHEN Jian-fei;SHI Hong-yan;SUN Dong-bo;WU Bo-ping;GAO Xiu-chun

2007, 38(5): 476-481. doi:

Abstract ( 906 )

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The primers and probes were designed and synthesized according to the sequences of transmissible gastroenteritis virus and β-actin, and then reaction parameters were optimized to develop a TaqMan fluorescence quantitative RT-PCR assay. Meanwhile,37 field samples were detected and the results were compared with that of routine RT-PCR and antigen rapid test kit. It was showed that the fluorescence quantitative RT-PCR assay could detect 15.3copies/µL of plasmid DNA and its specificity and reproducibility were very good，while the sensitivity of the routine RT-PCR was 1.53×103copies/µL. The result of field test also showed that its sensitivity was higher than that of the routine RT-PCR and TGEV antigen rapid test kit (chromatographic immunoassay).

Construction and immunological characterization of the Recombinant Adenovirus Expressing the E0 Antigen of Classical Swine Fever Virus Shimen strain

2007, 38(5): 482-487. doi:

Abstract ( 844 )

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Classical Swine Fever Virus （CSFV）E0 gene was amplified from the plasmid pMD18-T-E0 by PCR and cloned into the adenoviral shuttle plasmid pAdTrack-CMV. A two-step transformation protocol was employed for the construction of recombinant adenoviral plasmid pAdEasy-E0. The resultant recombinant DNA was transfected into 293 cells to yield packaged adenovirus. E0 gene was confirmed to be integrated into the genome of recombinant adenovirus by PCR, and the expression of E0 was detected in the 293 cells infected with recombinant adenovirus by Western blot. ELISA results showed that systematical immune responses to CSFV virus could be induced effectively in mice after twice immunizations through intraperitoneal route. The replication-defective recombinant adenovirus expressing (CSFV) E0 was constructed and could elicit favorable immune responses in mice.

Comparisons of Preventive Effects of Maternal Antibody on Immunosuppression Induced by Homologous and Heterologous Reticuloendotheliosis Viruses

SUN Shu-hong;CUI Zhi-zhong;SUN Ai-jun;ZHU Xiu-tong

2007, 38(5): 488-492. doi:

Abstract ( 862 )

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Egg-type Hyline breeders were vaccinated with cell-adapted reticuloendotheliosis virus (REV) strain REV-C99(p30), all chickens became REV-antibody positive 2w after vaccination. And all chicks (10/10) from vaccinated breeders were REV-maternal antibody positive within 7 days after hatch. To compare preventive effects of maternal antibody on immunosuppression induced by homologous and heterologous REV, both REV-maternal antibody positive and negative chicks were inoculated with homologous strain REV-C99(p3) of low passage and heterologous REV field stain HA9901. The results indicated that REV-specific maternal antibody was able to effectively prevent immunosuppression induced by homologous or heterologous REV strains as the hemagglutinatin inhibition antibody titers were compared after vaccination with inactivated vaccines against H5 and H9 avian influenza viruses.

Construction of defined mutations of avian pathogenic Escherichia coli strain and detection of tsh gene in avian isolates

CHEN Xiang;LIU Jing;MAIO Xiao-qing;WANG Xiao-quan;GAO Song;JIAO Xin-an;LIU Xiu-fan

2007, 38(5): 493-499. doi:

Abstract ( 2066 )

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Gentamycin-resistant gene (GM) was ligated with the flanking fragments of tsh gene amplified by PCR, and then the product was transferred into MCS of pUC18 via DNA recombination techniques. The resulting plasmid was named pUC18-tshFRGM. The fragment of interest was digested and subcloned into suicide vector pMEG375 to generate the plasmid pMEG375- tshFRGM. The plasmid was transformed into APEC E037 used as recipient strain containing tsh gene. ΔTsh-deleted E037 mutant strain was screened by homologous recombination. The animal infection results revealed that the pathogenicity of E037 (Δtsh) strain reduced. Successful construction of Δtsh-deleted E037 mutant strain has an important value and significance for elucidating the function of tsh gene and the role of tsh gene playing in the pathogenicity. The 50% lethal dose (LD50) of E037 and E037 (Δtsh) in commercial day-old chickens experimentally inoculated via intratrachea were 105.6CFU and 109.0CFU, respectively. In the chicken challenge model, the mutants were tested to determine the individual role of this system for virulence and persistence in chickens. Among 243 avian E.coli isolates, tsh was present in 68.7% (167 strains). 146 (87.4%), 21 (12.6%) and 0 (0%) of 167 tsh+ isolates were high, intermediate and low in pathogenicity, respectively. Among tsh+ isolates, 89.5%-100% of those high pathogenic isolates of O1, O2 or O78 serogroups hold tsh, while 53.3% in other isolates except O1, O2 and O78 serogroups possessed tsh (P<0.01).

Inhibition of CSFV’s propagation in PK-15 cells by shRNA expression vector

YE Gui-sheng;ZHANG Yan-ming;XU Hao

2007, 38(5): 500-505. doi:

Abstract ( 846 )

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Interference sequences of different positions of CSFV NS3 gene were designed,these sequences were annealed and cloned to interferece plasmid. Recombinant interferencing plasmids were named pGene-NS3-1, pGene-NS3-2, pGene-NS3-3 and pGene-NS3-Negative (negative control) by restriction analyzing and sequencing. These recombinant plasmids transfected PK-15 cells and G418 screened. CSFV were inoculated in cells,which cultured largely.Collected cells were analyzed for Real-time PCR and ELISA. The results of Real-time PCR indicated that mRNAs were silenced by pGene-NS3-1,pGene-NS3-2 and pGene-NS3-3 and inhibition ratio are 63%、46%、49%.The results of ELISA indicated that CSFV’s propagation were inhibited in PK-15 cells by pGene-NS3-1,pGene-NS3-2 and pGene-NS3-3 differently.

Secreting expression of porcine interferon-beta in Pichia pastoris and its anti-FMDV activity

Yao Qing-xia;Qian Ping;Cao Yi;Xu Zuo-fei;He Yan-nan;Si You-hui;Chen Huan-chun

2007, 38(5): 506-512. doi:

Abstract ( 1674 )

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To highly express secreted porcine interferon-beta (rPoIFNβ), the signal peptide was excised from the interferon-beta gene and the mature gene (mPoIFNβ) was cloned into the yeast-Escherichia shuttle vector pPIC 9K to construct secreting recombinant expressing plasmid of pPIC 9K-mPoIFNβ. The pPIC 9K-mPoIFNβ was linearized by SalⅠ and co-transformed with ssDNA into Pichia pastoris cells GS115 with LiCl. The GS115 was defective with histidine (His). The transformants were selected with MD culture plate and identified by PCR. And then, the multicopy recombinant Pichia pastoris strain was selected by G418 resistance. The selected strain could specifically secret 22kD and 26 kD mPoIFNβ proteins which was demonstrated by SDS-PAGE and Western blot. The mPoIFNβ proteins were about 29.4%–30.8% of total secreted proteins and the concentration was about 80 mg/L. The results of physicochemical property of the secreted protein indicated that the recombinant protein was insensitive to acid (pH2), partly sensitive to heat (56℃), and the antiviral activity could be neutralized by anti-PoIFNα serum not by anti-PoIFNγ serum. The antiviral activity of rPoIFNβ was tested by CPE50 method. The results showed that the rPoIFNβ was of high antiviral activity, which was 2.0×106U/mg against VSV in MDBK cells. The rPoIFNβ could suppress FMDV in IBRS-2 cells.

Determination of Residues of Zeranol and Related Compounds in Bovine Muscle Using Gas Chromatography-Mass Spectrometry

ZHANG Wei;WANG Jian-ping;SHEN Jian-zhong;LI Xiao-wei;YANG Jie-fu

2007, 38(5): 513-517. doi:

Abstract ( 1374 )

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A method for determination of zeranol and related compounds（talernol, zearalanone, -zearalenol）in bovine muscle by gas chromatography-mass spectrometry was described. The samples were extracted with methanol, condensed, diluted with water and extracted with ethyl acetate. Then hexane was added to remove the fat, The solution was extracted with KOH and then with a ether, cleaned up with NH2-SPE, After derivatization, the samples were analysed by gas chromatography-mass spectrometry. The linear range was 0.5-10.0ng/g and the limit of detection was 1.0 ng/g. Recoveries were 71.5%-81.4% with CVs of 1.4%-5.0% at spiked levels of 5.0 ng/g.

Localization of Neuropeptide Y Positive Neurons in Small Intestine of Wanxi White Goose

2007, 38(5): 518-520. doi:

Abstract ( 691 )

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