Acta Veterinaria et Zootechnica Sinica

Cloning of Porcine Suv39h2 cDNA and Its Expression in E. coli

LIU Li-na;PENG Jian;ZHENG Rong;LIU Min;JIANG Si-wen

2009, 40(5): 611-616. doi:

Abstract ( 824 )

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Based on the mRNA sequence of human Suv39h2 gene, a porcine fragment of its cDNA sequence was obtained using in silico cloning method. By RT-PCR method, a porcine Suv39h2 cDNA fragment of 1 291 bp was amplified from muscle tissue and inserted into pGEX-KG plasmid expressing glutathione Stransferase (GST) fusion protein. After confirmation by the sequencing and restriction enzyme analysis, the recombinant plasimids were transformed into E. coli BL21 competent cells. The optimal IPTG induction conditions were determined as follows: induction time was 4 h and IPTG concentration was 0.7 mmol·L^-1. The fusion protein was insoluble. Using Western blotting, we found a band with molecular weight of 66 kD, which indicated that porcine Suv39h2 was expressed in E. coli. This work laid a foundation for further work on functional study.

Relationship between Polymorphism of 5′Regulatory Region of Porcine MSTN and Growth Performance

WU Jun-hong;WU Yan-qun;ZHAO Xiao-feng;WU Jiu-sheng;XU Ning-ying

2009, 40(5): 617-621. doi:

Abstract ( 731 )

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The polymorphism of the 5′regulatory region of Myostatin gene was detected using PCR-RFLP method in 45 Jinpi F2 pigs.The results showed that there were 3 genotypes (AA, TT and AT) and their frequencies were 0.244, 0.311 and 0.444, respectively. Growth, carcass traits and Longissimus dorsi muscles analyzed using immunohistochemical were recorded. The results showed that the DraI polymorphism of MSTN gene had significant effect(P<0.05) on the muscle fiber diameter, the percentage of MHC type I fiber, the backfat thickness (shoulder)，the average daily gain from birth to 4 months old and the eye muscle lightness.

Association of the Polymorphism of Porcine SGCB Gene with Carcass Traits

FU Ya-yuan;LEI Ming-gang;PAN Gang;CHENG Lei;HE Jun-xian;LI Feng-e;ZUO Bo;XIONG Yuan-zhu

2009, 40(5): 622-626. doi:

Abstract ( 928 )

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The sequence of extron 4 of the porcine SGCB gene was obtained from Large White, Landrace and Meishan pigs. Sequence comparison revealed a A/G mutation which was detected by ARMS-PCR. The A and G alleles were balanced in the Large White, Landrace, Meishan pigs and Large White×Meishan F₂ population. Association analysis between the polymorphism and carcass traits was performed in Large White×Meishan F₂ population with 266 pigs. The genotype AA had significant effect on average lean meat percentage(P<0.01), the genotype GG had significant effect on loin-muscle height(P<0.01) in Large White×Meishan F₂population.

Polymorphism of Exon 1 in Hormone-sensitive Lipase Gene and Its Association with Carcass Traits in Two Guizhou Native Pig Breeds

WANG An-na;;RAN Xue-qin;WANG Jia-fu;XIA Xian-lin

2009, 40(5): 627-632. doi:

Abstract ( 1287 )

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The aim of the present study was to investigate the association of the polymorphism of HSL gene with carcass traits in native pig breeds in Guizhou province. Two native pig breeds in Guizhou province, Nuogu pig (n=30) and Luobo pig (n=50), were chosen with the crossbreed of Large White×Landrace (n=30) as control. The polymorphism of HSL exon 1 was detected by restriction fragment length polymorphism (RFLP) method. Three genotypes, AA, AB and BB, were detected from the HSL gene in three pig populations. It showed that A allele was dominant in the two native pig breeds with 60% in Nuogu pig, 89.5% in Luobo pig and the B allele was detected mainly from the European crossbreed with 91.7%. Compared with B allele, a single nucleotide mutation (G/A) was confirmed in A allele at the 442 bp of the coding region in exon 1 of HSL gene which resulted in the amino acid change from valine to isoleucine. Moreover, the lean percentage, backfat thickness and loin eye area of individuals with BB genotype were significant superior than that with AA genotype (P<0.01). These results indicated that the B allele committed to improve the lean percentage and loin eye area, and decreased the backfat thickness. It suggested that HSL gene could be a molecular marker for the selection of lean percentage in the native pig breeds.

Production of ω-3 Fatty Acid Desaturase Gene (sFat-1) Transgenic Embryos by Somatic Cell Nuclear Transfer in Pig

FENG Chong;;ZHOU Yan-rong;LONG Chuan;LIU Xiao;CHEN Hong-xing;PAN Deng-ke;YANG Bo-hui

2009, 40(5): 633-638. doi:

Abstract ( 768 )

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In this study, fetal fibroblast cells of Large White pig were transfected with sFat-1 gene from round-worm C. Briggsae by lipofectamine mediated transfection. After selected by 600 μg·mL^-1 G418 for 10 days and analyzed by PCR, RT-PCR, 11 positive transgenic cell clones were collected. Reconstructed embryos were combined by porcine oocytes which were matured for 42 h and transgenic cells. After culture, the cleavage percentage of embryos (76.6%±4.1% vs. 81.6%±3.1%) and blastula (10%±1.97% vs. 9.7%±1.4%) have no significant difference between the transgenic cloning embryos and nontransgenic cloning embryos (P>0.05). In assistant activation, CHX was used and make a higher (20.6%±0.89% vs. 10%±1.97%, P<0.05) percentage of blastula than the electrical activation, but the cleavage percentage was insignificant between them (72.4%±4.96% vs. 76.6%±4.1%, P>0.05). As a result, fetal fibroblast sFat-1 transgenic cell line of Large White pig can be obtained by lipofectamine mediate transfection, and the cell line used as nuclear donors make no significant difference in the development of the transgenic cloning embryos and non-transgenic cloning embryos; Then using CHX in assistant activation can significantly raise the percentage of blastula.

Analysis of SNPs in Promoter Region of Growth Hormone（GH）Gene in Minipigs

ZHENG Mao-en;PAN Deng-ke;FENG Shu-tang;LIU Xiao;YE Shao-hui;PU Ya-bin;HE Xiao-hong;ZHAO Qian-jun;GUAN Wei-jun;MA Yue-hui;

2009, 40(5): 639-644. doi:

Abstract ( 828 )

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The single nucleotide polymorphism (SNP) of growth hormone gene was investigated in six different pig breeds，including four minipig breeds which were Wuzhishan pigs，Bama pigs，Xiang pigs and Tibet pigs，and other two breeds which were Dalan pigs and Landrace pigs. Three pairs of primers for promoter region of GH gene were designed on the basis of database of pig genomic sequence and the SNPs were detected by PCR-SSCP method. The results indicated that three mutations were in the 5′flanking region. The analysis results showed that the frequencies of allele A and D in four minipig breeds were higher than that in other breeds at locus of 5′flanking region (P<0.05). According to the above results， difference of body size may have relationship with these SNPs of 5′flanking region and amino acid mutation of the leading peptide of GH in these pig breeds.

Effect of Different Treatments with Bovine Cumulus Cells as Donor Cells on Cloning Embryos

YAO Ya-xin;LI Xiang-chen;ZHANG Yong;GUAN Wei-jun;MA Yue-hui

2009, 40(5): 645-651. doi:

Abstract ( 1377 )

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In this study, the cumulus cells were dealed with Trichostatin A(TSA), Roscovitine(ROS), serum starvation and contact inhibition, we detect the change of level of acetylation, the cell cycle, and the development of cloning embryos after the treatments, which provided academic foundation for raising efficiency of cloning. Using the above methods, the cell morphology change and the motility rate were observed, then the levels of acetylation and cell cycle were detected through the indirect immunofluorescence and the flow cytometry respectively, and the motility rate were more than 94% all. TSA obviously enhanced the level of acetylation (P<0.05); ROS also made a high level of acetylation, but lower than the TSA treatment (P<0.05), the serum starvation treatment was lower than the control. The cumulus cells after treatments had good morphology, TSA made the cell cycle stop at the G0/G1 and the highest cleavage and blastula rate (P<0.01, （85.2±3.4）% vs （68.6±6.7）%;（30.2±5.7）% vs （10.4±8.3）%). These results indicated that the cumulus cells treated with TSA not only had a high level of acetylation and better morphology change compared with the other groups, but also had the highest percent of blastula, so it was more suitable method to deal with the donor cells.

Endocrine Regulation of Follicular Development in the Lay-incubationCycle of Magang Geese

LIU Rong-zhen;HUANG Yun-mao;LI Wan-li;TIAN Yun-bo;SHI Zhen-dan

2009, 40(5): 652-657. doi:

Abstract ( 710 )

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This study was conducted to investigate the endocrine regulatory mechanisms of follicular development in the lay-incubation cycle of Magang geese. Experiment 1: investigated the changes in palsma concentrations of PRL, LH, P4, and INB during the cycle, and the follicular development at incubation (d0), end of incubation (d10), onset of lay (d25), peak of lay (d40), end of lay (d55) and incubation (d70) in the layincubation cycle. Experiment 2, after being deprived of laying nest to terminate incubation behaviour on day 1 (d1), Magang geese were immunized intramuscularly on days 1, 23 and 45, with 1 mL of immunogen containing 1, 0.8, and 0.5 mg, respectively, of recombinant chicken PRL protein. The photoperiod under which the geese were kept was increased from 11L:13D to 16L:8D on day 33. In expreiment 1, with the termination of incubation, PRL concentration in plasma decreased, whereas the concentrations of LH, P4 and INB increased. PRL concentrations fell to lowest levels prior to the onset of lay and rose to high levels during laying to the peak when incubation initiated. LH concentration in plasma exhibited a bi-phasic pattern in the whole cycle. The patterns of P4 and INB concentrations were opposite to that of PRL, reaching to high levels at peak lay and to the lowest at incubation. Laying resumed on day 24 in the layincubation cycle, and approximately 10 follicles were recruited into hierarchical development before onset of lay, among which 8 developed to ovulation and were laid as eggs. Ninety percent of the geese exhibited incubation behabviour after laying one clutch of approximately eight eggs in approximately 30 days. Results of experiment 2 showed that immunization against PRL increased the clutch size by one egg (8.0 vs 7.0）, although initially retarded the rise of laying rate (P<0.05). Development of incubation behaviour was also delayed by one week in the PRL immunized geese, which still reached to 100% as in the control geese. Results of the two experiments indicated that the alternative secretions of PRL and LH coordinated the lay incubation cycle in Magang geese. As the results of follicular development, secretions of INB and P4, the latter through PRL secretion, determine the quota of follicular development and clutch egg size. PRL triggers the incubation behaviour and atresia of eggs at bottom on developing hierarchy, however, PRL may stimulate follicular development and egg laying around the onset of lay.

Analysis on the Polymorphism of Sequences of Pit-1 Gene in Goose

CHENG Jin-hua;ZHAO Wen-ming;QIAO Na;WANG Xue-bin;JIANG Qing-lin;ZHANG Kang-ning;CHEN Guo-hong

2009, 40(5): 658-663. doi:

Abstract ( 724 )

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Genetic polymorphism of goose Pit-1 gene fragment was analyzed in 6 goose populations. The primer was designed according to chicken Pit-1 mRNA and genomic sequence. Two insertion/deletion mutations were found in the sequence of Pit-1 gene, and 3 genotypes AA, AB and BB were detected in 6 goose populations. Compared with AA genotype, there were 3 and 13 bp insertions in BB genotype after position 132 and 145 bp，respectively. All the populations were in Hardy-Weinberg equilibrium at this polymorphic site (P>0.05). The genotype distribution in Landosie was significantly different with that in other goose populations (P<0.01). The genotype distribution in Zi goose and Shitou goose were significantly different with that in other populations（P<0.05）. Allele A was the dominant allele in Shitou and Landoise goose populations, while allele B was the dominant allele in Wanxi White goose, Siji goose and Zhedong White goose populations.

Analysis on the Association of Polymorphism of H-FABP Gene with Carcass Traits of Peking Duck

ZHU Xiang-yun;;YANG Xiao-gang;;WANG Jie;LIU Xiao-lin;HOU Shui-sheng;HUANG Wei;YU Jun-ying;ZHAO Ling

2009, 40(5): 664-669. doi:

Abstract ( 751 )

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In this paper, 300 Peking ducks were used to analyze H-FABP gene polymorphism by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP). The results showed that the polymorphism of the 3rd intron was caused by C/G mutation at 156 bp. In three populations(males and females), only the Z4 population resulted in genotypes which showed a significant difference in carcass weight, abdominal fat weight and eviscerated weight (P<0.05),the Z4(male) population resulted in genotypes which showed a significant difference in breast weight, leg weight,and skeleton weight. It implied that H-FABP could have a major effect on carcass performance or could be linked to genes that significantly affect carcass performance in Z4 population of Peking duck.

Recombination of CBH Ⅱ Gene with Non-antibiotic Selected Vector and Expression and Activity Detection in Lactobacillus

ZHAO Ying;SUN Zhe;LIU Yan;HU Hou-ying;CHEN Da-fang;LOU Yu-jie;ZHANG Hong-fu

2009, 40(5): 670-675. doi:

Abstract ( 1253 )

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Recombinant plasmids pMD18-T-CBHⅡ and pW425t, prokaryotic expression shuttle vector between E.coli and Lactobacillus, were digested with SmaI and SphI enzymes respectively. The purified CBHⅡ gene was subcloned into the expression vector pW425t. Thus,the recombinant pW425t-CBHⅡ was constructed, and then was transformed into the competence thyA genemutant Lactobacillus DOMLaS107. Treated lysates of bacterium were loaded directly on SDS-PAGE, and approximately 49.6 kD protein was observed, and recombinant Lactobacillus can produced clear hydrolysis halos on the Congo-Red-CMC plate.

Effect of Immune Challenge on Gut Development and GLP-2 Secretion of Piglets

CHE Lian-qiang;ZHANG Ke-ying;DING Xue-mei;CHEN Dai-wen

2009, 40(5): 676-682. doi:

Abstract ( 774 )

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Two trials were conducted to study Glucagon-like peptide 2 secretion in postweaning piglets and the effect of immune challenge on gut development and GLP-2 level. The first trial indicated that the GLP-2 level was reduced by the weaning anorexia, and then tended to recover with the increased feed intake. The second trial showed that the immune system was activated by the immune challenge, the feed intake (P<0.01), GLP-2 level (P<0.05), the villus height/crypt depth (P<0.05) were also decreased, while the mucosa lactase and sucrase activity, goblet cells tended to be decreased. The results indicate that immune challenge resulted in the anorexia and immune system activation, which trigger the decreased GLP-2 that may influence gut development.

Effects of Dietary Energy Level and Source on Blood Metabolites, Hormone Secretion and Follicular Fluid Composition in Gilts

ZHOU Dong-sheng;WU De;ZHUO Yong;WANG Yan-zhong;TAN Xian-yi;ZHOU Ping

2009, 40(5): 683-690. doi:

Abstract ( 757 )

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The objective of the study was to investigate the effects of dietary energy levels and sources on the blood metabolites, hormone secretion and the follicular fluid composition in gilt. 54 gilts with initial body weight of (59±4.2)kg were randomly allotted into six treatments. Treatments were low(L), medium(M), and high(H) feeding energy levels, which were 87.5%, 100% and 112.5% of recommendatory energy requirements by NRC, respectively, and dietary energy sources (starch, S or fat, F). Blood samples and follicular fluids were collected on d18 and d19 of 2nd estrus cycle. The results showed that circulating triglyeride, total cholesterol concentrations were higher in the fat group than that in the starch group (P<0.05), but blood glucose concentrations were similar between the fat and starch groups (P>0.05), dietary energy level had no significant effect on blood metabolites concentration (P>0.05). Gilts fed high energy level diet had a higher area under curve of the plasma insulin (Ins AUC), concentration of insulin growth factor-Ⅰ(IGF-I) and leptin than gilts fed the lower energy diet(P<0.05), but no significant difference between the fat and starch groups (P>0.05). Luteinizing hormone (LH) pulses were higher in gilts fed high energy level diet than that in gilts fed low energy level diet (P＜0.05), blood estradiol (E2) concentration was higher in fat group than that in starch group（P<0.05). Sizes of large follicles (diameter ≥3 mm) and concentrations of IGF-I and E₂ in follicular fluid were increasing significantly as the increasing energy level (P<0.05), but the numbers of large follicles and follicular fluid composition were not affected by dietary energy sources (P>0.05). The results indicated that gilts fed the high energy diet elevated blood metabolic hormones concentration IGF-I and LH secretion, and increased follicular fluid IGF-I, E₂ concentration and follicular sizes. However, gilts fed the dietary fat had a higher blood cholesterol and E₂ concentration.

Eukaryotic Expression and Subcellular Localization Preliminary Analysis of Porcine Epidemic Diarrhea Virus Nucleocapsid Protein

LV Mao-jie;FENG Li;SHI Hong-yan;CHEN Jian-fei;SUN Dong-bo;SHEN Shi-chuan;CUI Xiao-chen;WANG Cheng-bao;FAN Xiu-ping;

2009, 40(5): 691-696. doi:

Abstract ( 1177 )

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Using positive plasmid pMDT-18-N which contained the gene encoding nucleocapsid protein of porcine epidemic diarrhea virus strain CV777 as template, the N gene was amplified by the upper/lower primers each with the internal sites of Kpn Ⅰ and Xho Ⅰ, respectively. By digestion with restriction enzymes Kpn Ⅰ and Xho Ⅰ, the PCR product was then subcloned into eukaryotic expression vector pcDNA3.1(+) that digested with the same enzymes. After restriction enzyme digestion and DNA sequencing identification, the constructed recombinant plasmid was named pcDNA3.1(+)-N, deletion and insertion were not found in its sequence. The pcDNA3.1(+)-N was transiently transfected into Vero E6 cells, and the expression of N gene was detected by Western blot and indirect immunofluorescence assay with mouse antiserum against N protein. N protein subcellular localization were analyzed by confocal microscopy. Results verified that N gene could be expressed successfully in Vero E6 cells, and the N protein localizes both in the cytoplasm and the nucleus.

Analysis of the Antigenicity of Foot-and-Mouth Disease Virus Persistent Infection Yellow Cattle Isolates and Their Serum Neutralization Characteristic

SHEN Xiao-yan;CONG Guo-zheng;LIU Xiang-tao;CHANG Hui-yun;LIN Tong;SHAO Jun-jun;SHANG You-jun;XIE Qing-ge

2009, 40(5): 697-705. doi:

Abstract ( 733 )

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The yellow cattle were artificially infected with FMDV O/Akesu/58 strain to develop the carrier state. To reveal antigenic variation of FMDV persistent infection isolates, the isolates and serum were obtained monthly to study the antigenicity of the isolates and the neutralization characteristic of serum and antibody level. By RT-PCR, the VP1 gene of the isolates were amplified and the variation of antigenic gene were analysized during FMDV persistent infection. The neutralization activity of the isolates and themselves serum were tested by microserum virus neutralization assay and the antibody titre to structural protein were detected with liquid-phase blocking ELISA. At the same time, the relationship of them was analyzed. The results showed that the homology of VP1 nucleotide sequence of all the isolates was above 98%, no base deletion or insertion was found. Compared with O/Akesu/58 strain, the homology of VP1 nucleotides sequence of the isolates was 85%, amino acid sequences only being 90%.The VP1 gene of the isolates had several nucleotides mutations, including 16 coincident nucleotides mutations compared with their parental strain, only two point mutation leading to amino acid change(I56→T,A210→E),and there were 4 transversion mutations and 3 amino acid among the isolates. Moreover, it was found that the different time isolates and the corresponding serum could counteract and the neutralization activity were gradually declined with higher neutralizing activity, the lowest was 1∶80.The results were coincident with the detection of Liquid phase blocked-ELISA. These results illuminated，during the yellow cattle persistently infected FMDV, antigenic variation of the isolates were unremarkable and could be recognised by themselves cattle serum, moreover the serum and the isolates could specially counteract with high neutralizing antibody titre. But the relativity of serum antibody titre and obtaining isolates was vague.

Construction of an Infectious Full-length cDNA Clone of Foot-and-mouth Disease Virus Asia1/JS/China/2005 Strain

LI Ping-hua; BAI Xing-wen; LU Zeng-jun;SUN Pu;GUO Jian-hong;CAO Wei-jun;LIU Xiang-tao;YIN Hong;LIU Zai-xin

2009, 40(5): 706-711. doi:

Abstract ( 1224 )

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Three overlapping cDNA fragments covering the approximate FMDV full genome (7.5kb）of the 3′ end viral genome were amplified using long RTPCR and were cloned into pBluescriptSK+ vector with unique restriction sites, respectively. A fragment (about 0.7 kb ) including 15 C of the 5′ end viral genome was amplified by over-lap PCR and was cloned into pGEM-T vector. All positive clones were assembled into a low copy number vector pCDNA31/Zeo（+）which was removed T7 promoter in the vector sequence and constructed FMDV Asia1/JS/China/2005 full-length cDNA clone. RNA was synthesized in vitro using T7 polymerase, the infective virus was obtained by transfecting the RNA into BHK-21 cells. The rescued virus was identified by the RT-PCR, indirect immunofluorescence, electron microscope and sulk mice pathogenicity, the results showed infectious FMDV was rescued successfully. The full-length infectious cDNA clone will lays the basis for elucidating the mechanism of pathogenesis of FMDV and developing novel vaccines against FMD.

Safety Evaluation and Minimum Immune-doses Analysis of the Recombinant Fowl Pox Virus Expressing S1 Gene of Infectious Bronchitis Virus and IFN-γ Gene

SHI Xing-ming;WANG Yun-feng;WANG Mei;LAN De-song;REN Gang;LI Ji-song;ZENG Wei-wei;SUN Yan;LIU Sheng-wang;TONG Guang-zhi

2009, 40(5): 712-716. doi:

Abstract ( 1341 )

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This experiment was conducted to study the safety evaluation and minimum immune-doses of the recombinant fowl pox virus expressing S1 gene of infectious bronchitis virus and IFN-γ gene. The vaccines were chosen and diluted with the saline water. 7-day-old SPF chickens were inoculated in the endothelium under the wing at the dose of 1.0 ×10⁵PFU per chicken, local reaction appeared three days later, local swell became the largest at the 5th day. The inoculated chicken showed no sign of other discomforts,and inspirit, appetite and development were the same as controls. The results indicated that the vaccine was safe for chicken. The vaccine was diluted to 1.0 ×10¹~1.0×10⁴PFU, and the 28-day-old SPF chickens were inoculated as above, then challenged with infectious bronchitis virus LX4 strain 3 weeks after inoculation. The results showed that the chickens inoculated in the dose range of 1.0×10²to 1.0×10⁴PFU could induce local reaction, morbidity and mortality was reduced, and the protection rate was exceed 73%, but the inoculum of 1.0×10¹ PFU can induce local reaction partially (5/ 11), and the protection rate was 64%. These results indicated that there was a correlation between the local reaction and the efficiency, and the local reaction can reflect the efficiency of the vaccine. According to the fact of vaccine production and use, the optimum dose of the vaccine was 10³PFU.

Genetic Diversity of Clostridium perfringens Isolated from Healthy Chickens at Commercial Farms Revealed by AFLP and ERIC-PCR

NI Xue-qin;ZHENG Xiao-li;ZENG Dong;Joshua Gong;SONG Zhen-yin;

2009, 40(5): 717-724. doi:

Abstract ( 1114 )

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The objective of this research was to study the epidemiology of C. perfringens colonizing healthy birds, characterize the population diversity and observe how diversity changed between different areas. Isolates were obtained from fresh feces and cecal samples of healthy chicken in ten farms of 8 cities in Sichuan province. Each isolate was typed using amplified fragment length polymorphism (AFLP) and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). A total of 34 C. perfringens isolates of type A from 600 birds were typed. There found 12 AFLP genotypes and 8 ERIC-PCR genotypes. The analysis of genetic type and origin source revealed that isolates from different farms had different genetic types. The subtype of C. perfringen appeared to be simple within a single farm, and mixed with few other gene subtypes. The dominant gene type of C. perfringen was AFLP gene type Ⅷ or ERIC-PCR gene type 1. These results reveal that the diversity of C. perfringen of healthy chickens in Sichuan province is low and there is close correlation between the epidemiology and regions.

Prokaryotic Expression and Purification of Bioactivity Analysis of Chicken Granulocyte Macrophage Clony Stimulating Factor（GM-CSF）

TAN Bing;WANG Hong-ning;ZHANG Yi;ZHANG An-yun;FAN Wen-qiao

2009, 40(5): 725-729. doi:

Abstract ( 712 )

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The ChGM-CSF gene without signal peptide was amplified by PCR method and ligated into the expression plasmid pET-32a(+) to construct prokaryotic plasmid p32GM-CSF. ChGM-CSF recombinant protein was expressed by induction with IPTG in E.coli and purified with Ni-NTA column. MTT assay was used to detect bioactivity of rChGM-CSF. Polyclonal antibody was produced by rabbit injected with the purified protein. The result showed that expression plasmid p32GM-CSF was successfully constructed. SDS-PAGE demonstrated that the recombinant protein was expressed in form of inclusion body and was approximately 34 ku. Western Blot analysis showed that it could be specifically recognized by the rabbit sera to chicken GM-CSF. Results of MTT assay confirmed that it can enhance chicken bone marrow cell proliferation obviously. These results demonstrated that the E. coli-derived rChGM-CSF and polyclonal antibody against rChGM-CSF have some bioactivities. Our results would provide foundation for further research of biology characteristics and application of ChGM-CSF.

Construction and Characterization of Avian Pathogenic Escherichia coli Mutant with fimC Gene Deletion Mutation

SUN Qing-jie;LIU Xue-wei;LI Yi-jing

2009, 40(5): 730-737. doi:

Abstract ( 1465 )

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In order to determine the effect of fimC gene, the wild type avian pathogenic Escherichia coli (APEC) isolate O₂ was selected as the prototype of the (APEC) Type Ⅰfimbriae. Based on the original sequences of TypeⅠ fimbriae operon gene clusters in the GenBank, fimC deletion mutant was constructed. fimC gene, which missed 169 bp, was amplified by PCR and cloned into a suicide vector pCVD442. The recombinant plasmid pCVD442 ∷△fimC was transformed into a host cell SM10λpir, then conjugated from SM10λpir (pCVD442∷△fimC) into E. coli O₂ (NalR). The strain of E. coli O₂ fimC deletion mutant was constructed by homologous recombination. The O₂(△fimC) mutant obtained was further confirmed by fimC PCR amplification,sequencing and transmission electron microscopy.There were some differences in the biochemical reaction between the wild type and the O₂(△fimC) mutant, but no difference in the drug allergy test. Compared with the wild type isolate, the O₂(△fimC) mutant grew slowly during all stages of growth in vitro. The animal experiments indicated that adherence of bacteria to avian tracheal epithelial cell and pathogenicity of the mutant strain was much slighter than those of the wild strain. This work provides the important basis for us to understand the role of fimC in the molecular pathogenesis mechanisms and the interaction between the Type Ⅰ Fimbriae and susceptible host cell and other biological characterizations of the O₂(△fimC) mutant.

Expression of fimC Gene from Pili of Avian Pathogenic Escherichia coli(APEC) and Immunity Protection Effectiveness of fimC Protein Yolk Antibody

WANG Ya-jun;LI Yi-jing

2009, 40(5): 738-742. doi:

Abstract ( 1296 )

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This experiment was conducted to determine immunity protection effectiveness of fimC protein from Avian Pathogenic Escherichia coli(APEC) after fimC protein was expressed. fimC gene was amplified from APEC O2, and then was cloned into pMD18-T vector and sequenced. The results showed that fimC gene was 657 bp and encoded 218 amino acids. The sequence of fimC gene was analyzed by homology comparing with that of human, they shared 97.9% homology in nucleotide and 96% in amino acid. The fimC gene was sub-cloned from pMD18-T vector into the plasmid vector pET-30c to construct a recombinant expression plasmid pET-30c-fimC, which was transformed into E. coli BL21. Then a fusion protein about 25 kDa was expressed by inducing with IPTG, and the expressed protien was proved to be antigenic by Western Blot analysis. The laying hens were immunized with the expressed fimC protein to prepare high yolk antibodies of fimC. One-day-old healthy chickens were immunized with the yolk antibodies of fimC, and injected with APEC next day, results showed that the mortality of immunized group and the control group was 60% and 70% respectively. The results indicated that fimC yolk antibodies could provide protection to some extent against the attacks of APEC.

Establishment of Dairy Cow Mammary Gland Epithelial Cell Line

CHEN Jian-hui;TONG Hui-li;LI Qing-zhang;GAO Xue-jun

2009, 40(5): 743-747. doi:

Abstract ( 1177 )

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Tissue mass inoculation method was used in this study, and mammary epithelial cells of diary cow were successfully cultivated, mammary gland epithelial cells were identified using cytokeratin 18 immunofluorescence staining. The survival rate of cells inoculation，doubling time of cell population, growth curve，morphological and biological character were detected，and identification and establishment of normal cultured dairy cow mammary epithelial cell lines were set up，cell could be passaged 20 generations with a good behavior of proliferation. Through the cell passage and repeatedly freeze-thaw， dairy cow mammary gland epithelial cell lines were established, and a large number of mammary epithelial cells were acquired.

Characteristics of Lung Structure in Different Age Plateau Yak

HE Jun-feng;YU Si-jiu;CUI Yan

2009, 40(5): 748-755. doi:

Abstract ( 734 )

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Histology methods and transmission electron microscope technique were used to investigate the pulmonary morphological structure and adaptive changes in one-day-old yak, five-month-old yak and adult yak. The results showed that the percentage of media muscle thickness of pulmonary artery (MT%) were thicken, the mean value of one-day-old group, five-month-old group and adult group were 10.71%, 12.53% and 11.18%, respectively. There was a complete smooth muscle layer in bronchiole wall in one-day-old yak. In gas-conducting airways, the secretory granules of goblet cells were dense; there was sparse area in granules central. The secretory granules of Clara cells were dense secretory granules or a mixture of dense secretory granules and pale secretory droplets. In pulmonary artery, endothelial cells were cubic and proliferated, some of them intruded lumen. Smooth muscle cells are cubic, organelles increased. The thickness of blood-air barrier in yak was very thin. The thickness of blood-air barrier in different age group were 0.445(one-day old),0.506(five-month old) and 0.423(adult) μm, respectively. These results indicated that hypoxia affected the structure of lung in yak; it was significant in five-month-old group, but as the increasing of age, the affection was decreasing. The changes of structure affected with age indicate that the lung of yak has adapted to the hypoxia.

Effect of Melatonin on the Function of Liver Mitochondria in Goats with Endotoxemia

LIU Yu-qing;TANG Zhao-xin;GUO Jian-ying;SU Rong-sheng;LIU Jing

2009, 40(5): 756-762. doi:

Abstract ( 1276 )

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To investigate the effects of melatonin（MT）on the function of liver mitochondria in goats with endotoxemia，48 normal goats were divided randomly into four groups：saline（SL）group,LPS group,MT group and MT+LPS groupFour groups were treated with saline（SL）, LPS（1 mg·kg^-1）, MT（1 mg·kg^-1）and MT+LPS，respectively.After injection of drugs，livers were collected from 6 killed goats per group at the 3rd hour and 6th hour，respectively.Liver mitochondria and mitochondrial DNA （mtDNA） were extracted for evaluation of mitochondrial membrane potential（MMP）,activity of adenosine triphosphatase（ATPase）,content of Cardiolipin（CL）and level of 8-hydroxy-2′-deoxyguanine（8-OH-dG）in mtDNA.Results showed that：compared with SL group， MMP of LPS group was obviously declined，activity of ATPase was restrained， content of CL decreased greatly，level of 8-OH-dG increased significantly；but compared with LPS group，MMP of MT+LPS group was raised obviously， activity of ATPase was significantly recovered， content of CL increased greatly，level of 8-OH-dG decreased apparently.The experimental results indicated that melatonin could reduce liver mitochondrial damage induced by endotoxin， and could protect liver mitochondrial function in goats with endotoxemia.

The Metabolism of Flaxseed Lignans in Rats

ZHOU Wei;WANG Guo-jie;HAN Zheng-kang

2009, 40(5): 763-768. doi:

Abstract ( 721 )

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The rats, which had been adapted to flaxseed lignan (secoisolariciresinol diglusoside, SDG) for 7 d, were used as a model for non-ruminant animals. Based on the reversed-phase highperformance liquid chromatography (RP-HPLC), two experiments were conducted to study the main site of the transformation of SDG, the metabolism and absorption of lignans. Experiment Ⅰ: After SDG infusion, the concentrations of SDG, enterodiol (END) and enterolactone (ENL) in the chyme of gastrointestinal tract (GIT), feces and serum in rats with or without antibiotics were measured. Experiment Ⅱ: After the bile was collected through cannula in hepatocholedchus, the levels of lignans were analyzed. The results showed that antibiotics can significantly inhibit the transformation of SDG, it may indicate that the colon and caecum, which contain more germs in GIT, produced the most mammalian lignans, and the formations of mammalian lignans depend on the flora in GIT. SDG could be absorbed into blood circulation and its content was higher than that of END and ENL. Once the SDG and mammalian lignans were absorbed, they might undergo enterohepatic circulation.

Preparation of Chicken CDC25B-mRNA Probe and Its Detection in Duodenal Mucosa by in situ Hybridization

QIN Jun-hui;HOU Fang-liang;ZHANG Hui;BAO Hui-jun;ZHOU Qiang;LI Mei-ying;CHU Xiao-hong;CHEN Qiu-sheng

2009, 40(5): 769-774. doi:

Abstract ( 699 )

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In order to investigate the distribution of CDC25B-mRNA in the duodenal mucosa of chicken, the sense and anti-sense digoxigenin (DIG) labeled RNA probe were prepared and utilized. The fragment of CDC25B gene was obtained by RT-PCR through total RNA of chicken embyros. Amplified cDNA fragment was subcloned into pGM-T easy vector, and the plasmid was transformed into E. coli DH5α and screened by “whiteblue plaque selection”. The recombinant plasmid was identified by EcoR Ⅰ restriction enzyme digestion and sequencing, then CDC25B/pGM-T easy vector was linearized with the restriction enzyme of NcoⅠ and SpeⅠ respectively. The sense and anti-sense DIG labeled RNA probe were produced by SP6 and T7 RNA polymerase respectively and transcription in vitro according to the protocol of “DIG RNA Labeling Kit (SP6 /T7)”. The sense and anti-sense RNA probes were prepared successfully. The distribution of CDC25BmRNA in the duodenal mucosa of chicken was examined by in situ hybridization histochemistry( ISHH). There were many labeled cells distributing in the duodenal mucosa of adult chicken. Of these labeled cells, 8160%±963% and 36.21%±8.81% CDC25B-mRNA positive cells were distributed in the basilar part and middle portion of duodenal mucosa of adult chickens respectively. Most of these labeled cells were positive in the regions of the stem cell and proliferation. The positive signals were both in the cytoplasm and cell nucleus. The signals of ISHH were decreased from basilar part to upper in the crypt of lieberkuhn and disappeared in the inferior of villi of small intestine.The labeled cells were both negative in the lamina muscularis mucosae and muscular layer. In conclusion, the sense and anti-sense DIG labeled RNA probes for ISHH of CDC25B were prepared successfully in this experiment, which provided an approach to study further the location of CDC25B-mRNA in chicken. The results of ISHH confirmed the existence of CDC25B-mRNA and athletic proliferation activities in the duodenal mucosa of adult chicken.

Cloning and Sequence Analysis of the rDNA-ITS of Parabronema skrjabini

ZHANG Xiao-dong;YANG Xiao-ye;YANG Lian-ru;LI Lin-chuan;ZUO Hai-tao;NA Ren-hua;ZHAO Zhi-guo;WANG Jun-jie

2009, 40(5): 775-779. doi:

Abstract ( 1377 )

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The internal transcribed spacer （ITS1, 5.8S and ITS2）of ribosomal DNA nucleotide fragments of Parabronema skrjabini isolated from the camel of Inner Mongolia region were amplified by PCR using three pairs of conserved primers NC5, NC13, NC13r and NC2. The PCR fragments were purified and cloned into pGM-T vector. The inserts were successfully sequenced, and the results revealed that the first sample′s (P.sk1) ITS inserts were 837 bp in length and consisted of partial 18S, 28S and complete ITS1（298 bp）, 5.8S（157 bp） and ITS2 （281）rDNA sequences. The second sample’s (P.sk2) ITS1 inserts were 372 bp in length and consisted of partial 18S, 5.8S and complete ITS1（296 bp）. The third sample’s (P.sk3) ITS2 inserts were 484 bp in length and consisted of partial 5.8S, 28S and complete ITS2（284 bp）. ITS2 sequences shared 30.2%-60.1% homology with other nematodes. It was the first time that the complete sequence of ITS and 5.8S rDNA of P. skrjabini were reported. The results of this study laid a foundation for further studies.

Analysis on Polymorphisms of the Prion Protein Gene in Local Sheep Species of Inner Mongolia, China

WANG Yi-qin;QIN Zhen-kui;BAO Yong-gan;KOU Fu-jun;JING Wen-kui;QIAO Jun-wen;ZHAO De-ming

2009, 40(5): 780-784. doi:

Abstract ( 1152 )

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We investigated prion protein gene(PRNP) polymorphisms in three sheep breeds in Inner Mongolia, China. Blood samples were collected from 46 Ujumqin, 34 Sunite and 22 Mongolian sheep. The genetic DNA of PRNP of each sheep was extracted, amplified, and sequenced, and amino acid alignment was determined. Polymorphism was detected at 8 codons, among which M157I, Q220H and R223K have not been previously reported. The frequency of the amino acid residues ARQ/ARQ at codons 136, 154 and 171, which is associated with medium-high susceptibility to scrapie, was 74.5%, and the frequency of scrapie-resistant amino acid residues ARR/ARR was 7.9%. The highly susceptible amino acid residues VRQ/VRQ at these codons were not detected from the tested sheep. Of the 3 sheep breeds, Ujumqin sheep had the highest frequency (15.2%) of scrapie-resistant amino acid sequence, ARR/ARR at codons 136, 154 and 171, accounting for 87.5% sheep that carry these polymorphisms. Our findings are of special importance for both live sheep export and sheep breeding.

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