猪圆环病毒2型灭活疫苗中病毒抗原含量实时荧光定量PCR检测方法的建立

依据GenBank公布的猪圆环病毒2型Cap基因序列,在保守区域设计特异性引物和TaqMan探针,优化反应体系,建立评价猪圆环病毒2型灭活疫苗中病毒含量的实时荧光定量PCR检测方法,对方法的特异性、敏感性和重复性进行试验,并验证灭活剂用量和灭活时间对检测结果的影响。结果显示：该方法只对猪圆环病毒2型基因有特异性扩增,其他3种对照病毒基因的扩增结果均为阴性;检测灵敏度达到10^2.0 TCID₅₀/mL,比普通PCR方法高100倍;方法的重复性好,对同一样品进行10次检测,变异系数为2.28%;不同灭活剂用量和灭活时间对结果的影响较小,不会因各厂家使用的灭活剂用量和灭活时间不同,影响疫苗对比实验的公平性。该研究成功建立了一种评价猪圆环病毒2型灭活疫苗中病毒含量的实时荧光定量PCR检测方法,用于猪圆环病毒2型灭活疫苗样品中病毒抗原含量的定量,其结果可以反映不同猪圆环病毒2型灭活疫苗样品中抗原含量差异,为研究猪圆环病毒2型灭活疫苗病毒抗原含量评估方法提供了新的思路。     

Prokaryotic Expression and Antigenicity Analysis of Porcine Japenese Encephalitis Virus Envelope Protein Domain Ⅲ

In order to analyze the antigenicity of porcine Japanese encephalitis virus (JEV) E protein domain Ⅲ, which was expressed by pET-28a vector with His-tag and purified through Ni-NTA, the BALB/c mice were immunized with the purified protein.We identified the antigenicity of domain Ⅲ of E protein and the anti-mice and anti-porcine JEV E Ⅲ protein specific antibody titers by SDS-PAGE, Western blotting, indirect ELISA and IFA.SDS-PAGE results showed the expressed target protein existed mainly in the form of inclusion body.Western blotting, ELISA test results showed that the protein had good reactivity with anti-serum.The mice immunized with the purified JEV E Ⅲ protein generated 1×10⁵ anti-JEV E Ⅲ protein specific antibody titers by ELISA, and the porcine immunized with the porcine JEV generated 5.1×10⁴ anti-JEV specific antibody titers.The IFA results showed that JEV E Ⅲ protein anti-serum could identify JEV antigen.The above results showed that the recombinant JEV E Ⅲ had good antigenicity.These results provided important basis for development of diagnostic antigen for JEV.     

Isolation and Identification of BPIV3 Genotype C

To identify the infection agents from Ningxia Hui Autonomous region, where feedlot cattle indicated bovine respiratory disease complex (BRDC), the M gene of the bovine parainfluenza virus type 3 was amplified by RT-PCR.The PCR product was ligated to pMD18-T vector and cloned to E.coli DH5α.The positive clones were sequenced and compared with the reference strains in GenBank by the molecular biology software.Sequence alignment results showed that a BPIV3 strain was isolated from the samples and named NX49, the M gene of NX49 included 1 056 nucleotides.Evolutionary analysis showed that the NX49 belonged to BPIV3 C genotype and shared 99.4% nucleotide identity with that of the SD0835 isolated in Shandong province.The characterization of the NX49 demonstrated that it was sensitive to temperature, acid and organic matter.The presence of Mg²⁺ showed no protection against the treatment at high temperature.The HA test suggested that the NX49 enables to agglutinate the guinea pig RBC at 4 ℃ and the titer was 1∶4.The study isolated a BPIV3 genotype C strain successfully, which facilitate the study of molecular evolution and epidemiology of BPIV3 in China.     

Acute and Sub-chronic Toxicity Test on Rats of a New Kind of Antidiarrheal Chinese Herbal Medicine Compound Preparation for Livestock

In order to understand the security of a new kind of antidiarrheal Chinese herbal medicine compound preparation for livestock,acute and sub-chronic toxicity test were conducted.Acute toxicity test used the largest drug dose method,20 Wistar rats were orally treated with the Chinese medicine compound preparation.In the sub-chronic toxicity test,80 rats were randomly divided into 4 groups with 20 rats in each group and orally given a dose of 3 000,1 500,750 and 0 mg/(kg·BW）of Chinese medicine compound preparation once a day for 30 days.The general clinical status was observed,rats weight were measured and the dose was adjusted every week during the test,after the test measured blood routine index,biochemistry index,and preceded the gross anatomy observation,weighing each major organs and calculated the viscera coefficient,and proceded main viscera histopathological observation between the high dose group and the control group.The acute toxicity results showed that every rat would be alive gavaged with the lethal dose（LD₅₀）of compound preparation larger 5 g/(kg·BW).The sub-chronic toxicity autopsy showed that except heart,lung,and testicles in individual rats appeared mild bleeding in the high dose group,the other dose group organs found no abnormal change.The haematological index showed except mononuclear cell rate（P<0.05),and hematocrit declined significantly（P<0.05）in the high dose group,all the indexes of the other groups were in the normal range,there was no significant difference from the control group.The test suggested the Chinese medicine compound preparation was no toxicity under the condition of this test according to acute toxicity classification standard of exogenous chemicals by WTO,there was no effect on the growth and development of rats in the sub-chronic toxicity test,and there was no chronic toxicity at least 1 500 mg/kg feeding conditions in short-term repeated application.     

水氮调控对苜蓿与无芒雀麦混播草地生长和辐射利用的影响

针对我国内陆干旱区人工草地生产管理粗放及气候资源利用不充分等问题,探究合理的牧草种植与水氮供应模式,以充分挖掘区域牧草的生产潜力。采用3 a生(2018年播种)紫花苜蓿(简称‘苜蓿’)和无芒雀麦,分析种植模式(苜蓿与无芒雀麦混播,D1;无芒雀麦单播,D2)、施氮量(低氮量N1:60 kg·hm^-2;高氮量N2:120 kg·hm^-2)和灌水量(以灌水下限占田间持水量θ_f的百分比计,分枝期均充分灌水(75%～85%θ_f),现蕾和初花期轻度亏水W1:65%～75%θ_f、中度亏水W2:55%～65%θ_f、重度亏水W3:45%～55%θ_f,灌水上限均为85%θ_f)对牧草叶面积指数(LAI)、干物质累积量、累积截获光合有效辐射量(CIPAR)、辐射利用效率(RUE)、产量(Y)、耗水量(ET_a)、水分利用效率(WUE)和氮肥偏生产力(PFP_N)的影响。结果表明：(1)牧草LAI和...     

不同粒度猫尾草对羔羊体外发酵特性和微生物数量的影响

通过Rusitec-s体外发酵系统研究不同粒度猫尾草对羔羊体外发酵参数与微生物数量的影响,选取6种不同粒度(1.00、2.36、3.35、4.75、8.00和12.50 mm)的猫尾草作为试验饲粮的纤维来源,按照猫尾草的粒度将试验分为6个处理组,每个处理4个重复,利用Rusitec-s型人工瘤胃模拟装置进行48 h体外发酵,测定养分降解率、发酵参数及发酵液中6种微生物菌群的拷贝数。结果表明：与其他处理组相比,2.36 mm组显著提高了体外中性洗涤纤维降解率(IVNDFD)、丁酸、异丁酸、戊酸、异戊酸摩尔比和溶纤维丁酸弧菌(B. f)数量(P<0.05),且显著降低总产甲烷菌(TMe)数量(P<0.05)。总菌(TB)与氨态氮(NH3-N)浓度、乙酸摩尔比呈正相关(R²=0.84,P=0.038;R²=0.88,P=0.021);B. f数量与丙酸摩尔比呈正相关(R²=0.82,P=0.045),且与pH呈负相关(R²=-0.98,P=0.001);TMe数量与体外粗蛋白质降解率(IVCP...     

小麦品种无性系变异改良技术研究

针对晋阳345和晋麦73两个定型品种存在抗寒性较差的缺点,对其幼胚愈伤组织进行了4种处理:1、愈伤组织在MD培养基上继代2～3次;2、愈伤组织在紫外灯下约10cm照射2h;3、愈伤组织在低温2℃和常温25℃下交替培养30d;4、愈伤组织在紫外灯下照射2h后,再在低温2℃和常温25℃下交替培养30d。结果表明,4个处理后代R2主要农艺性状的变异率差异显著,且紫外灯照射幼胚愈伤组织的变异率比低温2℃和常温25℃交替处理幼胚愈伤组织高,其后代各种农艺性状的变异范围较大,变异率较高。但后者有利于改良抗寒性较差的定型小麦品种。     

The Application of Pre-splitting Blasting to Excavation on High Slope in Soft Rock Section

Based on analyzing about blasting mode of slope in the construction of road, the paper shows that pre splitting blasting is mostly suitable in the section of severely weathering soft rock with developed joints and fractures, introduced the laying out about the hole of pre splitting blasting that can apply in the high slope of the expressway in the section, and designed the parameter. The practice shows that it gained favourable effect by this method.     

Analysis of Project Audit''s Function and Civil Action

Audit takes a unique status and has obvious function in financial surveillance system.The Audit is used to superintend the factuality,validity and virtue of fiscal and financial income and expenses.In the renewed China Audit law the functions and status of official audit have been intensified,and the qualification and status of Audit department as executing office is confirmed.This paper analyzed the official construction project audit's function and legal status.     

车用493型柴油机冷却问题初探——机体冷却水流动CFD模拟

车用493型柴油机在我国主要是轻卡的配套动力,量大面广。为了在提高升功率的同时满足国家排放要求,很多研究工作主要着重于强化结构件、改进气道及工作过程、提高供给系统的喷油压力等,在冷却方面着力不够。实际上冷却不均衡,热应力集中同样会造成机体变形,导致颗粒物排放上升。冷却效果的好坏又在很大程度上取决于冷却水套内冷却介质流动以及分布的情况。本文从冷却分析入手,利用CFD商用流体软件STAR-CD对车用493型柴油机的冷却系统中的机体内冷却水的流动进行了三维数值模拟,给出冷却水套内冷却介质的流动场,分析它对换热的影响。