Acta Veterinaria et Zootechnica Sinica

Cloning and Prokaryotic Expression of Porcine Prothymosin α Gene

LIN Hua;GUO Wanzhu;HAN Guoquan;XU Zhiwen;YAN Qigui;WANG Yin

2009, 40(11): 0-1694. doi:

Abstract ( 736 )

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The porcine prothymosin α (ProTα) cDNA fragment was amplified from kidney of porcine by reverse transcriptionpolymerase chain reaction (RTPCR). Sequence analysis showed that the complete open reading frame (ORF) of ProTα gene includes 333 bp and encodes 109 aa. The sequence shared 99.4% homology with the two published ProTα genes. pET32mProTα was constructed by gene rearrangement, then it was transformed into E. coli Rosetta 2 (DE3). SDSPAGE electrophoresis analysis showed that the fusion protein of ProTα molecular mass was about 12 070 of molecular weight. MTT essay confirmed that it can enhance lymphocyte proliferation obviously.

The Effects of Different PRRSV Strains Infection on the Peripheral Blood Immune Cells of Swine

WAN Bo;QIAO Songlin;ZHANG Gaiping;YE Xiaomin;XIAO Zhijun

2009, 40(11): 0-1698. doi:

Abstract ( 667 )

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In this study, 2monthold piglets were inoculated with porcine reproductive and respiratory syndrome virus (PRRSV) variant strain HN0701 and classical strain BJ4，respectively. To investigate the pathogenicity of different PRRSV strains, the clinical symptoms was observed and the peripheral blood immune cells and serum PRRSVspecific antibodies were detected. The results showed that the high fever was induced by PRRSV variant strain. Peripheral blood immune cells in HN0701 infected piglets decreased significantly than that in BJ4 strain, which indicated the severer immune suppression induced by PRRSV variant. The result of PRRSVspecific antibody detection showed that both the PRRSV variant and BJ4 strain can induce antibody quickly.

Study on Primary Culture and Identification of Cecum Epithelial Cells from Chicken Embryo

GU Shaopeng;WANG Minxia;ZHAO Sufen;ZHENG Mingxue

2009, 40(11): 0-1704. doi:

Abstract ( 714 )

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To establish an in vitro model for studying the injury mechanism of E. tenella, in vitro culture techniques of cecum epithelial cells, the host cells of E.tenella in chicken, were studied. Primary cecum epithelial cells from chicken embryo were isolated by the digestion of trypsin, collagenaseⅠ, thermolysin, thermolysin/collagenaseⅠ and dispaseⅠ/collagenaseⅪ, respectively. The best method for separating chicken embryo cecum epithelial cells was screened out by determining the cell viability, proportion of cell aggregates, cell total yield and cell aggregates yield. Thereafter cell purification, culture and identification were performed. The results showed that the best methods for isolating and purifying cecum epithelial cells were digesting the cecum from chicken embryo with thermolysin, then removing single cells by lowspeed centrifuge and fibroblast cells by differential velocity adherent. The isolated and purified cells obtained using the above method stayed in logarithmic growth phase from the 3rd to 5th day and from the 10th to 11th day after planting, respectively, and could survive for more than 14 days. The cultured cells were identified as cecum epithelial cells by morphological observation, alkaline phosphatase staining and scanning electron microscope observation. The proportions for epithelial cells at the 4th, 7th and 11th day were 81.67%, 84.33% and 72.00%, respectively, which implied that the primary cecum epithelial cells with high purity could be obtained from chicken embryo by this method.

Secretoryexpression of Antimicrobial Peptide Abaecin from Bee in Bacillus subtilis

LI Li;QIAO Shiyan;ZHU Faming;AO Changjin

2009, 40(11): 0-1685. doi:

Abstract ( 1051 )

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A recombinant plasmid pGplat containing the coding gene of abaecin was constructed, and it was transformed into B. subtilis 1A751 to express antimicrobial peptide abaecin. The overlapextension PCR method was used to splice the recombinant plasmid pGplat. The constructed recombinant plasmid with multicopy expression cassette was transformed into Bacillus subtilis strain by electroporation, positive clones were screened and inoculated to LB medium for expression under induction of maltose. The sample of culture supernatant of B.subtilis 1A751 was treated by vacuum freezedrying. Western blot assays were applied to investigate the expression of the abaecin gene in B.subtilis 1A751. Bioactivity of abaecin in culture supernatant of B.subtilis 1A751 was tested with plate diffusion method. The results showed that the secretoryexpressed abaecin was identified by Western blot, and there is a strap at 3.9 kD. Bioactivity of abaecin was tested with plate diffusion method, and it had high bacteriostatic activity against Gramnegative bacteria as E. coli and Salmonella pullorum. These results indicated that bioactive abaecin could be expressed in B.subtilis 1A751, which lays a foundation for further study of new efficient peptide antibiotics and feed additives.

Differentiation and Isolation of Porcine Fetal Neural Stem Cells

ZHENG Yuemao;LAN Zhigang;ZHAO Xiaoe;LIU Jun;NIU Yajing

2009, 40(11): 0-1689. doi:

Abstract ( 690 )

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Abstract:This experiment was conducted to study the charicteristics of porcine neural stem cells. Brains were dissected from 30day porcine fetuses, enzymatically dissociated, and grown in the presence of epidermal growth factor, basic fibroblast growth factor etc. Neural stem cells were induced to differentiate in vitro. Expanded neural stem cells and differentiated cells were harvested for analysis using reverse transcriptionpolymerase chain reaction (RTPCR). Porcine neural stem cells could be grown as suspended spheres, and they expressed Nestin, βactin, DCX, Hes1, Oct4, Desmin, CD90, Nanog and Sox2. They were differentiated into astrocyte (GFAP), oligodendrocyte (GalC) and neuron (NF，NSE and MAP2). This study shows that porcine neural stem cells can be cultured over the long term and are able to selfrenew and differentiate into neurons, astrocytes, and oligodendrocytes in vitro.

Comparision of Pathogenicity of Six Different Reticuloendotheliosis Virus Strains to 1dayold SPF Chickens

SUN Shuhong;CUI Zhizhong;WANG Hui

2009, 40(11): 0-1661. doi:

Abstract ( 681 )

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This experiment was conducted to investigate the pathogenicity of different Reticuloendotheliosis virus (REV) strains on 1dayold SPF chickens. 1dayold SPF chickens were inoculated with six REV strains including REVC99, a cloned strain. The pathogenicity of REV from different strains was evaluated by the measurement of growing rate of chicks and the antibody titers to the Newcastle disease virus(NDV) and Avian influenza virus (H9AIV and H5AIV) after vaccination. The results showed that REV challenge at a dose of 3 000 TCID50/bird resulted in the significantly (P<0.05) suppressed HI titers of NDV, AIV(H5, H9) at the 4, 5 and 6week after vaccination compared to control group, whereas there was no significant difference between the 6 REV strains. The results indicated that REV challenge at 1day of age would retard the growth of chicks and suppressed the humoral immunity. The result suggests that REV challenge is one of the important reasons of immune failure.

Molecular Cloning and Bioinformatics Analysis of Swine leukocyte Antigen Class Ⅱ DR Genes in Gansu Hezuo Pigs

MO Sike;FANG Yongxiang;FENG Haiyan;JING Zhizhong

2009, 40(11): 0-1668. doi:

Abstract ( 1117 )

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Class Ⅱ DRA and DRB genes of swine leukocyte antigen (SLA) of Gansu Hezuo pigs were cloned, and their characteristics and polymorphism were analyzed to explore immunological basic parameters for enotransplantation from pigs to humans. SLADRA and SLADRB genes of Hezuo pigs were cloned by RTPCR, sequenced and submited to Genbank (accession numbers: FJ905823 and FJ9058258). Analysis results through BLAST in NCBI and related software in ExPASy suggested that this kind of inbred strain of pig would be the animal model of choice for the porcinehuman xenotransplantation after modification with gene engineering methods.

Effect of Dietary High Fluorine on the Cell Cycle and Apoptosis of Liver in Chickens

GONG Tao;CHEN Tao;BAI Caimin;PENG Xi;CUI Hengmin

2009, 40(11): 0-1680. doi:

Abstract ( 665 )

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300 onedayold Avian broilers were divided into four groups and fed on Control diet(F 23 mg·kg1) and High fluorine diets(F 400 mg·kg1, High fluorine group Ⅰ; F 800 mg·kg1, High fluorine group Ⅱ; F 1 200 mg·kg1, High fluorine group Ⅲ) for 6 weeks, the effect of dietary high fluorine on the cell cycle and apoptosis of liver in chickens were researched by the methods of flow cytometry (FCM) at 14,28,42 days old. The weight of liver was significantly decreased and the relative weight of liver was significantly increased in high fluorine group Ⅱ and Ⅲ when compared with control group. By FCM, the G0/G1 phase was significantly increased, and S phase, G2+M phase and PI(Proliferating index) decreased in diversity in high fluorine groups Ⅱ and Ⅲ at 14 days of age. The G0/G1 phase was significantly decreased, S phase and PI significantly increased, and G2+M phase was not changed in high fluorine group Ⅱ and Ⅲ when compared with those of control group at 28 and 42 days of age. The apoptotic rate was higher in high fluorine group Ⅱ and Ⅲ than that in control group at 14, 28 and 42 days of age. The result showed that fluorine at 800 and 1 200 mg·kg1 in diet could cause enhanced multiplication capacity of hepatocyte and increased apoptotic.

Effect of Sympathetic Nerve on Development of Endometrium in Mice During Early Pregnancy

ZHAO Jiaohui;CHEN Yaoxing;WANG Zixu;DONG Yulan;BAI Yongping;XIE Dian

2009, 40(11): 0-1674. doi:

Abstract ( 645 )

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The objective of the present study was to explore the effect of sympathetic nerve system on development of endometrium in mice during early pregnancy（Embryo day 19）. The adult female kunming mice were treated by intraperitoneal injection of 6OHDA (100 mg·kg1 body weight), which will damage the sympathetic nerve. After 6OHDA treatment, the levels of serum 17βestradiol, the development of uterine gland and the proliferation of endometrium cells during early pregnancy were investigated by means of RIA, histology and immunohistochemistry. The results were demonstrated as follows: (1) the levels of 17βestradiol in 6OHDA treated group were decreased by 63.4%, 35.7% and 25.5% in Embryo day 1 (E1) (P＜0.01), E5 (P＜0.05) and E7 (P＜0.05), respectively, but were significantly increased by 79.3% and 156.7% in E3 (P＜005) and E9 (P＜0.01); (2) the proportion of uterine endometrial glands in 6OHDA treated group was declined by 37.5%, 38.6%, 41.8%, 61.4% and 58.1% in E1, E3, E5, E7 and E9, respectively (P<0.01); (3) the proliferative rate of endometrial cell during early pregnancy was decreased by 27.2%, 30.2%, 40.7%, 27.2% and 27.7% in E1, E3, E5, E7 and E9, respectively (P<0.01). In conclusion, the sympathetic nerves can regulate the secretion of estrogen and reconstruction of endometrium, further then regulate the animals implantation and development in early pregnancy.

Effects of Recombinant Bovine IL2 on Immune Response of FMDV GH Loop Peptide in Mice

GE Lanyun;LI Meng;LI Shuang;LIU Siying;XU Zongxiang;ZHANG Runxiang; GAO Mingchun;MA Bo;WANG Junwei

2009, 40(11): 0-1657. doi:

Abstract ( 672 )

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To study the immune response induced by GH loop peptide of Asia1 footandmouth disease virus (FMDV) with BoIL2 recombinant plasmid as adjuvant, the immune effects in immunized mice were investigated. Recombinant plasmids pcDNA3.1pBoIL2 was constructed and the condition of the expression was identified in vitro. GH peptide was expressed in Rosetta(DE3)pLysS and were successfully purified through GST affinity purification. Five groups of Kunming mice each with 8 were inoculated with different combinations including PBS, pcDNA31pBoIL2 and/or GH peptide, subsequently, immune effects have been evaluated through indirect ELISA assay, the neutralization test, lymphocytes proliferation assay and Quantitive Realtime PCR. The results showed that in comparing with the group immunized with GH+pcDNA3.1, the group communication with pcDNA3.1pBoIL2 induced significantly humoral and cellular immune responses. The results of this study indicate that the coinoculation of GH loop peptide with rBoIL2 (as adjuvant) showed good immune effect.

Cloning of H1foo CDS from Trace Bovine Oocytes and Microinjection of H1foo mRNA into Oocyte

YUN Yan;WU Sujun;SUI Jinqiang;HU Yongce;LEI Anmin

2009, 40(11): 0-1637. doi:

Abstract ( 743 )

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This study was performed for the optimization of gene cloning from trace bovine oocytes and the construction of an eukaryotic expression vector of bovine H1foo gene. The efficiency of gene cloning by improved RTPCR was tested.The results showed that the percentage of successful amplification from single oocyte was above 80% (10/12),and 100% in both 3 and 5 occytes. The complete coding sequences of H1foo was also cloned from 5 bovine oocytes,which was totally identical with previous reports.H1foo CDS was inserted into pMD19T vector and confirmed by restriction enzyme digestion and sequencing,then the eukaryotic expression vector pVenusH1foo was constructed.The recombinant plasmid pVenusH1foo was transfected into Hela cells. H1foo was efficiently expressed in Hela cells identified by Fluorescence microscopy observation，RTPCR and Western Blotting. H1foo was localized accurately on chromosomes of the oocyte and polar body after H1foo mRNA transcripted in vitro was microinjected into bovine oocytes. The bovine H1foo gene eukaryotic expression vector was successfully constructed,and our study will lay a good foundation for further studying the functions of H1foo, especially its role on oocyte maturation and early embryo development.

Polymorphism of TLR6 Gene and Its Relationship with Somatic Cell Score in Holstein Cows

CHU Mingxing;LI Chunmiao;SHI Wanhai;AN Yongfu;CHEN Hongquan;DI Ran;FANG Li

2009, 40(11): 0-1629. doi:

Abstract ( 1181 )

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The objective of the present study was to elucidate the relationship between Tolllike receptor 6（TLR6）gene and mastitis resistance of Holstein cows. Polymorphism of all exons of TLR6 gene was detected in 208 Holstein cows by PCRSSCP and PCRRFLP. The relationship between polymorphisms of TLR6 gene and somatic cell score (SCS) in Holstein cows was analyzed by least squares linear model. Six polymorphic sites T853A, G855A, G1793A, C1859A, G1934A and A1980G were found; and T853A mutation resulted in an amino acid change of valine→glutamic, G855A mutation caused an amino acid change of aspartic acid→asparagine, A1980G mutation caused an amino acid change of isoleucine→valine. For T853A and G855A polymorphic sites, three genotypes (AA, AB and BB) were found in 208 Holstein cows by SSCP, and the frequency of AA, AB and BB genotypes was 0.308, 0.490 and 0.202, respectively. Holstein cows were in HardyWeinberg equilibrium at the sites. Least squares mean of SCS for BB was significantly lower than that for AB (P<0.01) or AA (P<0.01), least squares mean of SCS for AB was significantly lower than that for AA (P<0.01). For G1793A polymorphic site, two genotypes (CC and CD) were found by digesting the PCR products with restriction endonuclease BsiYⅠ, Chisquare test showed that Holstein cows were not in HardyWeinberg equilibrium. Least squares mean of SCS for CC was not significantly lower than that for CD (P>0.05). For C1859A polymorphic site, two genotypes (EE and EF) were found by digesting the PCR products with restriction endonuclease BstXⅠ, and the frequency of EE and EF genotypes was 0178 and 0822, respectively. Holstein cows were not in HardyWeinberg equilibrium at the site. Least squares mean of SCS for EE was not significantly lower than that for EF (P>0.05). For G1934A polymorphic site, two genotypes (GG and GH) were found by digesting the PCR products with restriction endonuclease BsiYⅠ, and the frequency of GG and GH genotypes was 0.356 and 0.644, respectively. Holstein cows were not in HardyWeinberg equilibrium at the site. Least squares mean of SCS for GG was not significantly lower than that for GH (P>0.05). For A1980G polymorphic site, three genotypes (II, IJ and JJ) were found by digesting the PCR products with restriction endonuclease BclⅠ, and the frequency of II, IJ and JJ genotypes was 0.260, 0.567 and 0.173, respectively. Holstein cows were in HardyWeinberg equilibrium. Least squares mean of SCS for JJ was significantly lower than that for IJ (P<0.01) or II (P<0.01), least squares mean of SCS for IJ was significantly lower than that for II (P<0.01). BB and JJ were the most favorable genotypes, AA and II were the most unfavorable genotypes for mastitis resistance. The results preliminarily indicated that both B and J alleles of TLR6 gene are two potential DNA markers for improving mastitis resistance in dairy cattle.

Effect of High Level of Zinc Oxide on Tight Junction Protein Expression in Intestinal Epithelial Cells and Intestinal Mucosal Barrier in Early Weaning Piglets

HU Caihong;QIAN Zhongcang;LIU Haiping;XU Yong

2009, 40(11): 0-1644. doi:

Abstract ( 779 )

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To study the effect of high level of zinc oxide on intestinal mucosal barrier and its mechanisms in early weaning piglets, 54 piglets, weaned at 21 d of age, were allocated into three treatments on the basis of similar BW, ancestry, and gender. Each treatment had three replications of six pigs (half males and half females). The experimental treatments diets were basal diet and basal diet +3 000 mg·kg1 Zn (ZnO), another treatment piglets were receiving sow milk with mother (control). Plasma Dlactate and endotoxin level, DAO activity, bacterial translocation in mesenteric lymph nodes and colon E.coli were measured to evaluate the intestinal permeability at 28, 35 d of age, and the level of Occludin and ZO1 expression were also detected. The results showed that plasma Dlactate and endotoxin level and DAO activity were significantly increased in piglets of basal diet compared to the control group (P<0.05) at 28 d. The three parameters remained high at 35 d. There were no significant differences between the high zinc and the control (P>0.05). At 28 and 35 d, bacterial translocation in mesenteric lymph nodes of piglets with high zinc was lower than that of piglets with basal diet(P>0.05). Colon E.coli of piglets with basal diet was increased compared to the control group (P<0.05) at 28 d, but there were no significant difference between the high zinc and the basal diet (P>0.05).The parameters did not differ among three groups at 35 d (P>0.05). Compared with the control, Occludin and ZO1 mRNA and ZO1 expression was significantly decreased in the basal diet group (P<0.05) and the high zinc (P>0.05), but the parameters of piglets with high zinc was greater than that of basal diet (P<0.05). It is suggested that early weaned piglets was in stresses, increasing intestinal permeability and decreasing the expression of tight junction protein Occludin and ZO1, and these damage may be relaxed by diet supplementation with high level of zinc oxide, through inhibiting bacterial adhesion in the intestinal mucosa and preventing the increase in epithelial cells permeability.

Cloning, Sequence Analysis and Expression of Two Transcript Variants of Wild Boar CAPN10 Gene

YANG Xiuqin;GUAN Qingzhi;YU Hao;GUO Lijuan;LIU Hui;LIU Di;

2009, 40(11): 0-1614. doi:

Abstract ( 684 )

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In order to further study the roles of CAPN10 in muscle growth and meat quality, the study was designed to clone the cDNA sequence of Wild boar CAPN10 with RTPCR and RACE methods, and to analyze its expression profile with semiquantitative RTPCR and competitive RTPCR. Two transcript variants of CAPN10 were obtained, and the sequence analysis showed that the two polypetides were identical in the former 604 amino acids, and had the characteristic domain of calpain family, CALPAIN_CAT structure, and the catalytic triad residues in the same position. Semiquantitative RTPCR analysis indicated that CAPN10 mRNA of Wild boar was expressed in 12 tissues examined and the mRNA level in muscles from Yorkshire pigs at 6, 9, 12monthold was much higher than that from crossbred pigs (P<0.05) at the same development stages. Competitive RTPCR results indicated that the two variants were all expressed in 9 tissues of Wild boar examined. It is suggested that there is association between the relative amounts of CAPN10 and meat tenderness.

Chromosomal Distribution of Endogenous Jaagsiekte Sheep Retrovirus in the Mongolian Sheep Genome

CHAI Ju;QI Jingwei;LIU Shuying;ZHANG Wenguang ;LAI Shuangying;LI Jinquan

2009, 40(11): 0-1620. doi:

Abstract ( 666 )

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This study was aimed to explore the distribution of chromosomes of the endogenous sheep pulmonary adenomatosis virus gene in Mongolian sheep. A pair of primers was designed according to the sequence of the enJSRV in the GenBank(DQ838493). The partial sequence of the gag gene was cloned and amplified by PCR, which was further marked by Dig to locate the distribution of the enJSRV in the Mongolian sheep chromosomes with the FISH(Fluorescence in situ hybridization). The result showed that the distribution of the enJSRV gag gene in each of the 6 chromosomes of Mongolian sheep during the metakinesis at 1q45, 1p23, 2q41, 3q23, 6q13, 9q22 and 14q25, and stronger fluorescence signal appeared on the 6th and 9th chromosomes. The results suggested that, on the 6th and 9th chromosomes, enJSRV genes were multiple copied at the two sites, and there were low copy number at 1q45, 1p23, 2q41, 3q23, and 14q25, which related to the endogenous evolution of the sheep pulmonary adenomatosis virus.

Genomewide Identification of Quantitative Trait Loci for Pork Firmness in a Large Scale White Duroc × Chinese Erhualian Resource Population

DUAN Yanyu;ZHOU Lihua;MA Junwu;GUO Beili;HUANG Weibing;QIAO Ruimin;HAN Pengfei;YAO Fei;HUANG Lusheng

2009, 40(11): 0-1587. doi:

Abstract ( 687 )

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To identify quantitative trait loci（QTLs）for firmness of M. Longissimus dorsi (LD) and M. Semimembranous (SM) in pigs, the related traits were measured in 490 F2 animals from a White Duroc × Erhualian resource population. A whole genome scan was performed with 194 microsatellites on 19 porcine chromosomes, and the QTL analysis was performed based on a leastsquare regression method. A total of 9 QTLs were identified，including 4 QTLs for firmness of LD on SSC1, SSC2, SSC7 and SSC10, those on SSC1 and SSC7 reached the 1% genomewide significant level. Five QTLs for firmness of SM were detected on SSC1, SSC7, SSC10, SSC12 and SSC14, the QTLs on SSC7 and SSC14 showed 5% and 1% genomewide significant effects respectively.

Yeast Twohybrid Study of Pig Calsarcin-3 Interaction Proteins

CHEN Dalei;ZHU Wenjuan;YANG Shulin;YANG Lianyu;CUI Wentao;CHU Mingxing;MA Yuehui;LI Kui

2009, 40(11): 0-1599. doi:

Abstract ( 705 )

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This work was conducted to study the regulation role of Calsarcin3 (CS3) and its interaction proteins on Calciumion signal transduction pathways and understand the molecular mechanisms of skeletal muscle fiber differentiation and development. The mRNA was extracted from the skeletal muscle of the adult Landrace and the purified dscDNA was obtained.The pGBKT7CS3 bait vector without toxicity and autoactivation was constructed with the Calsarcin3 gene of Landrace pig. dscDNA was amplified by long distance PCR. Finally, the Calsarcin3 interaction proteins were screened through hybridization of bait vector with dscDNA prey library. Through the alignment of interaction genes in GenBank, the function of Calsarcin3 was analyzed. In this study, five proteins interacting with CS3 were identified. These results indicated that the β capping protein muscle Zline(CAPZB) may regulate the growth of the actin filament by capping the barbed end of growing actin filaments with Calsarcin3.Actin,alpha 1(ACTA1) as a contractile protein may play a significant role in regulating the skeletal muscle activity with Calsarcin3.Titin(TTN) may be involed in the structure of the Zdisk by binding with Calsarcin3.

Construction of Eukaryotic Expression Vector with a Sitedirected Mutation of Porcine MSTN Propeptide Gene and Its Expression in C2C12 Cells

ZHANG Xiaowei;CUI Wentao;WU Xiaoxiong;YANG Shulin;MU Yulian;TANG Zhonglin;LI Kui

2009, 40(11): 0-1593. doi:

Abstract ( 784 )

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This research intended to construct eukaryotic expression vector with a sitedirected mutation of porcine MSTN propeptide gene, and verify its expression efficacy in C2C12 cells. By means of PCR amplification with DNA extracted from blood samples of Tongcheng pigs as templates, MSTN propeptide gene containing the first intron was cloned and then suquenced. It was then subcloned into vector pEGFPN1 without EGFP fragment and sitedirected mutagenesis was perfomed which successfully converted Asp into Ala at the 76th amino acid site in propeptide sequence.After being confirmed by R.E digestion and sequencing, the recombinant plasmid pEGFP()N1ProMstnD76A was transferred into C2C12 cells.After 48 h, the expression of target gene was detected at transcriptional level using RTPCR and Realtime PCR. The results showed that the recombinant expression plasmid pEGFP()N1ProMstnD76A was constructed successfully. After being transiently transfected into C2C12 cells, the high expression level of target mRNA was detected and the primary mRNA transcript was found spliced correctly. The present research will lay a good foundation for further studying the functions of porcine MSTN propeptide gene in vivo and provide useful molecular materials for preparation of transgenic pigs.

Cloning and Sequence Analysis of the Promoter Region of Porcine Lung Surfactant Protein A Gene

CHEN Shuming;JIN Dapeng;WANG Lixian;ZHANG Longchao;YAN Hua;CHENG Duxue

2009, 40(11): 0-1608. doi:

Abstract ( 713 )

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In order to explore the mechanism of porcine SPA gene transcription regulation by studying the promoter region of porcine SPA gene,the upstream sequence of Large White porcine SPA gene was attained by Genome Walking PCR,sequencing and sequences alignment analysis.The DNA sequence of 1 033 bp (DQ985806) was amplified.The ratio of G and C in the sequence was about 55%.The sequence analysis showed that there were a TATAbox at －33 bp and GCbox, CAATbox, GTbox and transcriptional initiator. Meanwhile, some transcriptional factor binding sites including Sp1,TF,NF κB,and so on were detected. In addition,a special repetitive sequence of 5′CATCACTGT3′ was also detected. The results showed that the cloned sequence (1 033 bp) may be considered as the promoter region of Large White porcine SP-A gene.

The Influence of Lairage Conditions for Finishing Pigs on Mental Performance, Blood index and Meat Quality

CHAI Jin;PENG Jian;XIONG Qi;ZHANG Changxin;MIAO Wen;LI Fenge;ZHENG Rong;JIANG Siwen

2009, 40(11): 0-1650. doi:

Abstract ( 1160 )

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A total of 60 crossbred (Large White × Landrace) pigs with halothane genotype NN (castrated males and females) were allotted into three treatments：3 h lairage with toys, 3 h lairage and 0 h lairage in a randomized complete block design and used to evaluate the influence of lairage conditions on mental performance, blood index and meat quality for finishing pigs at slaughter. Mental performance of the pigs was recorded by score standards during lairage. Blood samples were taken at exsanguinations to measure blood temperature, plasma cortisol, ACTH, glucose, lactate, plasma enzymes and hematological index. Postmortem meat quality measurements including muscle colour value (MCV)，electrical conductivity (EC), pH at 45 min and 24 h from Longissimus thoracis and Semimembranosus and drip loss were taken respectively. The result showed：① 3 h lairage group with toys demonstrated significantly higher mental performance than 3 h lairage group at 3 sampling times. All the pigs showed increasedly calm with the lairage time running off; ② 0 h lairage increased cortisol, ACTH and lactate (P＜0.05), while decreased lactate dehydrogenase(LDH), creatine kinase(CK) (P＜0.05). All biochemical index in serum were not influenced by toys during lairage (P＜0.05);③ Muscle colour value, electrical conductivity, pH at 45 min and 24 h from Longissimus thoracis（LM）and Semimembranosus（SM） and drip loss were not affected by any treatment (P＜0.05); ④ 3 h lairage with toys and 3 h lairage groups exhibited high red blood cell (RBC)， hemoglobin (HGB) when compared to 0 h lairage. Whereas, 3 h lairage with toys displayed higher white blood cell (WBC) levels than 0 h lairage. In addition, Lymphocyte (WSCC) was significantly higher in 3 h lairage compared with 0 h lairage. There were significant difference for hemocyte index between 3 h lairage with joys and 3 h lairage groups. We could conclude from the results that in local commercial conditions, the most adequate preslaughter lairage time was 3 hours for short travel. Pigs resting showed an increase in relieving stress and a recovery in immune competence. Hence, holding pigs in lairage with toys for a few hours after arrival at the abattoir may at least improve mental state.

Expression of Growth/Differentiation Factor 11 Gene in E. coli and Its Antibody Preparation

SUN Linlin;SUN Yi;ZANG Li;JIANG Yunliang

2009, 40(11): 0-1581. doi:

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The objective of the present study is to obtain specific antibody against porcine GDF11 protein to further reveal its function in pigs. The coding region of porcine GDF11 was cloned, expression vector was constructed and transformed, inducible expression and purification were carried out and antibody was isolated after GDF11 injected into mice. A GDF11 coding region sequence was amplified from total RNA isolated from porcine embryo kidney tissues by RTPCR and inserted into pMD18T. After being digested with BamHⅠand EcoRⅠ, the GDF11 fragment was inserted into the prokaryotic expression vector pGEX4T2 and sequenced, and the recombinant plasmid pGEX4T2GDF11 was obtained. The competent E.coli cell strain BL21(DE3) were transformed by the recombinant plasmid pGEX4T2GDF11 and induced by IPTG to produce fusion protein GSTGDF11 of 65 ku. The expressed fusion protein GSTGDF11 was digested with thrombin to release GDF11 protein of 42 ku which was subsequently used to immunize mouse. The antiGDF11 polyclonal antibody with high sensitivity (125 600) and specificity was obtained.

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