Acta Veterinaria et Zootechnica Sinica

Expression of Fibroblast Growth Factors (FGF2) and Fibroblast Growth Factor Receptors (FGFR1) in Organs of Cloned Cattle that Die within Hours after Birth

LI Shi-jie;;LI Dong-jie;DU Wei-hua;FAN Bao-liang;;DAI Yun-ping;LI Ning

2008, 39(10): 1289-1294. doi:

Abstract ( 1451 )

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Cloning by somatic nuclear transfer is an inefficient process in which many of the cloned animals die shortly after birth and display organ abnormalities. In an effort to determine the possible roles of FGFs played in neonatal death and organ abnormalities, we have examined expression patterns of fibroblast growth factor (FGF2) and their receptor (FGFR1) in six organs (heart, liver, spleen, lung, kidney and brain) of both neonatal death cloned bovines (n=9) and normal control calves (n=3) produced by artificial insemination (AI) using real-time quantitative RT-PCR. The effect of the age of the fibroblast donor cell on the gene expression profiles was also investigated. Aberrant expressions of FGFR1 were found in some studied tissues, but the expression of FGF2 had similar levels with the normal controls. The expression of FGFR1 showed a higher level in heart and liver of both cloned bovines. Because FGF systems play an important role in embryo development and organogenesis, the aberrant transcription patterns detected in these clones may contribute to the defects of organs reported in neonatal death of clones.

Association of Single Nucleotide Polymorphisms in Exon 17 of Porcine HK2 Gene with Meat Quality and Carcass Traits

WANG Jun;DENG Chang-yan;XIONG Yuan-zhu;ZUO Bo;LI Feng-e;LEI Ming-gang;ZHENG Rong;

2008, 39(10): 1295-1299. doi:

Abstract ( 883 )

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Hexokinase (HK) Ⅱ is a key enzyme member in the glycolysis reaction which expressed mainly in muscle.In this study,the polymorphisms of MspⅠsite （A171G） in the 17th exon of HK2 gene in 186 pigs of six pig populations were analyzsed by using PCR-RFLP，and the relationship between the HK2 genotypes and pork quality, carcass traits was evaluated.The results showed that the frequencies of the two alleles (A and B) were similar in all studied homozygous and heterozygous pig breeds, and allele B was dominant. In addition, no AA genetype was found in six breeds, the statistically associations of AB and BB with carcass length, rib numbers, and lion eye area were very significant different (P<0.01), but the significant differences (P<0.05) of their genetype effects were found in backfat thickness at shoulder, backfat thickness at thorax-waist, and average backfat thickness over three positions, lion eye height, lion eye width, fat meat percentage, lean meat percentage and, intramuscular fat. It needs more and comprehensive studies to verify whether the polymorphic site could be used as the candidate site affecting carcass and meat quality traits in markers assisted selection.

Study of SLA-DMA Gene Based on SSCP in Chinese Miniature Pigs

YU Hui;LI Bo;LI Hua;LI Bin;LI Xue-wei;LIU Wei-min;ZHAO Hai-quan

2008, 39(10): 1300-1305. doi:

Abstract ( 1451 )

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The polymorphisms of exon 2 and exon 3 in the SLA-DMA gene were studied based on the PCR-SSCP patterns and the sequences in Ganzi Tibetan pigs (T), Hezuo pigs (HZ), Bama pigs(BM) and Wuzhishan pigs(WZS), no allelic variation was recognized in the second exon, but two allelic variations were recognized in the third exon in four different miniature breeds of swine. By optimizing a low ionic strength solution (LIS) PCR-SSCP, cloning sequencing, bacterical colony PCR, asymmetric PCR and structure prediction of DNA single-strand, the complicated patterns about allele K and J based on PCR-SSCP in the exon 3 were confirmed. There was highly identity comparing the pattern of allele J with the predicted second structure of DNA single-strand. The frequencies of three kinds of genotypes JJ, JK, KK in the third exon of SLADMA gene were fit with Hardy-Weinberg equilibrium in four populations. The frequency of allele K was higher than that of allele J. Values of the heterozygosity and the polymorphism information contents in the exon 3 of SLA-DMA gene were showed low polymorphism in T, but moderate polymorphisms in BM, WZS,and HZ.

Genetic Variations of FosB and Their Association with Maternal Behaviours in an White Duroc × Erhualian Resource Population

YANG Zhu-qing;CHEN Cong-ying;REN Jun;GUO Yuan-mei;YANG Guang-cheng;MA Jun-wu;GAO Jun

2008, 39(10): 1306-1311. doi:

Abstract ( 972 )

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In this study, FosB was chosen as a candidate gene affecting sow maternal behaviours for its important role on process of animal maternal behaviours. Genetic variations of FosB and their association with sow maternal behaviours were analyzed in a White Duroc×Erhualian resource population. Single nucleotide polymorphisms (SNPs) were screened in two FosB amplicons of 488 and 806 bp by directly sequencing the PCR products. Three SNPs including FosB-EU163403-g.187A>T, FosB-EU163403-g.188A>C and FosB-EU163403-g.297C>G were identified. The association of the g.297C>G polymorphism with sow maternal behaviours was evaluated in the White Duroc×Erhualian intercross using PCR-Mbi I-RFLP. Transmittion disequilibrium test (TDT) results showed that there was no significant association between this polymorphism and sow maternal infanticide, nest building, nurturing, piglets squashing and trampling (P>0.364). The result was confirmed by Family-Based Association Tests (FBAT, P>0.236). Considering that maternal behaviours are complex traits and controlled by polygene, further analysis in a larger population and (or) in other candidate genes are worthwhile.

Study on the Relationship between Polymorphism of Microsatellite Markers and Growth Trait in Boer Goats

YAO Hong-wei;YANG Li-guo;SHEN Zhong;ZHANG Chun-yan;XU Yun-xiang;HUA Guo-hua;CHEN Shi-lin

2008, 39(10): 1312-1319. doi:

Abstract ( 873 )

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Eight microsatellite loci which in 2nd and 5th chromosome were selected according to the genetic map of male goat and related reports.The population including 250 pure Boer goats in this study.The results showed that, for the BMS1248, BMS1898, ILSTS034, LSCV22, LSCV37, OarFCB011, IDVGA64, INRA040, the number of effect alleles(Ne), heterozygosity (H) and polymorphism information content (PIC) in the Boer goats population were 10.057 6，0.900 6，0.892 0； 6.831 5，0.853 6，0.839 6； 8.101 8，0.876 6，0.864 0； 7.067 5，0.858 5，0.841 7； 7.730 7，0.870 6，0.857 3；5.912 5，0.830 9，0.810 1；6.134 7，0.837 2，0.817 2；5.957 8，0.832 2，0.812 2， respectively. The relating effects between markers and growth traits were analyzed in a generalized linear model, the results indicated that the 162/140 genotype of BMS1248 had positive significant effect on birth weight, birth length, birth height, birth chest; 119/101 genotype of BMS1898 had positive significant effect on birth weight, birth length, birth height, 111/111 genotype had positive significant effect on birth chest; 193/167 genetype of ILSTS034 had positive significant effect on birth weight,while 193/167 and 199/167 had positive significant effect on birth chest; 161/147 and 159/143 genotype of LSCV37 had negative significant effect on birth weight, while 161/147 genotype had negative significant effect on birth weight and birth length; 281/243 genotype of IDVGA64 had negative significant effect on birth weight and birth length; 246/216 genotype of INRA040 had positive significant effect on body weight and birth chest,while 246/216 and 238/238 had positive significant effect on birth height.

Identification and Genetic Analyses of Single Nucleotide Polymorphisms in Goose Myostatin Gene

ZHENG Yun;GONG Dao-qing;WU Wei;ZHAO Xu-ting;DUAN Xiu-jun;GU Zhi-liang

2008, 39(10): 1320-1328. doi:

Abstract ( 976 )

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Myostatin, a new member of the TGF-β super family, is specifically expressed in skeletal muscle cells and functions as a negative regulator of skeletal muscle growth. In this study, the single nucleotide polymorphisms (SNPs) of Myostatin gene was identified in various goose breeds including Zhedong White geese, Wulong geese, Yangzhou geese, Landish geese, Rheine geese, Huo geese, Jilin White geese, Shitou geese, Wanxi White geese, Zi geese, etc. by PCR-SSCP and DNA sequencing. The results showed that: （1） The 12 pairs of primers for goose Myostatin gene were designed based on goose Myostatin gene sequence. Three SNPs located in the promoter region of the Myostatin (T769G, C543T ) and the intron 1 (A1 632G) were identified in geese Myostatin gene, respectively. （2）Population genetic analyses indicated that frequency of allele A ( P3 ) in promoter region was over 0.77 and higher than that of allele B, which is a dominant allele. Frequency of allele D ( P4 ) among Yangzhou geese, Landish geese, and Shitou geese were higher than that of allele C, and the frequency of allele D were 0.625 0, 0.548 8 and 0.647 4 among the three breeds, respectively. But on the contrary, it presented that frequency of allele C is higher than that of allele D in other breeds.

Gene Cloning, Sequencing and Eukaryotic Expression Plasmid Construction of Chicken Calbindin-D28k

YU Lu;WANG Ya-qian;ZHANG Shi-yuan;SUN Huai-chang;WANG Zhi-yue;SUN Long-sheng;LI Yan-wu;LI Zhi-xue;ZHOU Lian-gao;YAN Gui-qin

2008, 39(10): 1329-1335. doi:

Abstract ( 1452 )

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Total RNA was extracted from small intestine of New Yangzhou laying hens at 200 day of age. RT-PCR was conducted subsequently based on published chicken Calbindin-D28k gene sequence (NM_205513.1). The expected amplified cDNA fragment was cloned into TA vector. Sequence analysis revealed that the cloned Calbindin-D28k gene, encoding 262 amino acids, was 789 bp in length which was 99.9% identity but only one nucleotide changed from G to T at site 760 bp compared with published sequence from GenBank database. The homology of Calbindin-D28k gene sequence between chicken and other species such as horse，human，mouse and frog was 79.3%、79.1%、77.4%、77.3%, respectively. Reconstruction plasmid of pCEP4-CaBP-D28k was carried out by inserting the fragment of CalbindinD28k gene into eukaryotic expression vector pCEP4, and the target segment in recombinant plasmid was characterized and confirmed by restrictive endonuclease assay as well as sequence analysis. This research provide a novel means to study regulation mechanism of calcium metabolism in chickens through pCEP4-CaBP-D28k.

Orientate-Derivation Character of Chicken Spermatogonial Stem Cells

LI Bi-chun;WEI Cai-xia;YU Fei;HE Xian-hong;NI Li-gang;CHEN Qiang;WANG Xiao-yan;XIAO Xiao-jun;XU Qi;LIU Tie-zheng

2008, 39(10): 1336-1342. doi:

Abstract ( 1563 )

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The aim of this study was to investigate the capability of spermatogonial stem cells(SSCs) differentiating into osteoblasts, neuron-like cells,and adipocytes in vitro. In present study, SSCs were isolated from testis of chicken embryo on 18^th-20^th hatching days, then cultured and subcultured to the 3rd passage in vitro. ① SSCs were Orientate-induced into osteoblasts by medium consisting of desamethason, glycerol 2-phosphate disodium salt hydrate and vitamin C, and differentiated cell were examined with Von Kossa’s stain,cytochemistry staining and immunohistochemistry stain. ②SSCs were Orientate-induced into neuron-like cells in the medium containing RA and IBMX. The differentiated cells were identified by toluidine blue and immunohistochemistry stain. ③SSCs were induced into adipocytes in the medium consisting of desamethason, insulin and IBMX. The differentiated cells were then identified by oil red-O and specific express PPARγ. The results showed that 80% SSCs were induced into osteoblasts during 15-21 d after cultured in induction medium. The osteoblasts formed Von Kossa stain positive nodules. Dark granules were found in the cell, suggesting the deposition of mineralized materials. The osteoblasts showed brown or deep dark after alkaline phosphatase stain and showed CollagenⅠpositive. About 85% cells was toluidine blue positive after 7 d when SSCs were induced into neuron-like cells. About 85% oil red-O positive cell formed during 14-21 d when SSCs were induced into adipocytes. The induced cells were highly expressed PPARγ. Therefore, we concluded that chicken SSCs have the capability of multi-differentiation in vitro.

Effects of Ovine Sperm Extract on the Development Competence of Homogeneous and Heterogeneous Embryos Reconstructed by Nuclear Transfer

SAI Wu-jiafu;PENG Xin-rong;LI Xiang-chen;AN Zhi-xing;ZHENG Yue-mao;QUAN Fu-sheng;ZHANG Yong

2008, 39(10): 1343-1348. doi:

Abstract ( 1463 )

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Ovine sperm extract (OSE) was used to activate ovine somatic cell nuclear transfer (SCNT)embryos and goat-ovine interspecies somatic cell nuclear transfer(ISCNT) embryos. The effects of different dose of OSE activation on the development capacity of the reconstructed embryos were compared according to the cleavage rate, blastocyst rate and blastocyst cell number. The results showed that OSE could effectively activate the SCNT and the ISCNT embryos and the optium concentration of OSE was 5 mg/mL, the activation effect was significantly（P<0.05） better than other groups. The effects of OSE activation or chemical activation (using ionmicin and 6-DMAP) on the development capacity of the reconstructed embryos were evaluated, the results indicated that there was no significant difference in blastocyst rate and blastocyst cell number when the ISCNT embryos were activated by using the OSE or the chemical activation method and there was no significant difference in blastocyst rate when the SCNT embryos were activated, however, there was a significant increase（P<0.05） in blastocyst cell number when the SCNT embryos was activated by using OSE compared with the chemical activation method（72.4 vs 65.2 ,P<0.05）.

Effects of Dietary Magnesium Asparatate Supplementation on Lipid Peroxidation Status in the Liver and the Thigh Muscles of Broiler Chickens

LIU Yong-xiang;LIU Yan-li;HUANG Yan-kun;GUO Yu-ming

2008, 39(10): 1349-1354. doi:

Abstract ( 1538 )

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The experiment was conducted to investigate the effects of dietary magnesium asparatate (MgAsp) supplementation on lipid peroxidation status in the liver and the thigh muscles of broiler chickens and its possible mechanisms. One hundred and twenty eight 22-day-old male AA broiler chickens were allocated into two treatments, each treatment with eight replicates of 8 chickens. The maize and soyabean meal are the main ingredients of diet in control group, and the diet supplemented with 1% MgAsp served as the diet of experimental group. At 42 days old, one of each replicate was slaughtered. Serum, thigh muscle and liver were collected. Results showed that, compared with the control group, the Mg concentration of serum, thigh muscle and liver in broilers fed with Mg supplementation diet increased by 22%, 21% and 29% (P<0.01), respectively; Malondialdehyde (MDA) contents of liver and the thigh muscle decreased by 24％(P<0.01) and 29％(P<0.01), respectively; Hepatic catalase (CAT) and total superoxide dismutase (SOD) activities increased by 25％(P<0.01) and 14％(P<0.05), respectively; The thigh muscle CAT activity increased by 37％(P<0.01); Dietary Mg supplementation significantly decreased reactive oxygen species (ROS) production in the thigh muscles(P<0.01); There was no difference in the total content of unsaturated fat acids between two groups (P>0.05). These results suggested that MgAsp supplementation in the diets based on maizesoyabean could decrease lipid peroxidation level in the liver and the thigh muscles of broiler chickens; Enhanced CAT activity and decreased ROS production in the thigh muscle lead to the decrease of lipid peroxidation level, however it is the enhanced hepatic CAT and SOD activities not ROS production that decreased lipid peroxidation level in the liver of broiler chickens.

Physical and Chemical Characteristics of Supplemental Organic Zinc Sources and Their Stabilities in vitro Fermentation Rumen

LIANG Jian-guang;LV Lin;LUO Xu-gang;DIAO Qi-yu;LIU Bin;

2008, 39(10): 1355-1366. doi:

Abstract ( 917 )

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Two experiments were conducted to test physical and chemical characteristics of organic Zn sources and their stabilities in vitro fermentation rumen. In experiment 1, physical and chemical characteristics of seven organic Zn sources were analyzed. The results showed that: Zn concentrations and amino acid composition of organic Zn sources varied greatly; Two organic Zn sources (ZnPro B and ZnAA B) had characteristic peaks of complexed Zn. The solubility of all Zn products was more than 90% in the buffer of HCl, citric acid and meutral ammonium citrate, and the solubility of Zn sources in pH2 buffer was ranged from 53.92% to 98.54%, and the solubility in water and pH5 buffer was changed from 6.04% to 98.54%; No complexed Zn was found for these organic Zn sources filtrates from pH2, 0.2 mol/L HCl-KCl, pH5, 0.1 mol/L KH₂PO₄- K₂HPO⁴buffer, or deionized water; Four organic Zn sources showed weak complex strength, and one organic Zn source showed moderate complex strength, and two organic Zn sources showed strong complex strength. In experiment 2, a completely randomized design involving a 6×4 factorial arrangement of treatments was used to evaluate the stability of organic Zn sources with different complex strength in rumen by in vitro rumen fermentation method. The results showed that: The organic Zn sources with different complex strength were stable in the rumen in vitro, and the rumen by-pass ratio of them were more than 92%. The rumen by-pass ratio of organic Zn source with strong complex strength was the highest (99.55%), and that of organic Zn source with weak complex strength was in the middle (94.85%), and that of organic Zn source with moderate complex strength was the lowest (92.16%).

PCR-16S rDNA Identification of Aerobe and Facultative Anaerobe from Rumen of Cows and Affected by the Dietary

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

2008, 39(10): 1367-1372. doi:

Abstract ( 854 )

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In order to study the effect of dietary on the bacteria in the rumen of cows, nine different concentrate diets were used to feed Holstein cows during the dry period. Partial aerobe and facultative anaerobe were isolated, identified and detected. Five health cows were fed continuously in nine periods. The results showed that 56 strains of bacteria were isolated from the rumen fluid by artificial medium, and the 16S rDNA genes of 7 strains of representative bacteria were amplified by PCR. These bacteria were S.bovis(A0625, A1212, 0131), B.cereus(0113), B.licheniformis(0091), B.pumilus(0083) and S.thermophilus(0123) respectively by sequence homogeneous analysis. At the same time, the quantities of S.bovis, Bacillus and S.thermophilus were increased as the increase of concentrate. The results demonstrated that the dietary of different concentrate has different effects on the quantity of aerobe and facultative anaerobe in the rumen, and the quantities of S.bovis was affected significantly. New evolutive branch of bacteria was not shaped by the phylogenetic analysis, however, there were several mutations in the nucleotide sequences of the S.bovis and S.thermophilus.

Histopathology of Ducks Experimentally Infected with a Highly Pathogenic Avian Influenza Virus，A/duck/Guangdong/220/2004（H5N1）

LI Yu-gu;ZHOU Quan-he;CUI Cong-ying;YE Yuan-lan;MA Yong-jiang;ZHANG Yuan;LI Chu-xuan

2008, 39(10): 1373-1381. doi:

Abstract ( 1335 )

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Ducks were experimentally inoculated with a highly pathogenic avian influenza virus, A/duck/Guangdong/220/2004(H5N1) by intravenous and eyes-nares-mouth-cloaca routes, then the micro and ultrapathology were investigated. The pathologic lesions characterized by systemic hyperemia, congestions, hemorrhages, thrombosis, edema were observed in multiple organs; And the necrosis and apoptosis occurred in the heart, liver, spleen, lung, kidney, pancreas, encephalon, thymus and bursa of Fabricius etc. The most prominent lesions were vasculitis, necrotic pancreatitis, atrophic and necrotic thymusitis and bursatitis, splenitis, tracheitis, hemorrhagic bronchointerstitial pneumonia, viral myocarditis, nonsuppurative encephalitis, local viral hepatitis, ulcerous enteritis, tubulointerstitial nephritis and mesangial proliferatial nephritis. It was suggested that the pathogenic mechanism of the virus is involved in necrosis and apoptosis, and the severe pathologic lesions in multiple organs result in duck death.

Expression of the Capsid Protein of Rabbit Haemorrhagic Disease Virus in Insect Cells and Its Protective Efficacy to Rabbits

WANG Fang;HU Bo;REN Xue-feng;FAN Zhi-yu;YANG Long-sheng;XU Wei-zhong;ZHANG Ze-bin;HE Kong-ang

2008, 39(10): 1382-1387. doi:

Abstract ( 910 )

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The objective of this study is to develop genetic engineering vaccine to rabbit haemorrhagic disease. RT-CR was used to amplify the capsid protein VP60 gene of rabbit haemorrhagic disease virus (RHDV). After being cloned and transformed, the recombinant shuttle vector, Bacmid-P60, was constructed and transfected into Sf9 insect cells, then a recombinant baculovirus, named rAcV-ac-P60 was obtained. The identification by IFA, SDS-AGE, Western blotting, HA and HI showed that the recombinant protein of VP60 was expressed in Sf9 insect cells. The expressed protein was applied to immunize three-month-old rabbits that without immunizing with RHD vaccine. The results showed that the antiRHDV antibodies were induced and developed after 21 days of immunization, the anti-RHDV titers in serum ranged from 26 to 27. When challenged with 2 10 HA units of infectious RHDV, the control rabbits died, while the immunized animals survived. The results indicated that the recombinant proteins expressed by recombinant baculovirus could be ideal candidate for RHDV vaccines.

ArcA Gene Transcription Level of Actinobacillus pleuropneumoniae in Anaerobic Condition

DUAN Li-li;WEN Xin-tian;CAO San-jie;HUANG Xiao-bo

2008, 39(10): 1388-1394. doi:

Abstract ( 815 )

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In this study，the ArcA gene mRNA levels of Actinobacillus pleuropneumoniae serotype 1 stimulated in anaerobic condition were measured by mimicking the anaerobic condition. Transcription levels of ArcA gene in anaerobic condition were determined by semi-quantification RT-PCR, and the housekeeping gene recF was used as an internal standard. The transcription level of ArcA was very low in the standard condition, but it was highly transcripted in anaerobic. ArcA might be a potential virulence gene of Actinobacillus pleuropneumoniae, and may play important role in pathogenesis.

REP-PCR Identification of Airborne Staphylococcus aureus Isolated from Chicken Houses and Their Spreading around the Farms

ZHONG Zhao-bing;CHAI Tong-jie;DUAN Hui-yong;MIAO Zeng-min;YAO Mei-ling;YUAN Wen

2008, 39(10): 1395-1401. doi:

Abstract ( 1422 )

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In order to study the transmission of S. aureus in chicken houses to their ambient air， the air samples， including indoor， upwind (10， 50 m) and downwind air (10， 50， 100， 200， 400 m) of 4 chicken houses were collected using Andersen-6 stages sampler and RCS. The S. aureus concentration of every sampling site was calculated. Chicken feces samples were collected according to the standard method and S. aureus was isolated. S. aureus was used as an indicator in this study.The repetitive extragenic palindromic elements PCR (REP-PCR) method was applied to generate genomic amplification products of S. aureus isolated from the chicken feces and the air. By analyzing the different concentration and genetic similarity of S. aureus in every sampling site， the transmission of bioaerosol from chicken houses to their ambient can be identified. The results showed that the S. aureus indoor concentration in 4 chicken houses were higher than those of upwind and downwind sites(P＜0.05 or P＜0.01)， but there were no significantly differences of S. aureus concentration among every downwind site (P＞0.05). The fingerprints and the phylogenetic tree indicated that a part of the S. aureus (38.7%) isolated from indoor air had the same REPPCR fingerprints with those isolates isolated from feces， the most of S. aureus isolated (55.9%) from downwind 10， 50， 100， 200 even 400 m had the same REP-PCR fingerprints with those isolated from indoor air or feces too， which indicated that some isolates isolated from downwind air and indoor air originated from the chicken feces. However， those isolated from upwind air had the low similarity coefficients (60%-87%) with those isolated from indoor air or feces. It was concluded that the isolates isolated from upwind air didn′t come from the chicken feces or indoor air. In a word， the S. aureus in chicken feces can be aerosolized and transmitted into the indoor and outdoor air， especially the downwind air of the chicken houses with the air exchange. This study revealed the transmitting rule of airborne S. aureus bioaerosol to the ambience.

Expression of 10 ku Protein Gene from Taenia solium and Its Preliminary Application

WU Guo-hua;ZHENG Ya-dong;JIA Wan-zhong;ZHANG Shao-hua;JING Zhi-zhong;CAI Xue-peng

2008, 39(10): 1402-1405. doi:

Abstract ( 1536 )

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Recombinant expression plasmids, designated as pGEX-4T-TSO10 and pGEX-4T-CE10, were constructed by insertion of the TSO10 gene from Taenia solium oncosphere and the CE10 gene from the larva, respectively, and identified by sequencing. The results of SDS-PAGE, Western-blot and lamellar scanning showed that both fusion target proteins expressed in E. coli were 35 ku in molecular weight and can be recognized by the positive serum from a cysticercosis patient, and that their contents accounted for 25% and 30% of total bacteria proteins, respectively. Furthermore, the results of ELISA indicated that the recombinant proteins are suitable for diagnosis of cysticercosis.

Cloning and Prokaryotic Expression of P27/30 Gene of Parthenogenetic Haemaphysalis longicornis

YANG Cai-ming;YANG Guang-you;ZHANG Xiao-qian;JIA Xiao-yong;YU Zeng-ying

2008, 39(10): 1406-1410. doi:

Abstract ( 835 )

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With reverse transcription polymerase chain reaction(RT-PCR), P27/30 gene was cloned from parthenogenetic Haemaphysalis longicornis of China. The length of P27/30 gene was 670 bp, it included complete ORF and encoded 201 amino acid residues，the deduced mass was 23.38 kDa．Its nucleotide sequence exhibited 99.85％ similarity to that of the P27/30 gene from Japan parthenogenetic Haemaphysalis longicornis. Results of RT-PCR analysis showed that the P27/30 gene could express in the eggs，larvae，nymphs，fame adult ticks and engorgement adult ticks．After subcloned into expressing vector pET32a（+），it could express in E．coli BL21(DE3) . The recombinant protein was about 24 kDa．With Western-blot analysis, the recombination express protein was approved to be able to recognize antibody of Haemaphysalis longicornis.

Digital Description and Identification of 7 Kinds of Helminth Eggs

HASI Su-rong;LIU Zhen-lian;AMU Gu-leng

2008, 39(10): 1411-1414. doi:

Abstract ( 796 )

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In order to realize the digitized description and the automatic identification of helminth eggs in domestic animal feces, the Pseudo-Jacobi-Fourier Moments were used in this study for describing and identifying 7 kinds of helminth eggs, such as F. hepatica egg, H. contortus egg and B. trigonocephalum egg, etc. The results showed that the microscopic characteristics of these 7 kinds of helminth eggs were successfully digitally described and identified by PseudoJacobiFourier Moments. The reconstructed images were clear enough to be identified and contained almost all the main information of original images of helminth eggs. The average identification rate was reached as high as 96.8%。

Effects of Somatostatin Fusion Gene Carried with PLGA Microspheres on Rabbits Growth

DAI Jian-wei;ZHANG Qian-qian;WANG Yan;LIU Song-cai;ZHANG Yong-liang

2008, 39(10): 1415-1420. doi:

Abstract ( 1287 )

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In this paper, we described the regulation of animal growth using plasmid DNA encoding the SS gene that fused with the hepatitis B surface antigen (HBsAg) gene. The fusion genes were successfully expressed in CHO cells, as proven by RT-PCR and ELISA assayed. Poly D, L-lactide-co-glycolic acid (PLGA) plasmid-encapsulating microspheres were prepared and injected directly into the anterior tibialis of rabbits. Daily weight gain and the levels of insulinlike growth factor-Ⅰ (IGF-Ⅰ), SS, and hepatitis B surface antibody (HBsAb) were monitored. From days 15 to 30 postinjection, the daily weight gain of the treated group was significantly increased (P<0.01) by 1068%. A significant increase in the IGF-Ⅰ concentration and decrease in the SS level of the treated group were also observed. The results indicated that the combination of plasmid-mediated positive immunization against SS led to more robust weight gain in rabbits.

Tissue Damages and Hsp47 Expression in Two Breeds of Piglets after Transportation

ZHU Lin;BAO En-dong;ZHAO Ru-qian;YAN Jian-yan

2008, 39(10): 1421-1425. doi:

Abstract ( 1442 )

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12 Pietrain (PIE) and 24 Erhualian (EHL) piglets (20 kg±1 kg) were randomly devided into 2 (transported and control) groups. 6 PIE and 12 EHL were transported for 2 h, the rest piglets were maintained under normal conditions as the control. By means of histopathological method, immunohistochemistry and enzyme linked immunosorbent assay, the tissue damages, the localization and the quantification of Hsp47 in the same tissues such as liver, kidney, stomach were detected in different breeds of transported piglets. After 2 h transportation there were obvious degeneration in the liver, kidney and stomach of transported piglets; Hsp47 were predominantly expressed in the endothelial cells of central vein and hepatic sinusoid in liver, in the endothelial cells of renal glomeruli in kidney, in the connective tissues of the mucosa and submucosa in stomach; The expressions of Hsp47 in kidney and stomach of PIE transported piglets were significantly higher than those in the control group (P＜0.05), but there was no obvious difference in EHL piglets. It seems there were no obvious Hsp47 changes in livers of both PIE and EHL piglets. The induction degrees of Hsp47 expression displayed different changes in the tissues of transported and control piglets between two different breeds. It revealed that there were different sensitivities to transport stress for two breeds. EHL piglets gave more advantage to antistress.

Expression of Recombinant Gallinacin-9 from Chicken in E.coli and Determination of Its Antimicrobial Activity

HAN Zong-xi;MA De-ying;LIU Sheng-wang;LI Yi-jing

2008, 39(10): 1426-1431. doi:

Abstract ( 869 )

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Gallinacin (Gal) -9 was cloned from tongue of chicks by RT-PCR and sequenced. The full length cDNA of Gal-9 consists of 201 bp, encoding 67 amino acids. The cDNA of chicken Gal-9 gene was sub-cloned into EcoR Ⅰ and Sal Ⅰ sites of pGEX-6p-1 vector to construct recombinant plasmid pGEX-Gal-9. The recombinant plasmid was transfected into E. coli BL21 and the transformed bacteria was induced with IPTG. It was demonstrated by SDSPAGE that a 32 kDa protein which was equal to chicken Gal-9 protein in molecular weight was expressed in E.coli BL21. The recombinant fusion protein was purified. The recombinant Gal-9 was expressed as inclusion bodies. The recombinant Gal-9 exhibit expected anti-E.coli and antiPathogenic streptococcus activity, as measured by inhibition zone assaying.

Regulation of the Local-uterine Cytokines by Selenium during Early Pregnancy in Goats

LI Yin-qian;WANG Jian-hua;WANG Ai-hua;JIN Ya-ping;SHEN Wen-zheng;MA Yong-jiang

2008, 39(10): 1432-1437. doi:

Abstract ( 796 )

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In order to explore the regulation of the local-uterine cytokine IL-2, IL-4, TGF-β1 and TGF-β2 by selenium(Se) during early gestation of goats, the pregnant goats were grouped into experimental groups Ⅰ, Ⅱ, Ⅲ and control group. Group Ⅰ, Ⅱand Ⅲ were treated with sodium selenite respectively at doses of 0.3, 0.5 and 0.7 mg(Se)/kg by oral administration weekly for three times and control group was similarly treated with distilled water. Uterine samples were collected to determine the content of IL-2,IL-4,TGF-β1 and TGF-β2 by ELISA. The results showed that (1)There were no differences at the levels of all the tested cytokine between two uterine horns of each goat during early gestation. The IL-2 levels in fluid of two uterine horns increased during 15-18 d after pregnancy and began to decrease on day 19 after pregnancy, and IL-4 remained in a continuously high level during 15-19 d after pregnancy. The TGF-β₁ had irregular high level in the same period and TGF-β₂ increased primely and then remained at a high level. (2) Administration of 0.3, 0.5 and 0.7 mg/kg selenium could significantly decrease the content of IL-2 in uterine fluid during 16-18 d after pregnancy and significantly increase the level of IL-4 in uterine fluid in a dose-dependent manner. (3)Goats of 0.5 and 0.7 mg/kg groups showed significantly high level of TGF-β₁ in uterine fluid during 16-18 d after pregnancy, and presented high level of TGF-β₂ in uterine fluid during 16-17 d after pregnancy but no significant effect on TGF-β₂ secretion on day 18 after pregnancy. These results indicated that selenium has a regulatory function on secretion of the local-uterine IL-2, IL-4, TGF-β₁ and TGF-β₂ during early pregnancy in goats.

Construction of Florfenicol-resistant Recombinants and Protein Location of FloR in Florfenicol-resistant E.coli

WU Bei-bei;DU Xiang-dang

2008, 39(10): 1438-1441. doi:

Abstract ( 819 )

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A positive plasmid that comprises the entire floR ORF and possible regulatory site was obtained and named plasmid pGEM-floR. The recombinant plasmids were successfully transformed into E. coli JM109. A significant resistance to florfenicol and chloramphenicol was found in this strain which was susceptive to tetracycline and gentamicin. Location of FloR protein was confirmed by immunoblotting that using the membrane fraction of florfenicol-resistant E. coli strains (pGEMfloR/JM109 and CVM1841) and the florfenicol-sensitive (negative-control) strains (pGEM-T/JM109) with the GST-FloR1 mouse antisera. The Western blot results showed that the antibody had specific binding to the FloR protein and confirmed that FloR protein was a membrane protein in florfenicol-resistant bacteria. The research shows that protein encoded by floR gene locates at the cellular membrane and confers crossresistant to flofenicol and chloramphenicol.

A Disposable Amperometric Enzyme Immunosensor for Rapid Detection of Avian Influenza Virus

ZHAO Guang-ying;WU Shu-chun;WANG Li-nan;CHEN Guo-bin;CUI Shang-jin

2008, 39(10): 1442-1448. doi:

Abstract ( 1392 )

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In order to develope rapid detection technology for avian influenza virus antigen, a disposable electrochemical immunosensor was constructed by coating the screen-printed carbon electrode with chitosan membrane and HRP-anti-AIV . Cyclic voltammetry was selected as electrochemical means to detect the products of the enzymatic reaction. According to the decrease percentage of reduction current, AIV-Ag could be detected. Under the optimum conditions of immunoreaction and electrochemical detection, AIV-Ag can be rapidly detected by sigmoidal curve method and form a linear calibration between 3.75 to 125 ng/mL with an associated detection limit of 0.56 ng/mL (S/N=3). The immunosensor had acceptable specificity, reproducibility (RSD＜7.10%), stability (the amperometric response was 91.46% of the initial response after two weeks) and accuracy (91.18% of the results obtained by the immunosensor were in agreement with those obtained by ELISA). In addition, four channels screen-printed carbon electrode was employed for fabricating AIV-Ag immunosensor, the results indicated that the four channels immunosensor had good consistency. Therefore, the immunosensor could be hopefully used to detect AIV-Ag rapidly.

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