Establishment of Indirect ELISA Method for Detecting Recombinant Truncated N Protein of Porcine Epidemic Diarrhea Virus

In order to establish an indirect ELISA to detect antibody of porcine epidemic diarrhea virus (PEDV).The experiment using the recombinant and purified truncated N protein as antigen expressed in E.coli BL21(DE3),the indirect ELISA was named rnPED-ELISA.The recombinant truncated N protein antigen showed no cross-reaction with the positive sera of other 7 kinds of swine diseases,CV%of intro-batch duplicativity test and inter-batch duplicativity test were less 13%;Sensitivity and specificity of rnPED-ELISA relative to SN were 93.33% and 90.00%,respectively;rnPED-ELISA compared with TSZ PEDV antibody diagnosis Kit,91.67% concordance was obtained.200 serum samples were detected by this method,the total masculine ratio was 69.5%.Therefore,this rnPED-ELISA based on recombinant truncated N protein antigen had good sensitivity and specificity,could afforded a simple and rapidmeans for assessment of vaccination in the field and investigation of PED epidemiology.     

Indirect fluorescent antibody techniques for demonstration of trout viruses and corresponding antibody

Two variations of the indirect fluorescent antibody technique (FAT) have been utilized in the work concerning two important virus diseases of trout, viral haemorrhagic septicaemia (VHS) and infectious pancreatic necrosis (IPN). A “two layer” indirect FAT allowed demonstration of the respective viruses in cell cultures and a “three layer” indirect FAT allowed demonstration of trout antibody to the viruses. Antibody, by means of the latter technique, could be demonstrated only in artificially immunized trout.     

Ⅰ群禽腺病毒4型IgG抗体间接ELISA检测方法的建立

利用纯化的 Ⅰ 群禽腺病毒4型重组Hexon蛋白,通过各反应条件筛选,建立了 Ⅰ 群禽腺病毒4型IgG抗体间接ELISA检测方法.结果显示,抗原最佳包被浓度为1μg/mL;最佳封闭液为1% 牛血清白蛋白;待检血清最佳作用浓度是400倍稀释,37℃孵育60 min;酶标抗体的最佳作用条件为稀释度1:8000,37℃孵育60 min;最佳显色时间为10 min.用所建方法对160只临床鸡只进行检测,同时与拭子PCR检测对比,一致性较好,表明该方法可有效用于临床检测.     

羊种布鲁氏菌wzt基因的原核表达及间接ELISA方法的建立

为建立检测血清中布鲁氏菌抗体的间接ELISA方法,本试验采用PCR技术从羊种布鲁氏菌QY1菌株中扩增得到wzt基因片段,连接到pET-30a载体上,构建质粒pET-30a-wzt,将鉴定正确的质粒转化E.coli BL21（DE3）感受态细胞,经原核表达系统对其进行表达,表达产物用SDS-PAGE和Western blotting进行分析后,用亲和层析镍柱纯化wzt重组蛋白备用。以wzt重组蛋白为检测抗原,逐步优化条件后建立布鲁氏菌间接ELISA检测方法。结果显示,试验成功构建了pET-30a-wzt原核表达载体,并在BL21（DE3）宿主菌中表达;SDS-PAGE和Western blotting结果表明,重组蛋白约为35 ku,表达形式为上清,条带单一、无杂带,有很好的反应原性和特异性。ELISA优化试验确定了最佳包被浓度为15 μg/mL,血清最佳稀释度为1：80,酶标抗体的最佳稀释度为1：5 000;通过检测24份阴性样品确定临界值,当样品D_{450 nm}值≥ 0.30为阳性,样品D_{450 nm}值<0.30时为阴性;特异性试验表明,该方法不与小肠耶尔森菌、大肠杆菌发生交叉反应;批内及批间变异系数均<10%;用该方法对120份血清样本进行检测,并与虎红凝集试验进行相符性验证,符合率为96%。本试验建立的间接ELISA方法为布鲁氏菌病的检测提供了可靠的技术手段。     

Determination of net energy content of soybean oil fed to growing pigs using indirect calorimetry

The objectives of this experiment were: (i) to determine the net energy (NE) of soybean oil (SBO) fed to growing pigs using indirect calorimetry (IC); and (ii) to evaluate the effects of inclusion rate of SBO on heat production, oxidative status and nutrient digestibility in growing pigs. Eighteen growing barrows were allotted to three diets based on completely randomized design with six replicate pigs (period) per diet. Diets included a corn‐soybean meal basal diet and two test diets containing 5% or 10% SBO at the expense of corn and soybean meal. During each period, pigs were individually housed in metabolism crates for 14 days, including 7 days to adapt to feed, metabolism crate and environmental conditions. On day 8, pigs were transferred to the open‐circuit respiration chambers for measurement of daily O₂ consumption and CO₂ and CH₄ production. During this time, pigs were fed one of the three diets at 2.4 MJ metabolizable energy/kg body weight (BW)^0.6/day. Total feces and urine were collected and daily total heat production (THP) was measured from days 9 to 13 and fasted on day 14 to evaluate their fasting heat production (FHP). The results show that trends of decreased apparent total tract digestibility of neutral detergent fiber (linear, P = 0.09) and acid detergent fiber (linear, P = 0.07) were observed as the content of dietary lipids increased. The average THP for the three diets were 1326, 1208 and 1193 kJ/kg BW^0.6/day, respectively. The FHP of pigs averaged 843 kJ/kg BW^0.6/day and was not affected by diet characteristics. A reduction of the respiratory quotients in the fed state as the inclusion level of SBO increased was observed. In conclusion, the NE values of SBO we determined by indirect calorimetry were 33.45 and 34.05 MJ/kg dry matter under two inclusion levels. THP could be largely reduced when SBO is added in the feed, but the THP of SBO included at 5% in a corn‐soybean meal diet is not different from the THP of SBO included at 10%.     

检测牛乳κ-酪蛋白的间接竞争ELISA法与间接ELISA法比较

试验旨在建立一种快速简单检测牛乳κ-酪蛋白（κ-CN）含量的酶联免疫吸附（ELISA）方法,为解决牛乳蛋白掺假问题提供一定的技术支持。以牛乳κ-CN为包被抗原,以酶标抗体（HRP-IgG）为检测抗体分别建立间接竞争ELISA法和间接ELISA法,并对两种检测方法进行分析比较。结果表明：对于间接竞争ELISA法,κ-CN抗原包被浓度为2.5 μg/mL,线性范围为62.5~1 000.0 ng/mL,变异系数< 2%,回收率在98.46%~101.68%;对于间接ELISA法,κ-CN抗原包被浓度为1.56 μg/mL,线性范围为0.098~3.125 μg/mL,变异系数< 1%,回收率在99.10%~101.06%。比较两种检测方法,间接竞争ELISA法检测耗时较短,抗体应用量较少,而间接法不需要包被成本较高的目标标准蛋白,相关系数也较高,但其检测体系组成成分较多,且需包被待测样品,较难组成ELISA试剂盒体系,不宜用于现场检测。因此,大规模制备ELISA试剂盒体系用于快速检测蛋白含量时可采用间接竞争法,不仅抗体应用量少、节约成本且快速、简单,可更好地用于科研实践。     

地方流行性牛白血病间接gp51-ELISA抗体检测试剂盒的研制

利用重组牛白血病病毒gp51蛋白作为包被抗原研制了gp51-ELISA诊断试剂盒.试剂盒主要成分包括抗原gp51包被96孔板、HRP标记的兔抗牛IgG和牛白血病阴性、阳性标准血清.对试剂盒进行了特异性、灵敏度、重复性及保存期试验.结果表明,试剂盒特异性好,灵敏度是AGlDT的4～8倍,批间和批内的变异系数分别小于10%和15%,于一20℃保存至少能保持6个月检测结果稳定.与法国Synbiotics公司同类试剂盒进行比对试验,诊断敏感性、特异性和符合率分别为75%、96.1%和94.6%.对采集于山东济南的164份奶牛血清和进口澳大利亚奶牛的398份血清进行检测,抗体阳性率分别6.10%和4.77%.     

犬细小病毒VP2基因的原核表达及间接ELISA方法的建立

根据GenBank上发表的犬细小病毒（CPV）VP2蛋白基因序列，设计并合成1对引物，通过PCR扩增VP2基因全长。将其克隆到pET一30a载体中，构建了原核表达载体pET—VP2，转化大肠杆菌Rosetta，表达并纯化了重组蛋白，Western—blotting证明该重组蛋白具有免疫原性。利用重组蛋白为抗原，通过方阵试验确定了包被抗原的最佳包被量为15．8ng／孔，血清的最佳稀释倍数为1：100，建立了检测CPV血清抗体的间接ELISA方法。     

青霉素酶特异性抗血清间接ELISA方法的建立

以青霉素酶为抗原,免疫健康獭兔,自行制备青霉素酶特异性抗血清(PcAb),并建立间接酶联免疫吸附试验(ELISA)的测定方法,优化试验条件,测定制备的青霉素酶特异性抗血清效价。结果表面抗原最佳包被浓度为10μg/mL,酶标二抗最佳稀释度为1∶2 000,阳性血清最佳稀释度为1∶2 000,抗原最佳包被条件为4℃过夜,血清最佳反应时间为37℃1 h,酶标二抗反应最佳时间为37℃1 h,与底物作用的最佳时间为20 min。     

猪流感病毒HA基因的原核表达及其ELISA检测方法的建立

根据H1N1亚型猪流感病毒血凝素基因序列设计并合成特异性引物,从本室保存的H1N1亚型猪流感病毒中扩增信号肽和跨膜区缺失的血凝素基因,并将其克隆到原核表达载体pET-28a中,在大肠埃希菌中诱导表达.对表达蛋白进行SDS-PAGE和Western blot的结果表明,HA基因在大肠埃希菌中获得表达,产物具有免疫学活性,表达蛋白分子质量约为55 ku.表达产物经过纯化作为包被抗原建立了检测H1亚型猪流感抗体的间接ELISA方法.该方法具有较高的特异性和敏感性,重复性良好.应用该方法对2006年2 584份临床猪血清进行检测,结果显示多个地区检测猪流感抗体出现阳性,平均为20.5%.在6月、7月和12月分别出现抗体水平高峰,分别高达25.4%、25.0%和35.5%.