Preparation and Identification of Canine Parvovirus NY Strain VP2 Protein Polyclonal Antibody

This study was aimed to prepare canine parvovirus (CPV) VP2 protein polyclonal antibody.The recombinant expression vector pET28a-CPV-VP2 was constructed and transfromed into E.coli BL21 (DE3),the expression of recombinant proteins was induced by IPTG from which the fusion protein was identified by SDS-PAGE.The target protein was purified and emulsify with adjuvant,the prepared immunogen was inoculated into rabbit by subcutaneous injections to prepare of VP2 protein specific polyclonal antibody.The immuno-activity,titers,neutralization titers of the prepared polyclonal antibody were determined by immunoperoxidase monolayer assay (IPMA).The results showed that the expressed recombinant protein VP2 (rVP2) existed in the form of inclusion body with a molecular weight of 72 ku.The prepared polyclonal antibody titer was 1 600 dilution,the virus titer was 10⁷ TCID₅₀/mL,the neutralizing titer was 1∶2 884.The antibodies showed specific reaction with CPV.In conclusion,rVP2 specific polyclonal antibody showed wonderful immunocompetence,specificity and neutralizing activity,providing foundation for the development of genetic vaccine and clinical therapeutic method.     

Study on Expression Difference of IGFBP-5 Gene in Different Tissues of Kazakh and Yanqi Horses

The study aimed to explore the mRNA expression pattern of insulin-like growth factor binding protein-5 (IGFBP-5) gene in different tissues of Kazakh and Yanqi horses.The expression of IGFBP-5 gene in different tissues of heart,liver,spleen,lung,kidney,small intestine,large intestine,cecum,intercostal muscles,longissimus dorsi muscles,brachialis muscle and gluteus in two horses were detected by Real-time quantitative PCR and compared the mRNA expression in the same tissues of two breeds.The results showed that the expression of IGFBP-5 in longissimus dorsi muscle and brachialis muscle of two breeds were significantly higher than other tissues including heart,liver,spleen,lung,kidney,small intestine,large intestine and cecum (P<0.05),and was the lowest in large intestine.The expression of IGFBP-5 in kidney,small intestine,large intestine,cecum,longissimus dorsi muscle and brachialis muscles of Yanqi horse were higher than in the same part of Kazakh horse,and among those in longissimus dorsi muscle and large intestine of Yanqi horse were extremely significantly higher than in the same part of Kazakh horse (P<0.01),and in small intestine and cecum of Yanqi horse were significantly higher than in the same part of Kazakh horse (P<0.05).The test was for further researching the biological function of IGFBP-5 gene,and it could provide a theoretical basis for genetic improvement of production performance of horse in our country.     

Cloning and Bioinformatic Analysis of L1 Gene of BPV from Guizhou Province

In order to study and analyze L1 gene of bovine papillomavirus（BPV）in Guizhou province,the L1 gene of BPV-GZ01 strain was amplified,cloned and sequenced using bioinformatic softwares and methods,and the secondary structure,tertiary structure,B-cell preponderant epitope,conserved domains analysis, transmembrane domain and signal peptide of L1 gene were predicted.The results showed that the length of L1 gene was 1 494 bp,encoding 497 amino acids.The L1 gene of BPV-GZ01 strain shared an amino acid identities of 98.6%,99.4%,98.4%,94.4% and 91.3%,and a nucleotide identities of 99.1%,99.8%,99.4%,87.6% and 82.8% with those of BPV2,BPV2-SW01,BPV2-AKS01,BPV13 and BPV1 strains,respectively.The results of phylogenetic tree analysis indicated that there was a close relationship between BPV-GZ01 and BPV2-SW01 strains.The prediction of secondary structure of L1 protein indicated that the random coil,extended strand and alphahelix took a higher percentage.The L1 protein was supposed contain 6 potential antigen epitopes.And no transmembrane domains and no signal peptide were found.The tertiary structure of L1 protein was curved spiral structure.These results provided a theoretical basis for immunologic diagnosis and further research of nucleic acid vaccine of BPV.     

Cloning of Inhibin-α Gene and its mRNA Expression Pattern in the Ovary of Congjiang Xiang Pig

To reveal the relationship between inhibin-α(INHA) gene and the reproductive traits of Congjiang Xiang pig, INHA gene was cloned and sequenced taking the genomic DNA of Congjiang Xiang pigs as templates by polymerase chain reaction(PCR) method.The polymorphisms of INHA gene were tested in Congjiang Xiang pig populations with high-litter size and low-litter size using allele-specific PCR(AS-PCR) method.The expression profile of INHA gene in ovaries was detected from Congjiang Xiang pigs with high-litter yiled or low-litter yiled by Real-time PCR method.The complete coding region of INHA gene was 1095 bp in length, which coded for 364 amino acid residues.Compared with the known sequence, two candidate sites, G359A and A373G, were found out from exon 2 region of INHA gene in Congjiang Xiang pig.After investigation for the two sites in a large population, the frequency of alleles between two populations was not significant and without obvious relativity with the litter yiled of Congjiang Xiang pig.However, the INHA mRNA level in the ovary of Congjiang Xiang pig with high-litter yiled was higher than that with low-litter yiled.It suggested that INHA gene was much conserved, INHA gene expression level might be concerned for the regulation of ovary growth and follicle development in Congjiang Xiang pig breed.     

Cloning and Sequence Analysis of HA and NA Genes of One Strain of H6N6 Subtype Avian Influenza Virus Isolated from Guizhou Province

To investigate the epidemic situation of H6N6 subtype avian influenza virus (AIV) in Guizhou province,A/duck/Guizhou/013/2014 was isolated from Sansui duck in live poultry market of Guizhou in 2014,the hemagglutinin (HA) and neuraminidase (NA) genes of DK/GZ/14 were subjected to clone and sequence analysis.The results showed that HA gene had the highest nucleotide homologies (97.5%) with the duck-origin H6N6 subtype AIV isolated from Eastern China in 2009,and the strains of HA gene proteolytic cleavage sites was P-Q-I-E-T-R-G,which accordeol with the molecular characteristic of low pathogenic AIV (LPAIV).However,NA gene of A/duck/Guizhou/013/2014 had the highest nucleotide homologies (98.2%) with the duck-origin H6N6 subtype AIV isolated from Fujian in 2007.The phylogenetic tree showed that A/duck/Guizhou/013/2014 and Hunan strains located in the same branch,while three duck-origin H6N6 subtype AIV isolated from Guizhou in 2007 and A/duck/Guizhou/013/2014 located in the different branch for HA and NA genes in genetic evolution,which suggested that A/duck/Guizhou/013/2014 was far with the local H6N6 subtype.The results also clearly indicated that duck-origin H6N6 subtype AIV had genetic diversity in duck population in Guizhou.     

Development of A Dual Real-time PCR for the Rapid Detection of Listeria monocytogenes

To establish a rapid assay for Listeria monocytogenes(LM) detection,a Real-time PCR method was developed targeting iap gene of LM.The results showed that the test for 15 bacteria strains,only LM was positive,indicated that the method had high specificity.In addition,the sensitivity of Real-time PCR was 6.5 CFU/mL.Stability and reproducibility of the test showed that the coefficient of variation for the same sample repeat the Ct values were less than 2%.Furthermore,a total of 3 positive samples for LM were detected from 139 clinical samples by the method,which was in accordance with the testing result by GB 478930-2010 standard detection protocol.Therefore,the Real-time PCR method provides a novel rapid,sensitive and good repeatability detection method for LM infection.     

Expression Analysis of THBS3 Gene in Different Tissues and Skeletal Muscles at Different Periods from Landrace and Tongcheng Pigs

To study the expression pattern of THBS3 gene in different tissues and during skeletal muscle development, the THBS3 gene expression in different tissues and skeletal muscles during prenatal periods (33, 45, 65, 70 and 90 d) and postnatal periods (0, 9, 30, 60, 120 and 160 d) from Landrace and Tongcheng pigs were detected by Real-time quantification PCR.The results showed that THBS3 gene widely expressed in all tissues examined, exhibiting similar spatial expression patterns with expression peaks in lung in both pig breeds except in stomach and intestine.Moreover, although THBS3 gene showed a significant higher expression level in gestation than after birth in Landrace and Tongcheng pigs (P<0.05), it exhibited different expression patterns between Landrace and Tongcheng pigs, the expression peak was detected at gestation day 45 in Landrace pig, while was detected at gestation day 65 in Tongcheng pig.The results suggested that THBS3 gene involved in skeletal muscle growth and development in pigs, as well as the regulation of asynchronization of skeletal muscle development in different pig breeds.     

猪繁殖与呼吸综合征病毒分离株E11105 N基因的序列分析与原核表达

为研究PRRSV N蛋白的结构、功能以及N蛋白在病毒致病中的作用,以临床分离PRRSV毒株E11105为研究对象,采用Primer Premier 5.0设计一对特异性引物,经RT-PCR扩增出N基因片段,利用相关分子生物学软件对N基因序列进行分析;将N基因克隆连接到pColdⅠ原核表达载体上,经PCR、双酶切鉴定及序列测定后,得到重组质粒pColdⅠ-N,将pColdⅠ-N转入大肠埃希菌BL21(DE3)感受态细胞中,IPTG诱导表达后用SDS-PAGE蛋白电泳及Western blot验证分析。结果显示,分离株E11105N基因与北美洲型代表株VR-2332、欧洲型代表株Lelystad virus(LV)、中国2006年暴发的高致病性PRRSV代表株JXA1、中国代表株CH-1a的核苷酸序列同源性分别为93.3%、34.7%、99.2%、95.4%,氨基酸序列同源性分别为94.4%、15.3%、94.4%、99.2%;系统进化树显示,E11105株N基因与美洲型代表株VR-2332、中国高致病性JXA1毒株的亲缘关系较近;分离株E11105N基因所编码蛋白不存在跨膜区;二级结构主要以α-螺旋和无规则卷曲为主,分别占20.33%和63.41%;预测该蛋白可能存在5个较为明显的B细胞优势抗原表位。SDS-PAGE蛋白电泳结果表明,重组N蛋白主要存在于菌体沉淀中,分子质量约为16.7ku;Western blot结果显示,带His标签的重组表达蛋白能被His单克隆抗体识别,显色后条带约为16.7ku,与SDS-PAGE蛋白电泳的条带大小一致。     

广东水禽源大肠埃希菌耐药性及氨基糖苷类耐药基因研究

为了调查广东地区水禽源大肠埃希菌对氨基糖苷类药物耐药现状和耐药基因的流行情况,探索大肠埃希菌的氨基糖苷类耐药基因型与耐药表型之间的关系,本研究采用琼脂梯度稀释法测定251株广东地区水禽源大肠埃希菌对氨基糖苷类药物的耐药性和采用PCR方法检测耐药基因。药敏试验结果显示,大肠埃希菌对链霉素耐药较严重,鸭源和鹅源的耐药率分别为81.58%和75.43%,对庆大霉素和卡那霉素较敏感,耐药率分别为25%和54.86%,对阿米卡星最为敏感,其中鸭源的菌株耐药率仅为1.32%。PCR方法检测显示,aadA1和aph(3′)-1检出率较高,为84.6%和91.9%,表明携带aadA1和aph(3′)-1耐药基因的水禽源大肠埃希菌在广东地区呈流行趋势。耐药基因型与耐药表型相关性分析结果显示,耐药基因rmtB的携带与4种药物(庆大霉素、阿米卡星、卡那霉素和壮观霉素)耐药株的产生具有显著相关性,表明耐药基因rmtB对大肠埃希菌耐药株的产生起重要的作用。本试验结果可为指导广东地区水禽大肠埃希菌病的临床用药提供理论基础和研究大肠埃希菌的耐药基因提供相关的数据。     

应用分子靶点药物治疗犬肥大细胞瘤的研究进展

犬肥大细胞瘤因其恶性率和转移率都较高,并且常规化疗药物副作用较强,患犬生存期和生存质量显著下降,因此急需新型的药物进行治疗。近几年,治疗肿瘤的分子靶点药物凭借其疗效好、副作用低的特点,受到越来越广泛的关注,特别是在治疗犬肥大细胞瘤的应用中效果显著。论文重点介绍了欧美小动物临床最常用的伊马替尼、托赛拉尼、马赛替尼这3种以异常KIT为靶点的药物及其药物作用机理及药效,最后介绍了由c-kit基因二次突变所导致的耐药问题及其预防措施,期望能对我国小动物临床医师和科研工作者有所启发。