Acta Veterinaria et Zootechnica Sinica

Invasionassociated Factors of Apicomplexa

QI Nanshan;SUN Mingfei;LIAO Shenquan;WU Caiyan;LV Minna;YUAN Jianfeng;YU Jingshu;LI Xiangrui;CAI Jianping

2012, 43(2): 167-174. doi:

Abstract ( 364 )

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Apicomplexa is a protozoan class of obligate intracellular parasites, including Toxoplasma gondii, Cryptosporidium spp., Plasmodium spp., Babesia spp. and Eimeria spp., many of them are important pathogens to human and animal. They have conserved subcellular structures and invasion mechanism. The results of lots of researches demonstrate that the invasion mechanisms are mediated by many invasionassociated factors, including the proteins secreted from micronemes, rhoptries and dense granules. The researches about these proteins make great progress with the development of bioinformatics and biotechnologies. In this paper, we review the latest progress of invasionassociated factor combining with our research results in recent years.

Cloning, Sequence Identification and Expression Analysis of NAP1L5 in Porcine Placenta at Different Gestation Stages

GU Ting;ZHAO Shuhong;LI Changchun

2012, 43(2): 175-179. doi:

Abstract ( 393 )

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The aim of this study was to obtain the information about sequence structure, tissue expression and biology function of porcine NAP1L5 gene. In the present study, the porcine NAP1L5 was cloned by RTPCR, the sequence was analyzed by bioinformatics method, and its relative mRNA expression was detected by quantitative RTPCR in porcine placenta at different gestation stages. Sequence analysis showed that the CDS region of the porcine NAP1L5 gene included 579 nucleotides, encoding 193 amino acids, and the sequence similarity of NAP1L5 between the pig and cattle was 89%. The amino acids sequence included the nucleosome assembly protein. The result of quantitative RTPCR indicated that the expression of porcine NAP1L5 increased with the placenta development and there were significant difference between d26 and d50 of gestation stage (P<0.01). These results suggest that NAP1L5 is probably related to the nutrition absorption and early development of porcine fetus, and make a foundation for the further study on the function of NAP1L5 gene in porcine.

A Comparative Study of Metabolites and Hormone Concentrations in Different sized Follicles in Hu Sheep

YING Shijia;WANG Changlong;JIA Ruoxin;WU Yongcong;WANG Ziyu;NIE Haitao;ZHANG Guomin;HE Dongyang;WANG Feng

2012, 43(2): 180-185. doi:

Abstract ( 481 )

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This study was conducted to investigate the metabolites, metabolic hormones and reproductive hormones concentrations in differentsized follicles in Hu sheep during the luteal phase. 11 multiparity Hu sheep with 40 kg in body weight were used. On the 12th day after estrus synchronization, all ewes were slaughtered and follicular fluid was collected according to differentsized follicles. The effects of follicle size on serum ammonia, NEFA, urea, insulin and progesterone concentrations were not significant. Compared with follicles ≤2.5 mm in diameter, the follicular fluid glucose and estradiol concentrations in follicles >2.5 mm in diameter were increased (P<0.05), and the testosterone and glucagon concentrations and LDH activity were decreased (P<0.05). The estradiol concentration in follicular fluid was positively correlated with glucose (P<0.05) and progesterone (P=0.051) concentration and negatively correlated with LDH (P<0.01), glucagon (P<0.05) and testosterone (P<0.01) concentration. In conclusion, follicle development was coregulated by intrafollicular metabolites and hormones.

Correlation Analysis of Microsatellite DNA Markers with Some Substantial Economic Traits in Liaoning New Line Cashmere Goat

JIN Mei;WANG Yanjie;ZHANG Tingting;PIAO Jun;DIAO Xuetao;HE Zhenrui;ZHANG Xiaofang;KANG Huiqin

2012, 43(2): 186-195. doi:

Abstract ( 405 )

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For selection and breeding of multiple objective traits in production practice, the economic traits of Liaoning new line cashmere goat were analyzed by 125 microsatellite loci on the whole 29 chromosomes. The association among three economic traits (body weight, cashmere yield and cashmere fineness) and the marker genotypes was analyzed. The results showed that there were 111 microsatellite loci showing high polymorphism (PIC>0.5) and 4 microsatellite loci showing intermediate polymorphism(0.25<PIC<0.5),and 10 loci include BM1312 showing monomorphism. Effects of 115 polymorphic microsatellite loci on economic traits were also analyzed. The genotypes which favorably affect the three economic traits(body weight, cashmere yield and cashmere fineness) were found. Especially, DE at MoM064 (112117 bp) was favorable genotype for cashmere fineness and cashmere yield.This research provides an experimental basis for selection and breeding of multiple objective traits in production practice.

Expression of β3Adrenergic Receptor Gene in Ovine Adipose Tissues

WU Jianliang;QIAO Liying;LIU Jianhua;GAO Zhongyuan;LIN Peipei;LIU Wenzhong

2012, 43(2): 196-203. doi:

Abstract ( 434 )

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The aim of this study was to study the expression of β3andrenergic receptor gene in ovine adipose tissues. In this study, South African Mutton Merino (SAMM) and Shanxi Dam Line (SXDL) ovines were used to study the distribution and expression of ADRB3 in subcutaneous, omental, small omental, retroperitoneal, mesenteric and perirenal fat tissues of sheep by immunohistochemical technique and realtime polymerase chain reaction. The result showed that ADBR3 was located in the membrane of adipose cell. ADRB3 mRNA and protein were detected in all studied tissues, the lowest expression of ADRB3 mRNA and protein were detected in subcutaneous fat (0.159 and 0.139, respectively), and the highest expression in retroperitoneal fat (2.911 and 2.225, respectively), and higher expression level were found in great omental and retroperitoneal fat than that in subcutaneous fat (P<0.05). Significant difference (P<0.05) in expression level of ADBR3 protein was detected between the two sheep populations. The variation in expression of ovine ADRB3 in different adipose tissues reflected the difference between breeds, which suggested there was less genetic stability in Shanxi Dam Line. These findings are consistent with the known function of ADRB3 in mediating lipolysis and homeostasis in adipose tissues. It may provide theoretical foundation for breeding of new sheep breed using ADRB3 gene as a candidate gene.

Structural and Functional Analysis of Fatty Acid Synthase Gene Promoter of Goat

LI Jun;GE Ting;LUO Jun;SUN Yuting;SHI Hengbo;ZHU Jiangjiang;HAO Juan;ZHAO Wangsheng

2012, 43(2): 204-210. doi:

Abstract ( 411 )

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The objective of this study was to analyze the structure and function of fatty acid synthase gene（FASN）promoter of goat, to further reveal the transcriptional regulatory mechanism of FASN gene. In this study, the promoter of goat FASN gene was obtained by PCR. Seven promoter fragments in different length were obtained by deletion and cloned into luciferase reporter gene expression vectors. Then, the vectors were transfected into goat mammary epithelial cells and MCF7 cells, their expression activity were determined by using the dualluciferase reporter assay system. The promoter sequence of 2 589 bp of FASN gene was obtained. The bioinformatics analysis for sequence showed that there were a TATAbox at -41 bp and an Ebox at -74 bp of transcription initiation site (+1). Luciferase reporter assays demonstrated that the core region of the promoter was from -293 bp to -79 bp conferring basal transcriptional activity. Meanwhile, some transcriptional factor binding sites including Sp1, NFY, USF and SREBP were identified in this core region. The results indicated that the transcriptional factors such as Sp1, NFY, USF and SREBP might be involved in the transcriptional regulation of FASN gene.

Construction of a Suppression Subtractive Hybridization cDNA Library to Screen Differentially Expressed Genes from Duck Liver Infected Duck Hepatitis Virus

LI Xiu;XU Qi;ZHANG Yang;BI Yulin;ZHAO Rongxue;CHEN Changyi;DUAN Xiujun;CHEN Guohong

2012, 43(2): 211-219. doi:

Abstract ( 360 )

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This research aimed to detect and identify genes associated with duck viral hepatitis(DVH) by construction of suppression subtractive cDNA library, and explore their mechanism by function cluster analysis. Using suppression subtraction hybridization(SSH), a different expression SSHcDNA library of 3dayold and fullsib ducklings infected artificially by DHV was constructed and the same dayage ducklings were injected with the same quantity of saline. Of which 563 clones were sequenced, and 299 differentially expressed sequence tags (ESTs) were obtained. After dislodging the redundant cDNA sequence and clustering splicing, the software BLAST in NCBI of GenBank was used to do the nucleic acid and protein homology comparisons and functional analysis. The results showed that 70 different genes had highly homologous with ESTs(E value <e-10,match length>150 bp,match rate>80%), most of which were related to the synthesis of cell components, signal transduction and the biological control process of pathological conditions. The occurrence and development of DVH was a complex multistep process involving multiple genes. The results provide a basis for further study of the molecular mechanism of DVH.

Analysis of Transcription Regulation in the Promoter Region of PIT1 Gene in Goose

ZHAO Rongxue;ZHAO Wenming;XU Qi;DUAN Xiujun;DONG Biao;SUN Guobo;BI Yulin;LI Xiu;ZHANG Yang;HUANG Zhengyang;CHEN Guohong

2012, 43(2): 220-225. doi:

Abstract ( 419 )

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The aim of this study was to study the possible regulation mechanism of PIT1 promoter by searching for the cisregulatory elements, transacting factors and basic transcription units of the transcription regulation. The goose promoter of PIT1 gene was cloned by genome walking and subcloned into the luciferase expression vector pGL3Basic directly. A series of promoter missing mutants were constructed (-1 485/-1 bp，-1 293/-1 bp，-1 014/-1 bp，-775/-1 bp，-561/-1 bp，-353/-1 bp，-206/-1 bp), and identified by restrictive endonuclease enzyme cutting, PCR and sequencing. The recombinant plasmids were detected by SteadyGlo LuciferaseAssay system after transient transfecting GH3 cell. This study indicated that the PIT1 gene promoter cloned had obviously promoter activity. All of them, -561/-1 bp had highest activity, and many possible transcription factor binding sites such as (PIT1，POU3F2，myogenin and GR) were predicted. The pGLPIT1 promoter was constructed successfully by series missing method. The promoter cloned in the study had activity, the positive and negative regulation areas and core promoter region were found. Furthermore, the result provided the foundation for analyzing the promoter activity and transcriptional regulation mechanism.

Expression of Protooncogene cfos in Tarim Wapiti’s Antler at Different Growth Stages

HAN Chunmei;LI Shijun;TANG Jiwei;MA Wancai;REN Ke;LI Jie;WU Yanfeng;GAO Qinghua;

2012, 43(2): 226-231. doi:

Abstract ( 371 )

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To study the effect of oncogene cfos on velvet antler growth, antler tips were collected from 3 adult Tarim wapitis with 30 and 60dayold growing antlers, respectively, and the samples were dissected into four zones, including epidermis/dermis, mesenchyme, precartilage and cartilage cell layers. Locations of cfos expressions in the four zones of antler were determined by two immunohistochemical methods while quantitative analysis of cfos expression was conducted with fluorescent quantitative PCR. The results demonstrated that the expression of cfos was discovered in inner root sheath of hair follicle, hair matrix of dermal layer, and ring smooth muscle of artery while its weak expression was found in the basal cells between the papillary layer of dermis and basal layers. There was no detectable expression in veins, nerve and other subsidiary organs, and the cells expressing cfos were not detectable in mesenchyme, precartilage and cartilage cell layers by two methods. Analysis of quantitative PCR revealed that cfos expressed in all layers at different growth stages. Higher expression level of cfos was observed in dermal layer than that in mesenchymal cells layer, precartilage cells layer and cartilage（P<0.05）. And low expression of cfos was found in mesenchymal cells layer, precartilage cells layer and cartilage. Little variation was found between the expressions of cfos in mesenchymal and precartilage cells layer in the 30and 60dayold antlers while cfos expression in epidermis and cartilage layers decreased with the increase of age. The results suggest that oncogene cfos participated in the regulation of the proliferation and differentiation of stem cells in dermal layer during the rapid growth of the antlers, and cfos may promote the differentiatiing of osteoblast into bone cells in cartilage cells layer.

Effects of Dietary Cadmium on Production Performance，Antioxidative Capacity and the Retention of Laying Hens

SUN Tao;DAI La;TANG Feijiang;XIE Peng;ZOU Xiaoting

2012, 43(2): 232-241. doi:

Abstract ( 397 )

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This experiment was conducted to investigate the effects of dietary cadmium on production performance, antioxidative capacity and the retention in laying hens. One hundred and ninety two 40weekold Babcock B300 laying hens were randomly divided into four groups with three replicates per group and 16 laying hens per replicate. The diet of control group was a basal cornsoybean meal diet. The diet of experimental groups was the addition of cadmium(CdCl2·2.5H2O) for 5，10 and 20 mg·kg-1, respectively into basal diet. The experiment lasted for 9 weeks. The results showed that: 1) In 13, 46 and 79 weeks, the laying rate, ADFI of three experiment groups were lower than that of control group, but the differences were not significant; In 79 weeks, the ADFI of all groups were lower than that in 13 weeks and decreased by 5.89%, 6.77%, 9.61% and 12.71%(P<0.05), respectively; during the experiment period(19 weeks), laying rate of experiment groups were lower than control group and decreased by 1.86%, 2.27% and 2.68%(P>0.05), respectively, the differences of average egg weight, ADFI and feedegg ratio were also not significant(P>0.05) between the two periods; 2) Cadmium content in eggs increased with the cadmiumadded level increased, and the differences among different groups were significant(P<0.05), but the all values less than 30 μg·kg-1 ,and the cadmium in yolk is higher than that in egg white; 3) After cadmium added, differences of the content of cadmium in liver, kidneys and ovary among different groups were significant(P<0.05), the content of cadmium from high to low is kidneys, liver, ovary, and the concentration of cadmium in kidneys of 20 mg·kg-1 group could reach to 0.261 mg·kg-1; 4) MDA content in serum in experiment groups were significantly higher than that in control group and were 1.47, 2.24 and 2.68 times, respectively more than that of control group; activity of total SOD and CuZnSOD of experiment groups significantly lower than that of control group and decreased by 8.30%(P>0.05), 7.29%(P>0.05), 16.92%(P<0.05) and 11.61%, 14.94%, 16.37%(P<0.05), respectively; NO content in serum in experiment groups were decreased by 11.19%(P>0.05), 26.72%(P>0.05) and 46.68%(P<0.05), respectively compared to the control group. These results indicate that cadmium can accumulate in organisms and injury the body through oxidant stress, consequently lead to recession of production performance of laying hens.

Effects of Hainanmycin on Ruminal Fermentation Pattern, Methane Production and Micro Flora

LIU Wei;XIN Hangshu;LIU Caijuan;WEN Qinan;TAN Weizhuo;ZHANG Yonggen

2012, 43(2): 242-249. doi:

Abstract ( 339 )

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The aim of this study was to investigate the effects of different levels of supplementation with Hainanmycin on rumen fermentation parameters, methane production and microflora in vitro. The results showed that the addition of Hainanmycin could inhibit significantly the gas production (P<0.05) and the concentration of ammoniacal nitrogen (P<0.05), and pH enhanced linearly remarkably as the addition level of Hainanmycin increased (P<0.05). Compared to the control, the 10 mg·kg-1 Hainanmycin supplementation in the diet increased the pH by 4%. The content of propionate increased and the concentration of acetate, butyrate and A/P decreased significantly (P<0.05). The methane production was inhibited quadratically by the addition of Hainanmycin in the diet (P<0.05). The percentage of ruminococcu flavefaciens, fungi and protozoa accounting for the total bacterial 16S rDNA reduced significantly in experiment groups compared with the control (P<0.05), and methanogen and ruminococcus albus were not altered by the treatment. It was concluded that the supplementation of Hainanmycin in the diet could influence ruminal fermentation pattern, inhibit the methane production, and affect microflora significantly in vitro. And the methane production could reach the lowest level theoretically when the supplementation level of Hainanmycin was 7.2 mg·kg-1.

Effects of Lactobacillus rhamnosus on Antioxidative Functions and Cytokines Secretion in Caco2 Cells

HUANG Yi;HUANG Qin;LI Weifen;YU Dongyou;ZHOU Xuxia

2012, 43(2): 250-254. doi:

Abstract ( 341 )

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In this study, the effects of Lactobacillus rhamnosus on antioxidative and nonspecific immunologic functions in Caco2 cells were investigated. Caco2 cells were treated by PBS(group A), E. coli K88(group B) and L. rhamnosus (group C), respectively. Caco2 cells in group D were preincubated with L. rhamnosus and then infected with E. coli K88. The results showed that TAOC concentration decreased（P＜0.01）, but SOD activity enhanced in cell culture supernatant（P＜0.01）in group B. However, the adverse situation appeared in group C and, SOD activity in cell lysate increased（P＜0.05）. APRIL secretion were induced（P＜0.01）, however, IL8 secretion were inhibited（P＜0.01）both in group B and group C in different degree. IL10 production was stopped in group B, and was promoted in group C（P＜0.01）. Compared with group B, APRIL production significantly decreased（P＜0.01）, whereas levels of IL8 （P＜0.05）and IL10（P＜0.01）increased in group D. These results suggested that Caco2 cells were sensitize to respond to L. rhamnosus by an improved response in antioxidative function. In addition, inflammatory response against pathogenic bacteria in enterocyte attenuated, moreover, immune response reinforced, which indicated the activities for immunoprotection and immunoadjuvant in L. rhamnosus.

Development of a Rapid Gold Immunochromatographic Strip Test for the Diagnosis of FootandMouth Disease Virus

REN Weiwei;LIANG Zhong;ZHI Xiaoying;QI Guangyu;LIU Xiangtao;CAI Xuepeng;JIANG Tao

2012, 43(2): 255-262. doi:

Abstract ( 473 )

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The aim of this study was to develop a sensitive, rapid and simple immunity colloid gold chromatographic analysis method for examination of footandmouth disease, which can make the differential diagnosis with other infectious diseases. The purified antiFMDV Asia 1 type coupling 12S antigen rabbit antibody were coupled with colloidal gold. The purified antiFMDV A, O type coupling 12S antigen rabbit antibody and goat antirabbit IgG were wrapped onto nitrocellulose membrane as the test line (T line) and the control line (C line), respectively. The FMDV general diagnosis kit was then performed. The seriate results indicated that sensitivity of the test kit reached 0.98×104LD50. No cross reaction was found with SVD, VS, VES antigen by cross tests. In the clinic test, a total 90 field samples were detected with the test list. The result of corresponding rate for positive and negative samples were 96.70% and 100%. These experiments demonstrated that the established gold immunochromatographic strip test kit was sensitive, specific for detecting FMDV and was potentially useful for first time and differential diagnosis.

Genomic Characterization of a Variant of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

YANG Xiaorong;YIN Shuoyan;PAN Meng;ZHOU Lei;GE Xinna;CHEN Yanhong;GUO Xin;YANG Hanchun

2012, 43(2): 263-269. doi:

Abstract ( 408 )

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In order to monitor the variation of highly pathogenic porcine reproductive and respiratory virus (PRRSV) prevailing in recent years, the complete genome of a strain of PRRSV SD0901 isolated from one pig farm with clinical outbreak of PRRS was sequenced and analyzed by RTPCR amplification. The results showed that the genome size of the virus was 15 230 nucleotides, excluding the poly (A) tail. Comparative analysis of the whole genomic sequences revealed that the virus shared 98.6%98.7% identity with the representative strains of highly pathogenic PRRSV, and exhibited additional one amino acid deletion and insertion at the position 468 and 585586 respectively, besides 30amino acid deletion within its Nsp2coding region. In addition, one amino acid variation was found in GP2, GP3, GP4 and Mcoding regions of its genome, respectively. Phylogenetic analysis showed that the virus formed an individual branch although it belonged to the same subgroup as highly pathogenic PRRSV strains. These findings indicate that the virus is a variant of highly pathogenic PRRSV, implying that the variation of highly pathogenic PRRSV have occurred during its prevailing. Our study provides valuable genomic data for monitoring and analyzing genetic variation and evolution of highly pathogenic PRRSV.

Expression of Compound Multiepitope Gene of Classical Swine Fever Virus in E. coli and the Assessment of Its Immunologicity

TIAN Hong;HOU Xiangmin;WU Jinyan;CHEN Yan;SHANG Youjun;LIU Xiangtao

2012, 43(2): 270-274. doi:

Abstract ( 360 )

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Genetical engineer T, B cell epitopes of Classical swine fever virus (CSFV) were developed by employing molecular biological tools, and their immunogenicity has been primarily evaluated by immunizing experimental animals. A DNA fragment which encoding a tandem repeat protein of T, B cell epitopes of CSFV were designed and chemically synthesized, and it was cloned into pGEX6P1 vector in turn to form a recombinant plasmid pGEXBT500. A chimeric protein was obtained after transforming the pGEXBT500 into Escherichia coli BL21 (DE3) host cell and induced by IPTG. The western blotting analysis showed that the expressed products have a good reactogenicity, which can react specially with CSF positive sera. Inoculation with 100 μg recombinant protein induced strong neutralizing antibody response in rabbits. Our study indicated that the expressed T, B epitopes can act as the carrier protein for CSFV peptide epitopes, and this recombinant protein is a potential multiepitope peptide vaccine candidate to prevent CSFV infection.

Isolation and Identification of Group A Porcine Rotavirus

KU Xugang;ZHANG Kun;LIU Yuxi;HE Qigai;

2012, 43(2): 275-281. doi:

Abstract ( 378 )

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Porcine rotavirus can induce serious diarrhea in piglet, but most researches are on human. We successfully isolated and identified group A porcine rotavirus (GAR) virus, which make a contribution to pathogenesis, molecular biology study on porcine rotavirus. GAR were detected by RTPCR test from different feces samples of diarrhea, those positive samples were treated by trypsin, then was inoculated to MA104 cell line to isolate strain TMa. After routine RTPCR test and electron microscope observation, we sequenced and analyzed VP6, VP7 and VP8 genes of strain TMa. Through homology analysis, we found VP6, VP7 and VP8 are, respectively, identical to Beijing human strain LL3354 from China (96.0%), Indian human strain RMC321 (95.1%), wild boar strain GUB71 from Japan (97.0%). Strain TMa have a high similarity to human and wild boar rotavirus, we infer that GAR may spread from human to animal, and there exist gene recombination.

Identification and Structure Features of Opacity Factor Genes from Two Streptococcus suis 2 Strains Originated from China and Japan

FENG Yali;;RAN Xueqin;WANG Jiafu;QIAN Long

2012, 43(2): 282-289. doi:

Abstract ( 362 )

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Streptococcus suis is a swine pathogen which causes serious inflammation of piglets. It can infect people in contact with diseased pig or its byproducts. The mechanisms of S. suis pathogenesis are poorly understood. Taking two S. suis serotype 2 strains 606 isolated from China and 607 from Japan as samples, opacity factor of S. suis (ofs) genes were amplified from the genomic DNA of these bacteria by PCR method. The ofs genes contained 3 016 bp which encoded for 938 aa based on alignment analysis. The identity of nucleotide sequence of ofs gene from SS2 strain 607 was 100% to the reported SS2 genes while seven nucleotides in ofs of SS2 strain 606 were different and resulted in three amino acids substitution. One of the substitutions located at the Nterminal region of OFS protein in which glycin (without charge) was changed to be aspartic acid (with negative charge). It was proposed that the change would have an effect on the OFS ability to opacify serum of mammals. The other two substitutions were found from the repeats in Cterminal region of OFS in SS2 strain 606. Furthermore, the length and amino acid constitute of three repeats in OFS of SS2 strain 606 and 607 was very divergent from that of fibronectinbinding protein A (FnBA) of Streptococcus dysgalactiae. It was suggested that the pathway of OFS protein binding to fibronectin of host cells might be different from that of FnBA. However, OFS proteins did carry the typical structural elements of MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) with a large Nterminal region, specific repeats and LPXTG domain elements in the Cterminal region. It could be presumed that OFS of SS2 strain 606 and 607 function as an adhesin combined on the surface of bacteria and bind the fibronectin with its repeats at the Cterminus. The correlation between the OFS with moderate size (938 aa) and the high virulence of streptococci implied that ofs gene was a pivotal virulent gene of SS2 strain 606 and 607.

Development and Primary Application of Hybridoma Cell Lines Secreting Monoclonal Antibodies Against LPS Antigen of B. Melitensis

WANG Yan;WANG Xinwei;CHEN Lu;ZHAO Jun;YANG Xia;WANG Shan;WANG Chuanqing;Di LiBaiEr·AmuDi

2012, 43(2): 290-298. doi:

Abstract ( 376 )

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The aim of this study was to prepare the specific monoclonal antibody (McAb) against LPS of B．melitensis, and to develop a sandwich ELISA for detecting B．melitensis. The LPS from B. melitensis strain 16M was purified by using hot phenol extraction method, and identified by SDSPAGE. Six to eightweekold female BALB/c mice were immunized with inactivated thalli of B. melitensis (strain 16M) in Freund’s complete adjuvant and LPS in Freund’s incomplete adjuvant successively. Three hybridoma cell lines secreting McAb against LPS were obtained and named as strains 5H3，6B8 and 3H7, respectively. The ELISA titers of cell cultures supernatant and ascites fluids of all three hybridoma cell lines ranged from 1∶1 000 to 1∶5 000, and 1∶10 000 to 1∶160 000, respectively. Immunoglobulin subclass tests differentiated corresponding mcAbs from the three hybridoma cell lines as IgG3 (3H7) and IgM (5H3 and 6B8). The specificity assay showed that mAbs from 6B8, 5H3 and 3H7 had noncrossreactivity with antigens from Colibacillus O157, Salmonella pullorum, Flexneri Shigella and LPS of Gallibacterium anatis, and only reacted with the inactivated thalli of B. melitensis (strain 16M). RoseBengal plate and tube agglutination assays indicated that the acquired mAbs could react with standard antigen of B．melitensis, which further demonstrated that the mAbs from 6B8, 5H3 and 3H7 had strong specificity. A sandwich ELISA for detecting B．melitensis was developed by using the mcAbs produced from hybridoma cell lines. Simulative samples and clinical samples were tested with the developed sandwich ELISA. Results showed that this ELISA had very high accuracy. Specific hybridoma cell lines secreting monoclonal antibodies against LPS Antigen of B. Melitensis were successfully developed, and provide a basis for establishing rapid diagnostic method of Brucella Melitensis.

Construction of Defined Mutations of PRN of Rabbit Bordetella bronchiseptica and Property Research

LI Hongguang;WANG Fang;JIANG Ping;FAN Zhiyu;HU Bo

2012, 43(2): 299-305. doi:

Abstract ( 377 )

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We constructed pertactin (PRN) deletion mutant strain of rabbit Bordetella bronchiseptica (Bb) to study the function of pathogenesis of Bb, and to provide a theoretical basis for researching live attenuated vaccine against Bb. The 550 bp fragment of PRN1（upstream of PRN）and 440 bp fragment of the PRN2（downstream of PRN）were amplified by PCR respectively. The two fragments together with Gentamycin (GM) gene were linked and inserted into suicide vector, and the recombinant suicide vector was named as pMEG375PRN1GMPRN2. The recombinant suicide vector was transformed into host strain SM10. The transformed SM10 was mated with recipient strain Bb on the solid phase membrane, and the recombinant suicide vector was transformed into the recipient bacteria. According to homologous recombination and the principle of resistance screening, mutant strain was generated and was named as Bb△PRN. We compared Bb△PRN with wild type（WT）strain on genetic stability, growth characteristics, hemolytic activity, cell adhesion properties, immunogenicity. The results showed that the mutant strain stabilized genetically and grew slower than the wild type. There was no significant difference between them on hemolytic activity and adhesion on Hep2 cells. Comparing with parent strain, the virulence of mutant stain was attenuated about two fold. The assay of immunogenicity showed that Bb△PRN could produce a strong immune capacity. All of results indicated that mutant strain had good immunogenicity, and it could provide a theoretical basis for researching live attenuated vaccine against Bb.

Immunological Response Analysis of Chinese Merino Sheep with Different MBL Genotype Infected with Mycoplasma pneumonia

ZHANG Hui;ZHAO Zongsheng;ZHAO Feng;YU Peng;BAN Qian;WANG Zunbao

2012, 43(2): 306-313. doi:

Abstract ( 353 )

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The aim of this study was to analyze the immunological response to Mycoplasma pneumonia in Chinese Merino Sheep with different MBL genotype. MBL levels were measured in the four different individuals, 50 healthy Chinese Merino sheep with different genotypes were artifically infected with M. pneumonia under the same raising condition, while 20 sheep were the control group, the content of TNFα, IFNγ, C1, C3 in the serum were quantitatively assayed by ELISA on 1 day before infection (-1 d), and on 1 (the day of infection), 7, 14 and 21 day. The results showed that: 1)A and B type had low concentration of MBL, while C type’s MBL level was higher; 2) One week after infection, the percentage of IFNγ in the MBL A and MBL B was significantly lower than that of control group(P＜0.05), two weeks later, the level of C3 was lower and the TNFα was significantly higher(P＜0.05). Compared with the MBL A and MBL B at one week after infection, the level of IFNγ was significantly higher in the group of high concentration of MBL, two weeks later, the level of C3 increased and the level of TNFα was significantly lower (P＜0.05), the percentage of C1 had no significant difference among the groups (P＞0.05). Conclusion: there maybe some correlation between the lower concentration of MBL plasma and mycoplasma pneumonia, while there were some difference among the level of TNFα, IFNγ, C1, C3, the low MBL ones were likely to have more severe inflammatory response.

Effects of LBP on the Expression of Caspase3, Bcl2 and Bax Proteins in Spermatogenic Cells of Mice Pretreated with Bisphenol A

LI Xiaocai;SUN Xiaona;ZHONG Xiuhui;MA Aituan

2012, 43(2): 314-318. doi:

Abstract ( 333 )

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The aim of this experiment was to study the effects of LBP on the expression of Caspase3, Bcl2 and Bax proteins in spermatogenic cells of mice. Fifty adult male Kunming mice were randomly divided into five groups, which were A, B, C, D and E, with 10 mice in each group. Except the mice in group A which were injected with olive oil as control group, the mice in other four groups were intraperitonealy injected with 20 mg·kg-1 BPA for 7 days. Meanwhile, the mice in groups C, D, E were given different doses of LBP (50, 100 and 200 mg·kg-1) by intragastric administration consecutively for one week, the mice in control (A) group and model (B) group were treated with physiological saline. Histopathological changes of the testicular tissue were observed, immunohistochemical technique was applied to detect the expression of Caspase3, Bcl2 and Bax proteins. Results showed that BPA can enhance the expression of Caspase3 and Bax proteins (P<0.01), diminish the expression of Bcl2 (P<0.05). After supplement with different doses of LBP, the expression of Caspase3 were all significantly decreased in C, D and E groups than that in the model group (P<0.01). With the dose of LBP increasing, the expression of Bax was significantly decreased and the expression of Bcl2 gradually increased than that in the model group especially in the 200 mg·kg-1 LBP group (P<0.01). Compared with the model group, Bcl2/Bax ratio also increased obviously in groups B, C and D. Results suggest that LBP can restrain spermatogenic cell apoptosis through regulating the expression of apoptosisrelated genes, thus alleviate the reproductive damage induced by BPA on adult male mice.

Cloning and Analysis on Tissue Expression of GPR41 Gene in Dairy Goat

SUN Yuting;LUO Jun;ZHU Jiangjiang;ZHAO Wangsheng;LI Jun;ZHONG Yu;HAO Juan

2012, 43(2): 319-323. doi:

Abstract ( 369 )

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he aim of this study was to lay a foundation for understanding the physiological functions of dairy goat GPR41 gene by cloning GPR41 gene and analyzing its tissue expression level. Based on the published nucleotide sequence of GPR41 gene of bovine, human and mouse in GenBank, a pair of special primer was designed. The dairy goat GPR41 gene was amplified by RTPCR, the tissue specificity of dairy goat GPR41 gene were analyzed by realtime fluorescence quantitative PCR. The results showed that the coding region length was 978 bp, coding 325 amino acids. The sequence homology analysis of dairy goat GPR41 gene indicated that the homology of GPR41 of dairy goat was 96%, 78% and 74% compared with that of bovine，human and mouse, while the amino acid sequence homology was 97%,76% and 74% , respectively. The expression analysis of GPR41 gene revealed that dairy goat GPR41 gene mRNA is expressed abundantly in intestine tissue and, to a lesser degree, in mammary gland, but lowly in heart and kidney. These results suggest that GPR41 may play an important role in intestine and mammary gland tissue.

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