猪圆环病毒2型重组Cap蛋白在昆虫杆状病毒中的表达

猪圆环病毒2型(PCV2)基因组包含2个开放阅读框架(ORFs),其中ORF1编码病毒复制相关蛋白(Rep),ORF2编码病毒衣壳蛋白(Cap).为了在昆虫细胞表达Cap蛋白,本研究采用PCR扩增PCV2-ORF2编码基因,将PCR产物插入到昆虫杆状病毒转移载体上,经酶切反应及DNA序列分析得到验证.重组质粒与昆虫杆状病毒线性基因组混合,转染到昆虫细胞(Sf-21)进行基因重组,经3次病毒蚀斑克隆,获得高效表达Cap蛋白的重组杆状病毒,毒价可达1.28×108pfu/mL.采用SDS-PAGE凝胶电泳分析表明,重组Cap融合蛋白分子量为32.8 ku,占总蛋白含量的17.2%.免疫印迹试验分析表明,重组Cap蛋白与PCV2阳性血清产生特异性反应,证明该重组蛋白具有良好的免疫活性反应.本研究为进一步进行该病毒分子诊断、亚单位疫苗以及分子生物学等研究奠定了基础.     

杆状病毒表达系统融合表达猪圆环病毒2型Cap蛋白与口蹄疫病毒VP1蛋白及免疫原性评估

猪圆环病毒2型(porcine circovirus type 2,PCV2)Cap蛋白是病毒的主要结构蛋白,能诱导宿主产生特异性免疫应答。口蹄疫病毒(foot-and-mouth disease virus,FMDV)VP1蛋白是病毒的主要免疫原性蛋白,能刺激机体产生中和抗体。利用杆状病毒表面展示系统,将这2种病毒蛋白与猪CD40配体(CD40 ligand,CD40L)进行融合表达并展示在杆状病毒表面,构建了rv Ac-Cap、rv Ac-VP1和rv Ac-Cap-VP1 3种重组杆状病毒。通过SDS-PAGE和Western blotting检测3种重组杆状病毒感染的Sf9细胞总蛋白,证明3种融合蛋白成功表达。纯化的重组杆状病毒颗粒以1×108pfu/只的剂量免疫接种BALB/c小鼠,通过间接ELISA检测血清中Cap和VP1的抗体浓度,证明表面展示有Cap和VP1的重组杆状病毒能激发小鼠产生较高水平的抗体,与PCV2和FMDV商品化疫苗免疫组的抗体浓度差异不显著(P0.05),表明该重组杆状病毒具有较好的免疫原性。     

表达猪圆环病毒2型Cap蛋白的重组杆状病毒的构建及鉴定

利用Bac-to-Bac杆状病毒表达系统成功表达了猪圆环病毒2型Cap蛋白,并对该蛋白的生物活性进行鉴定。PCR获得Cap基因并克隆入pFastBacTMⅠ载体质粒,重组质粒pFastBacⅠ-Cap转化DH10Bac感受态细胞,通过蓝白斑筛选获得重组杆粒rBacmid-Cap。以脂质体法将重组杆粒转染对数生长期的Sf9细胞,获得重组杆状病毒。电镜下可见典型的杆状病毒,SDS-PAGE显示,重组病毒表达的Cap蛋白约28ku,间接免疫荧光试验证明,Cap蛋白良好表达并具有免疫反应性,为猪圆环病毒亚单位疫苗的研究奠定了基础。     

猪圆环病毒2型ORF2基因在Sf9细胞中表达及免疫原性研究

利用Bac-to-Bac杆状病毒表达系统表达猪圆环病毒2型(PCV2)核衣壳(Cap)蛋白。将优化合成的PCV2 ORF2基因克隆到杆状病毒转移载体p Fast HTA中,并将鉴定正确的重组质粒p Fast HTA-Cap2转化至大肠杆菌感受态细胞,经蓝白斑筛选得到含有目的基因的重组杆状病毒质粒(r Bac-Cap2),转染至Sf9细胞,获得重组杆状病毒,对感染重组杆状病毒的细胞培养物进行重组蛋白的表达,进行SDS-PAGE和Western blot分析,并对表达的重组蛋白进行小鼠免疫试验及其病毒血清中和试验。SDS-PAGE分析表明,优化合成的PCV2 ORF2基因得到表达,蛋白分子质量大小为33 ku;Western blot证实重组蛋白能够识别抗PCV2阳性血清,表明重组蛋白具有反应原性;小鼠免疫试验结果显示,该蛋白能刺激机体产生特异性抗体,具有较好的免疫原性;病毒血清中和试验证实,抗PCV2 Cap血清抗体具有中和病毒的活性,中和效价为1∶42。该蛋白在杆状病毒系统中的成功表达,为PCV2感染的诊断及亚单位疫苗的研制奠定基础。     

表达猪圆环病毒2型Rep蛋白的重组杆状病毒的构建与鉴定

猪圆环病毒2型(PCV2)是近年来新发现的引起断奶仔猪多系统衰弱综合征的主要病原,含有两个主要的阅读框ORF1和ORF2,分别编码复制相关蛋白(Rep)和核衣壳蛋白(Cap)。为了探究ORF1基因结构及其与功能的关系,本研究利用Bac-to-Bac杆状病毒表达系统将PCV2的ORF1基因克隆到杆状病毒转移载体pFastBacTMDual中,获得重组转移载体pFBD-ORF1,再将其转化进含穿梭载体Bacmid的感受态细胞DH10Bac中,经蓝白菌落筛选获得含有ORF1基因的重组穿梭载体rBacmid-ORF1,经脂质体介导转染昆虫细胞sf9,获得重组杆状病毒rAc-Rep,SDS-PAGE和Western-blotting分析可见大小约为36ku的特异性条带,表明rAc-Rep在sf9中成功表达了PCV2Rep蛋白。     

3个猪圆环病毒1型衣壳蛋白型特异性抗原表位的鉴定

猪圆环病毒1型(PCV1)感染猪肾细胞PK-15,血清学和遗传学研究结果表明,PCV1广泛存在于家猪中。本研究用杆状病毒系统表达的重组PCV1衣壳蛋白(PCV1-Cap)诱导产生单克隆抗体(mAbs)和多克隆抗体(pAbs)。用PEPSCAN分析来鉴定mAbs和pAbs识别的PCV1-Cap表位,结果精确定位了对PCV1-Cap特异的3个B细胞线性表位,包括85 GGTNPLP91、162 FTPKPELDKTIDWFH-PNNK180和219 YVQFREFILKDPLNK233。本研究结果将有助于PCV1和PCV2的抗原差异调查和鉴别诊断。     

猪圆环病毒2型Cap蛋白在杆状病毒表面的展示

本试验将猪圆环病毒2型(PCV2)去核定位区ORF2基因克隆到杆状病毒表面展示转座载体pBacSC中,转化DH10Bac大肠杆菌感受态,经抗性及蓝白斑筛选得到含ORF2基因的重组杆状病毒DNA,以脂质体介导法将此重组DNA转染Sf9昆虫细胞,获得重组杆状病毒。对重组病毒进行Western-blot分析和免疫金电子显微镜检测。结果表明,PCV2 Cap蛋白在重组杆状病毒中获得表达;免疫金电子显微镜观察表明,重组蛋白展示在杆状病毒囊膜上。本试验成功构建表面展示PCV2 Cap蛋白的重组杆状病毒,为下一步应用重组杆状病毒作为亚单位疫苗奠定基础。     

猪圆环病毒3型新疆株Cap蛋白的原核表达及纯化

本研究旨在通过构建pGEX-4T-1-Cap原核表达载体,制备猪圆环病毒3型（PCV3）重组核衣壳（Cap）蛋白抗原。将密码子优化的PCV3 Cap全基因插入pGEX-4T-1载体,构建重组质粒pGEX-4T-1-Cap,转化大肠杆菌BL21（DE3）感受态细胞中,筛选出最佳诱导条件,通过谷胱甘肽琼脂糖树脂纯化重组Cap蛋白。结果表明,成功构建重组质粒pGEX-4T-1-Cap;在IPTG终浓度为0.1 mmol/L、16 ℃诱导20 h的条件下,可获得大量可溶性重组Cap蛋白,SDS-PAGE结果表明分子质量在51.6 ku,与预期相符;Western blotting分析表明经纯化的Cap蛋白与鼠抗GST单克隆抗体、PCV3兔源阳性血清呈阳性反应,进一步证实了重组蛋白的表达。本研究表达并纯化了PCV3 Cap重组蛋白,为新型基因工程亚单位疫苗的研制和ELISA血清学诊断方法的建立奠定了基础。     

猪圆环病毒Cap蛋白的分泌表达及其免疫原性研究

探索利用杆状病毒表达系统(Bac-to-Bac expression system)分泌表达猪圆环病毒2型(PCV2)Cap蛋白及将其作为亚单位疫苗的应用价值。将PCV2 Cap基因克隆到已插入蜂毒信号肽(Melittin)的转移载体pFastBacⅠ中,将鉴定正确的重组质粒(pFastBAC-Cap)转化至DH10Bac大肠杆菌感受态细胞,经抗性及蓝白斑筛选得到含Cap基因的重组杆状病毒DNA(Bacmid-Cap),转染提取的Bacmid-Cap DNA转染至sf9昆虫细胞,获得重组杆状病毒(rBac-Cap),应用无血清培养的High Five细胞进行重组蛋白的表达及条件优化。重组蛋白通过聚丙烯酰胺凝胶电泳分析(SDS-PAGE)证明:在重组杆状病毒感染的High Five昆虫细胞中获得分泌表达,并可产生较高浓度的重组蛋白;Western blotting结果显示:重组蛋白可被猪PCV2的特异性抗体所识别,表明重组蛋白具有反应原性;应用重组蛋白免疫BALB/C小鼠试验结果显示:该蛋白可刺激小鼠产生较高水平的特异性抗体。该研究成功分泌表达了PCV2 Cap蛋白,该蛋白能够刺激机体产生免疫应答,为PCV2亚单位疫苗的研制打下基础。     

猪圆环病毒2型Cap蛋白家蚕表达体系的构建与鉴定

为了用家蚕杆状病毒表达系统表达猪圆环病毒2型(PCV2)Cap蛋白,将不同株系的PCV2 Cap蛋白基因、人工改造的Cap蛋白突变体基因克隆到家蚕杆状病毒转移载体,用共转染技术成功构建了重组杆状病毒.将重组杆状病毒感染家蚕,成功获得了大量Cap蛋白的表达产物.结果表明,纯化蛋白主要为病毒空衣壳粒子,推算PCV2空衣壳在...     

Ruminal protein degradation and protein value of feeds

The relationship between dry matter (DM) degradation and crude protein (CP) degradation in the dairy cow's rumen was determined with a view to defining the protein value of feeds for ruminants. The nylon bag technique was applied for these studies. For all the feeds investigate (green fodder and preserves from cocks-foot, ryegrass, alfalfa/grass and meadow grass, as well as alfalfa, extracted soybean meal) a significantly positive relationship was found to exist between the levels of DM and CP degradation (r = 0.73 to 1.0). The regression coefficient b1 (CP degradation as regressor) was found to average 0.87. The positive relationship between DM degradation and CP degradation implies that microbial protein amount and unfermented feed protein at the duodenum are negatively correlated. Model calculations show that, on account of the compensation between microbial protein and feed protein at the duodenum, in feeds with a CP concentration below 200 g/kg DM, the extent of ruminal protein degradation does not exert a marked influence on duodenal protein passage. The partial calculation of the duodenal protein supply on the basis of undegraded feed protein and microbial protein, as practiced in the new models of protein evaluation, leads to systematic errors unless the relationship between DM degradation and CP degradation is considered.     

Hypolipidaemic effects of potato protein and fish protein in pigs

This study was performed to assess the effects of potato protein and fish protein on concentrations of lipids in plasma and lipoproteins and the expression of genes involved in lipid metabolism in pigs used as an animal model. Therefore, 27 young male pigs with an average body weight of 22 kg were fed diets supplemented with protein extracted from potatoes (containing 849 g protein/kg dry matter), Alaska Pollack fillet as a source of fish protein (containing 926 g crude protein/kg dry matter) or casein which was used as control, for 3 weeks. Diets were formulated to supply identical amounts of each protein to the pigs by the three protein sources, namely 116 g/day in first week and 150 g/day in the second and third week. Pigs fed potato protein had lower concentrations of cholesterol in plasma and LDL than pigs fed casein (p < 0.05); no effect was observed on concentrations of HDL cholesterol and triglycerides. Pigs fed fish protein had lower cholesterol concentrations in plasma, LDL and HDL, and lower triglyceride concentrations in triglyceride-rich lipoproteins than pigs fed casein (p < 0.05). mRNA concentrations of genes involved in bile acid synthesis and cholesterol uptake were higher in pigs fed fish protein than in pigs fed casein (p < 0.05); no effect on these genes was observed in pigs fed potato protein. Expression of genes involved in lipogenesis and fatty acid oxidation was not altered by fish protein. In conclusion, this study shows that fish protein and potato protein lower plasma cholesterol concentrations in pigs. The hypocholesterolaemic effect of fish protein might be in part caused by a stimulation of bile acid synthesis; the reason for the hypocholesterolaemic effect of potato protein requires further elucidation.     

Influence of supplemental protein source and protein concentration on ruminal and intestinal digestion in heifers

Six pregnant Holstein heifers fitted with ruminal cannulas and T-type duodenal cannulas were used in a 6 x 6 latin square design experiment to determine whether diets formulated on a rumen undegraded CP (UDP) equivalent basis would provide a more accurate estimate of protein quality for ruminants. Six diets (barley [B]/brome-alfalfa hay-based) were formulated to contain three concentrations of CP (14.0%, 16.5% and 19.0%) and three protein sources (canola meal [CM], meat and bone meal [MBM] and soybean meal [SBM]). The six diets were B, 14% CP, CM, 16.5% CP; SBM, 16.5% CP; MBM, 16.5% CP; CM, 19% CP; and SBM, 19% CP. The diets were formulated so that the 16.5% CP diets were equivalent on a CP basis, whereas the MBM16.5, CM19 and SBM19 were equivalent on a UDP basis. Diets were compared with regard to protein degradability in the rumen and protein flow to, and digestion in, the intestine. Animals fed the CM and SBM diets had higher (P less than .05) ruminal levels of branched-chain VFA than the control diet. Ruminal ammonia nitrogen (N) concentrations were affected (P less than .05) by supplemental protein source and concentration (8.8, 10.9, 11.2, 11.2, 13.2 and 17.7 mM for B14, CM16.5, SBM16.5, MBM16.5, CM19 and SBM19, respectively). Ruminal OM digestion was affected (P less than .05) by protein source MBM16.5, which was lower than protein source in all other diets. Total N flow to the small intestine for the three diets formulated on a UDP equivalent basis was 224.0, 225.6 and 241.1 g N/d for MBM16.5, CM19 and SBM19, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of protein compositions and surface protein epitopes of Anaplasma centrale and Anaplasma marginale.

Protein composition was compared and epitopes were analyzed among the isolates of Anaplasma centrale and A. marginale by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting using bovine antisera and monoclonal antibodies, and enzyme-linked immunosorbent assay. Common and unique proteins were found among the isolates. All isolates tested had a major surface protein with an apparent molecular weight of 38 to 40 kilodalton which had slight molecular size variations between species. This protein was also a dominant immunogen to the host. At least two species-common epitopes, one of which might contain carbohydrate(s), were present on the major surface protein. One species-specific epitope was identified on the major surface protein of A. marginale isolates.     

Energy and protein metabolism in pregnant sows fed two levels of dietary protein

The present study was performed to quantify the energy and nutrient metabolism of pregnant sows fed high (HP) or low (LP) dietary protein [18.3 vs. 13.5% of dry matter (DM)]. A total of nine sows (four on HP and five on LP diet) were subjected to balance and respiration trials four times during their second pregnancy (approximately on days 30, 61, 80 and 104 of gestation). The digestibility of protein (83.0 vs. 79.9) (p < 0.01) and energy (84.9 vs. 83.7%) (p < 0.05) was higher for the HP diet. Daily intake of metabolizable energy (ME) and retained energy (RE) were similar for the two groups, with an average of 28.37 MJ ME and 3.94 MJ RE, respectively. Heat production (HE) measured according to the respiratory quotient (RQ) and carbon-nitrogen (CN) method was similar (464 vs. 454 kJ/kg 0.75/day, respectively). Sows fed HP retained more energy in protein (3.33 vs. 2.00 MJ/day) (p < 0.001) and tended to retain less energy in fat (1.59 vs. 2.50 MJ/day) than LP sows. Retained nitrogen (N) (22.3 vs. 13.4 g/day) (p < 0.001) and utilization of N (retained/digested) (45.2 vs. 38.1%) was higher for HP sows compared with LP sows. In late pregnancy, retained N, retained fat, HE and oxidation of carbohydrates increased, while oxidation of fat was reduced to zero. In conclusion, both diets provided adequate N for retention in maternal tissue and conception products. In spite of the lower utilization of N in LP sows, the N excretion was depressed by 5.6 g/day compared with HP sows, because of the lower N intake.     

寡肽在蛋白质营养中的作用及功能大豆寡肽蛋白饲料的开发

1传统蛋白质消化、吸收理论的局限性传统的蛋白质消化、吸收理论认为,蛋白质在肠道中最终都被消化成氨基酸,然后通过肠壁被机体吸收利用。根据这一理论,蛋白质仅为动物机体提供氨基酸,即蛋白质的营养就是氨基酸的营养。为使畜禽获得最佳生产性能,     

不同蛋白质来源的日粮对瘤胃发酵特性及蛋白质消化的影响

以4只安装永久性瘤胃瘘管的白山羊作为采集瘤胃液的供体,用体外法研究了4种不同蛋白质来源（玉米-豆粕型、玉米-豆粕-棉粕型、玉米-豆粕-菜粕型、玉米-棉粕-菜粕型）的日粮对不同培养时间的发酵底物的干物质和蛋白质消化率、产气量、瘤胃pH值和NH3-N质量浓度的影响。结果表明：经24 h人工瘤胃培养后,干物质和蛋白质的消化率由高到低顺序依次为：玉米-豆粕型>玉米-豆粕-棉粕型>玉米-豆粕-菜粕型>玉米-棉粕-菜粕型。发酵底物产气量由高到低依次为：玉米-豆粕-菜粕型>玉米-豆粕-棉粕型>玉米-豆粕型>玉米-棉粕-菜粕型。瘤胃NH3-N质量浓度由高到低依次为：玉米-豆粕-棉粕型>玉米-豆粕-菜粕型>玉米-豆粕型>玉米-棉粕-菜粕型。 各组的瘤胃pH值基本一致,处于正常瘤胃生理范围。