肉食兽细小病毒通用PCR诊断技术的建立

根据肉食兽细小病毒核苷酸序列高度同源的特点，设计合成了1对引物，以犬细小病毒、貂肠炎病毒和猫泛和白细胞减少症病细胞培养物为DNA模板，进行PCR特异性片段扩增，扩增片段大小为0．6kb。结果表明，扩增产物与设计的2个引物之间的序列大小一致。通过通用性、特异性与敏感性试验及对临床检样品检测，证明本法对肉食兽细小病毒通用。且具有快速、特异和高度敏感的特点。     

肉食兽细小病毒核酸探针的制备及其初步应用研究

将提纯的貂肠炎病毒(MEV)中间复制型DNA(RF-DNA),以HinaⅡ酶切,回收0.7kbC片段并克隆至质粒pBR322中,构成重组质粒pBM,经转化大肠杆菌RRⅠ并扩增后,提纯pBM,酶切电泳回收克隆的C片段,以[α-32P]dATP标记C片段和pBM,用光生物素标记pBM,分别制成放射性同位素和光生物素核酸探针。采用打点杂交技术检测了5种肉食兽细小病毒的细胞培养物和非细小病毒科5种病毒的细胞培养物,同时检测了貂和犬的粪便样品33份。结果表明,32P标记的C片段和pBM探针与光生物素际记的pBM探针均具有相同的杂交特异性,与5种肉食兽细小病毒及感染细小病毒的貂、犬粪样呈杂交阳性反应,与其他科病毒及健貂、犬粪样呈阴性。同位素32P和光生物素标记探针分别检出1 pg和10 pg貂肠炎病毒RF-DNA,敏感性比血凝试验分别离100倍和10倍。     

用反转录—聚合酶链反应检测猪繁殖与呼吸综合征病毒的研究

根据猪繁殖与呼吸综合征病毒美洲型标准毒株的ＯＲＦ６及部分ＯＲＦ７的序列,设计合成了一对引物２６３７４ ＰＳ／２６３７５ＰＲ,用反转录－聚合酶链反应技术对６株ＰＲＲＳＶ北京分离株进行了检测。结果该引物对６株病毒均能扩增出与预期大小相符的ＲＴ－ＰＣＲ产物,并与ＡＴＣＣ ＶＲ－２３３２株扩增产物的大小相当,而欧洲型标准毒株未获扩增片段。     

区别鹅细小病毒和番鸭细小病毒的PCR-RFLP方法的建立

根据GenBank登录的鹅细小病毒（GPV）和番鸭细小病毒（MDPV）非结构蛋白（NS）基因特征,本研究设计1对特异性引物对GPV和MDPV基因组DNA进行PCR扩增,目的片段大小均为810 bp,并对PCR产物进行切胶回收。用EcoRⅠ酶对GPV和MDPV特异性胶回收产物进行酶切鉴定,结果显示MDPV经EcoRⅠ酶切后琼脂糖凝胶电泳检测片段为2段,大小为530和280 bp;而GPV经EcoRⅠ酶切后琼脂糖凝胶电泳检测片段大小不变。本研究建立了一种快速区别GPV和MDPV感染的检测方法,可对番鸭感染水禽细小病毒的情况进行快速鉴别诊断。     

鸡贫血病毒国内分离株的酶切图谱多态性分析

用套式PCR 扩增了鸡贫血病毒(CAV)长度为687 bp 的特异片段,以Hae Ⅲ、Hinf Ⅰ、Hpa Ⅱ分别酶切,通过聚丙烯酰胺凝胶电泳分析了其限制性酶切片段的多态性。对几株国内外分离株的分析表明,TK5803和山东分离株SJ1、SJ2 应属于Todd 的限制性内切酶酶切图谱多态性分析法(RFLP)分类中的第2 组;吉林JL1 株和哈尔滨CL1 株相同,但与其他任何毒株均不相同,大连DL1 株也不同于任何现有毒株,因而都不能归属于Todd 划分的7 个小组;荷兰弱毒株与目前发现的所有毒株均不同,具有其特征性的酶切图谱。     

一株新分离犬细小病毒灭活疫苗的制备及免疫效果研究

JL0911株犬细小病毒是由军事兽医研究所流行病与病毒病防控技术实验室分离得到的一株新型犬细小病毒,其主要抗原位点上的氨基酸序列较国内先前分离到的其他株有较大差异,不能被归入目前已有的CPV-2a、CPV-2b、CPV-2c三个类型。本研究旨在利用JL0911株犬细小病毒研制灭活疫苗,并评价其免疫原性。试验采用10^5.5 TCID₅₀/mL的犬细小病毒JL0911,以甲醛灭活后,加入1/4体积的纳米佐剂,制备了犬细小病毒灭活疫苗。取1.5 mL上述疫苗,通过肌肉注射免疫普通家犬,免疫前后不同时间均采集血清,在F81细胞系上测定犬细小病毒的中和抗体。结果显示,免疫后14 d,试验犬血中细小病毒中和抗体效价较免疫前有显著提高,最高可由0提高至2⁹,表明本试验分离的JL0911株犬细小病毒具有生产犬细小病毒灭活疫苗的潜在价值。     

对流免疫电泳检测犬细小病毒免疫复合物中抗体的研究

通过对流免疫电泳的条件优选,实现了解离后犬细小病毒复合物中抗原抗体的分,达到了检测免疫复合物中抗体的目的。在应用检测试验中,通过与酶档ＳＰＡ染色法、血凝抑制试验及常规对流免疫电泳法比较,证实了该种检测方法的可靠性、敏感性和实用价值。     

鸡新城疫免疫鸡群强毒感染与HI抗体水平关系的研究

应用RT-PCR方法对鸡新城疫4种不同免疫程序鸡群NDV强毒的感染进行监测。结果显示,鸡新城疫弱毒疫苗和油乳剂灭活疫苗同时接种的免疫鸡群NDV强毒的感染率最低。同时应用HA-HI试验对这4群鸡进行平行抽样检测其抗体效价,发现鸡群免疫水平整齐且平均HI效价在11 log2以上时,免疫鸡群基本不感染鸡NDV强毒。     

犬Ⅱ型腺病毒DNA的快速提取及限制性酶切分析

按常规方法培养大肾传代细胞(MDCK),待细胞长成单层后,以2%的量接种犬Ⅱ型腺病毒弱毒株(MV-CAV_2).当绝大多数细胞出现病变时(约35～40h),直接从感染细胞培养物中提取CAV_2 DNA.以该法提取CAV_2 DNA,不仅操作简便、耗费少,而且产量也比常规的先纯化病毒再提取DNA的方法高2～3倍.该法可普遍用于从感染细胞中提取与蛋白共价结合的所有病毒核酸.采用电泳洗脱法纯化上述CAV_2 DNA,以Eco RI、Bam HI、Pst I、Sac I、Nsi I和Sph I酶切,电泳分离,建立了该病毒DNA的6种限制性内切酶图谱.本实验结果为该病毒DNA的分子克隆奠定了基础.     

酒精阳性牛乳的发生及其与热应激的关系研究

本文检测了牛乳及牛血清中的无机离子浓度和乳酸脱氢酶、肌酸激酶活性,同时, 检测了牛血清皮质醇含量和3种抗氧化指标。分析表明:酒精阳性乳患牛血清、全乳中无机离 子代谢表现紊乱,酶活性发生改变,机体总抗氧化能力下降。并从热应激角度对其进行了综合 分析。     

4种抗原定量方法观察牛支原体培养特征

为探索在疫苗研制过程中牛支原体抗原收获时间及抗原定量替代方法,将牛支原体08M株接种于含10%马血清的Thiaucourt's培养基,在110 h内同时监测其颜色变化单位（color change units,CCU）、菌落形成单位（colony forming units,CFU）、菌体蛋白浓度和核酸含量的变化,绘制相应曲线。活菌计数结果（CCU和CFU）显示,牛支原体生长可分为明显的4期,10 h进入对数期,30 h进入稳定期,活菌数最高可达1.0×10⁸ CCU/mL和7.7×10⁷ CFU/mL,75 h进入衰亡期;蛋白浓度从15 h开始迅速增长,至35 h蛋白浓度最高,为72.06 μg/mL,此后维持在58.38~70.65 μg/mL;核酸含量从15 h开始增长,至25 h后Ct值维持在15.32~17.84。结果表明,牛支原体蛋白含量在稳定期初期与活菌数具有良好的相关性。因此,在牛支原体灭活疫苗生产中,稳定期初期是最佳抗原收获时间,可用蛋白浓度法代替活菌计数法进行抗原定量。     

Observation of the Growth Trends of Mycoplasma bovis with Four Different Antigen Quantitation Methods

To explore the antigen harvest time of Mycoplasma bovis (M.bovis) and the antigen quantitation alternative method,M.bovis 08M strain was inoculated in the Thiaucourt's medium.Four growth curve plottings were made by measuring color change units (CCU),colony forming units (CFU),protein concentration and nucleic acid levels.Both the results of CCU and CFU counts showed that the growth of M.bovis was divided into four phases,the logarithmic phase appeared after being cultrured 10 h,the stationary phase appeared at 30 h with the highest number of viable cells up to 1.0×10⁸ CCU/mL and 7.7×10⁷ CFU/mL,and the decline phase started at 75 h.The protein concentration of M.bovis increased rapidly from 15 to 35 h,reached 72.06 μg/mL at 35 h,then maintained at 58.38 to 70.65 μg/mL.The nucleic acid levels of M.bovis increased rapidly from 15 h,and the cycle threshold (Ct) values were maintained between 15.32 to 17.84 after 25 h.These results indicated that there was a good correlation between the protein concentration and viable count of M.bovis at the early stationary phase,which was the best time period to harvest antigen.The protein concentration determination could be an alternative method to quantify antigen contents of M.bovis when preparing inactivated M.bovis vaccine.     

四组中药组方治疗绵羊传染性胸膜肺炎效果观察

羊传染性胸膜肺炎又称羊支原体性肺炎,由支原体所引起的一种高度接触性传染病,其临床特征为高热,咳嗽,胸和胸膜发生浆液性和纤维素性炎症,主要危害1～3月龄的羔羊,常呈急性和慢性经过,病死率较高,是绵羊养殖中较为常见的呼吸道疾病。临床上大多采用抗生素进行治疗,但常常发生耐药情况而导致疗效差的情况。近年来,我们采用早期应用中草药辅以较为敏感的抗生素进行治疗,取得了良好的治疗效果。本文就对常用的4组中药组方开展疗效观察,筛选出了一组疗效最佳的中药组方,以期为临床治疗提供解决方案。     

Relationship between the expression of key lipogenic enzymes,fatty acid composition,and intramuscular fat content of Limousin and Aberdeen Angus cattle

The amount and fatty acid composition of intramuscular fat (IMF) are major factors in determining the nutritional value of beef. Understanding the mechanisms regulating IMF formation is important for designing strategies for manipulation of IMF quantity and quality. The aim of this study was to investigate the contribution of key lipogenic enzymes towards intramuscular fat formation in Aberdeen Angus and Limousin crossbred steers. This study reports the presence of 150 kDa acetyl-CoA-carboxylase α (ACCα), 150 kDa fatty acid synthase (FAS), 37 kDa stearoyl-CoA desaturase (SCD), 50 kDa Δ6-desaturase (Δ6d), and 50 kDa Δ5-desaturase (Δ5d) immunoreactive bands in cattle muscle (as detected by Western blot analyses). A significant positive relationship was established between ACCα protein content and IMF content; FAS protein content and IMF, as well as between expression of these enzymes and the products of their reactions, saturated fatty acid. IMF level was also positively related to the expression of Δ6d and Δ5d proteins. However, no relationship was found between IMF and the amount of SCD protein. It is suggested that the rate of saturated fatty acids and PUFA biosynthesis might have a significant impact on IMF deposition in cattle.     

Relationship between lodging resistance of oats and the basal internode stem characteristics and lignin synthesis北大核心CSCD

Lodging is one of the important factors affecting yield and quality in oat crops. This research studied the physiological mechanisms of lodging resistance in oats,with a view to guiding future breeding. Mengyan 1 and Bayan 6 oat were included in this study as lodging-resistant varieties,while Dingyan 2 and Qingyin 2 were included as lodging-susceptible varieties. Data were collected on the morphological characteristics of basal elongation and differences in the contents of acid detergent fiber,neutral detergent fiber,cellulose and lignin and activities of key enzymes,for these oat cultivars with different lodging susceptibilities,so as to investigate the relationship between the lodging resistance of oats and the morphological characteristics and lignin content and synthase activity in the second-phase elongation at the base of stem. It was found that the amount of lodging among resistant cultivars was lighter and the lodging rate was lower than that of cultivars. The lengths of the second internode at the stem base of lodging easy cultivars were 34. 17% higher than those of lodging resistant cultivars. The internode diameter,stem wall thickness,stem weight,density,diameter,force and stem puncture force values of lodging resistant cultivars at the second internode of the stem base were,respectively,29.11%,26. 18%,25.80%,27.97%,34.61%,36.23% and 36. 60% higher than those of lodging-susceptible cultivars. The stem lignin and cellulose contents of cultivars resistant to lodging were significantly （P<0. 05） higher than those of cultivars susceptible to lodging. Lignin content was significantly （P<0. 01） positively correlated with the internode breaking force and puncture strength of the second internode at the stem base （r=0.9862 and r=0.9638,respectively）,and significantly negatively correlated with the observed lodging rate（P<0.05,r=−0.9407）. Lignin synthetase tyrosine ammonialyase,phenylalanine aminotransferase,cinnamyl alcohol dehydrogenase and 4-coumaric acid of CoA ligase in stems of lodging-resistant cultivars showed higher activities,resulting in accumulation of lignin at the internodes of the second elongation at the base of stem,which increased the lignification of cell walls and the mechanical strength of stems,and thus enhanced lodging resistance of the oats. © 2022 Editorial Office of Acta Prataculturae Sinica. All rights reserved.