新型鸭呼肠孤病毒DH13株结构蛋白σB的原核表达及免疫原性分析

本研究旨在利用原核表达系统表达新型鸭呼肠孤病毒（NDRV） DH13株重组σB蛋白,并检测其免疫原性,为NDRV基因工程疫苗研制等提供物质材料。采用RT-PCR方法扩增获得NDRV σB基因,构建原核表达重组质粒pET-30a-σB和pET-32a-σB,将2个重组质粒转化大肠杆菌BL21（DE3）感受态细胞,利用IPTG诱导获得相应的重组蛋白,通过SDS-PAGE分析重组蛋白的表达形式。纯化无His的σB蛋白,并以此蛋白作为免疫原免疫新西兰白兔,制备兔源多克隆抗体。利用Ni²⁺柱亲和层析法纯化含His的σB蛋白,以此蛋白作为检测原,通过间接ELISA方法测定兔源多克隆抗体效价;Western blotting鉴定多克隆抗体与重组蛋白的特异性识别能力。结果显示,PCR扩增获得大小约为1 100 bp的σB基因片段。SDS-PAGE结果显示,高效表达出了2种重组σB蛋白,大小分别约为41和46 ku,均以包涵体形式存在。间接ELISA结果显示,制备的多克隆抗体效价达1∶204 800,能特异性地识别原核表达的重组蛋白,表明重组无His的σB蛋白具有良好的免疫原性。Western blotting特异性鉴定结果显示,含His的σB蛋白和无His的σB蛋白均与制备的兔源多克隆抗体发生特异性反应,表明原核表达得到的重组σB蛋白具备良好的反应原性。本试验利用原核表达系统成功地表达出了重组σB蛋白,并证实了重组NDRV σB蛋白具有良好的免疫原性及反应原性,该结果将有助于后续对NDRV σB蛋白生物学功能的鉴定、NDRV诊断用抗原的制备及NDRV新型疫苗的研制。     

瘦肉型猪组合配套杂交效果研究

为探索瘦肉型猪组合配套杂交方法和效果而进行试验。试验采用HN121（温氏杜洛克猪）、HN212（法系皮特兰♂×温氏杜洛克♀）为父本,以HN204（温氏大白♂×美系长白♀）为母本,对HN323（HN121×HN204）和HN424（HN212×HN204）两个组合配套进行研究。结果表明,HN424组合与HN323组合相比母猪的窝产仔数、窝产活仔数、仔猪初生窝重、21日龄仔猪成活率、21日龄窝重等繁殖性状差异不显著（P>0.05）;HN424组合仔猪的体重、背膘厚降低5.63%（P<0.05）,瘦肉率提高4.95%（P<0.05）,眼肌面积增大33.08%（P<0.01）,肉色评分降低7.19%（P<0.05）,大理石纹评分降低11.30%（P<0.01）,滴水损失率提高10.63%（P<0.01）,其他遗传性状差异不显著;HN424组合与HN204二元母系相比,日增重提高9.26%（P<0.05）,背膘厚减少16.41%（P<0.01）,料肉比减少8.02%（P<0.05）;HN323组合与HN204二元杂交母系相比,日增重提高7.20%（P<0.05）,背膘厚减少11.43%（P<0.01）,料肉比降低5.35%（P<0.05）。说明采取四元杂组合和三元杂交组合均对“大×长”（HN204）二元杂交有显著的遗传改良作用,“皮×杜×长×大”四元杂组合可显著提高猪的瘦肉率、眼肌面积,但肉色、大理石纹、滴水损失率等肉质指标变差,建议在“皮×杜×长×大”四系组合配套杂交中加入能改良肉质基因的猪种。     

渗灌对日光温室番茄栽培环境的影响

研究了渗灌和漫灌对日光温室番茄栽培环境及番茄产量的影响。结果表明,渗灌下,土壤密度比漫灌低7.59%,差异极显著;土壤总孔隙度比漫灌高9.72%,差异显著;土壤黏粒和粉粒高于漫灌,而砂粒和细粗砾低于漫灌;观测时段内,渗灌下平均气温、10cm处土壤平均温度较漫灌分别高1.55℃、0.86℃,地表平均温度比漫灌低0.14℃;渗灌下温室空气相对湿度较漫灌低11.86%,以10:00—20:00差值最大;渗灌下番茄产量比漫灌高8.60%,且差异显著。     

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.     

Effects of Supplemental Complex Water Soluble Vitamin on Production Performance,Blood Biochemical Parameters and Oxidation Resistance of Dairy Cows

The objective of the study was to investigate the effects of supplemental complex water soluble vitamins on production performance, blood biochemical parameters and oxidative resistance of dairy cows in spring and summer.The experiment was designed by 2×2 factorial randomized blocks design.Eighty healthy dairy cows with similar parity and body weight were randomly divided to four groups, with 7 days of adaptation and 70 d experimental phase.The dairy cows in group Ⅲ and Ⅳ were fed a basal diet supplemented with complex water soluble vitamins in spring.The dairy cows in group Ⅱ and Ⅳ were fed a basal diet supplemented with complex water soluble vitamins in summer.The results showed that the ADFI and daily milk yield of dairy cows receiving water soluble vitamin in spring were extremely significantly higher (P<0.01);The ADFI of dairy cows receiving water soluble vitamin was significantly higher in summer (P<0.05) and daily milk yield were extremely significantly higher (P<0.01);The butterfat rate was extremely significantly increased and SCC was extremely significantly decreased in spring and summer (P<0.01);Supplementation of water soluble vitamin was also significantly increased LYM (P<0.05).In summer, T-AOC, T-SOD were extremely significantly increased (P<0.01), and TP, CK, LDH, TP, HDL and ALB were extremely significantly decreased (P<0.01) by water soluble vitamin supplementation than that of control group.In conclusion, complex water soluble vitamins could increase the production performance and non-specific immune function, improve the oxidative resistance and relieved the heat stress of the dairy cows.     

Isolation and Identification of Pathogenic Bacteria Causing Dairy Cow Mastitis in Liaoning and Drug Sensibility Test of Pathogenic E.coli

In order to understand the main bacterial pathogen species causing dairy cow mastitis in Liaoning, as well as the characteristics of drug sensitivity of the pathogenic E.coli,the milk samples from 75 dairy cows with clinical manifestations for mastitis in certain large-scale dairy farm in Liaoning were collected.The bacteria in milk were cultured and isolated with biochemical methods and in vitro drug sensitivity tests were processed with the isolated E.coli strains.The results showed that the main bacterial pathogen for dairy cow mastitis were E.coli（separation rate 58.7%),S.aureus（64.0%）and S.agalactiae（54.7%),and multiple infection including double and triple infection were identified.The drug sensitivity tests on the isolated E.coli indicated that the E.coli isolates were highly resistant to sulfonamides（resistance rate>85%）and chloramphenicol（resistance rate>30%),and they were relatively low resistant to ampicillin（9.5%),ciprofloxacin（9.5%),ceftiofur（7.1%）and ofloxacin（4.8%).The results was able to provide reliable theoretical basis for prevention and control of dairy cow mastitis in Liaoning area.     

光温调控对切花菊"优香"养分利用及品质的影响

光照与温度影响着切花菊的花芽分化进程,是切花菊畸形花形成的主要环境因子。如何在夏季高温期有效调控设施内的光照与温度对减少畸形花率、提高产品出成率及保鲜期具有重要意义。现以解决企业关键技术需求为出发点,以夏秋菊品种"优香"为试材,在设施生产条件下,利用温湿度自动监控系统、光温调控设施进行了遮光、降温处理,旨在为高温期的切花菊生产光温调控技术提供支撑。结果表明:采收期遮光与降温处理(8 127kg/hm~2)与未调控处理(6 270kg/hm~2)相比,地上部干物质累积量差异不显著,但切花菊的吸磷量显著增加了7.1kg/hm~2,氮和钾的吸收量差异不显著。经光温调控后,切花菊畸形花率由未调控下的42.5%降低至20.1%,外观品质基本一致,清水瓶插寿命延长了2~3d。基于自动温湿度监控系统的切花菊设施生产光温调控技术对切花菊外观品质影响不大,且在未影响切花菊正常干物质累积及养分利用的前提下,可有效降低花朵畸形花率、提高保鲜期和优级花的产出。     

梨新鲜度与其挥发性成分的关系研究

以砀山酥梨为试材,采用顶空固相微萃取-气相色谱-质谱联用(HS-SPME-GC-MS)技术,分析了砀山酥梨在(4±1)℃条件下挥发性成分的变化,检测了砀山酥梨可溶性固形物、硬度、酸度等新鲜度指标,探讨了砀山酥梨新鲜度与其挥发性成分之间的关系。结果表明:新鲜砀山酥梨在(4±1)℃条件下的保鲜时间为42d,其特征性气味成分主要为正己醇、1-辛醇;不新鲜砀山酥梨的特征性气味成分为2-甲基丁基乙酸酯、硅酸四乙酯、异戊醇、正己醇、1-辛醇、香叶基丙酮,且砀山酥梨由新鲜变为不新鲜时的阈值分别为1.57、2.74、1.39、15.15、211.13、2.36μg/L。试验结果为通过气味判断砀山酥梨的新鲜度提供了理论参考。     

贵州魔芋软腐病菌多基因分子鉴定及其致病力分析

为明确贵州魔芋软腐病菌种类?致病力及分布特点, 采用组织分离法对贵州主要魔芋种植区软腐病样进行病原菌分离, 对icdA, mdh, mtlD, proA, rpoS 等5个管家基因进行了扩增?序列测定, 分别用单基因和多基因联合系统发育树对病菌进行鉴定, 同时采用组织块接种方法测定了不同菌株的致病力?通过组织分离法共分离魔芋软腐病菌株47株; 采用5个管家基因进行分子鉴定, 将病菌分别鉴定为海芋果胶杆菌Pectobacterium aroidearum?胡萝卜果胶杆菌Pectobacterium carotovorum和方中达迪基氏菌Dickeya fangzhongdai 3个种, 其中海芋果胶杆菌P.aroidearum为贵州魔芋软腐病主要致病菌, 占分离菌株的70%, 广泛分布在多个地区; 其次为方中达迪基氏菌D. fangzhongdai, 占分离菌株的28%, 也普遍存在于贵州各魔芋种植区; 胡萝卜果胶杆菌P. carotovorum最少, 占分离菌株的2%?致病力测定结果表明, 菌株间致病力存在一定的差异, 其中海芋果胶杆菌不同菌株之间致病力差异较大, 低?中?高致病力菌株都有, 方中达迪基氏菌差异较小, 仅有中?高致病力菌株?本研究确定了贵州魔芋软腐病菌种类?致病力及在贵州的分布特点, 首次报道了海芋果胶杆菌?方中达迪基氏菌是贵州魔芋软腐病的主要病原菌, 进一步加深了对魔芋软腐病及其发生流行的认识, 为软腐病的科学防控提供了科学依据?