猪瘟病毒石门株在ST细胞连续传代后E0基因变异分析

为了解猪瘟病毒(CSFV)石门(Shimen)株连续传代后变异情况,根据已发表的CSFV E0基因序列(AF092448.2),设计合成引物,以在ST细胞中连续传代培养的CSFV石门株细胞毒总RNA为模板,每5代通过RT-PCR方法扩增病毒的E0基因,测定其核苷酸序列和氨基酸序列,用DNA Star软件分析比较原代、5代、10代、15代、20代、25代接毒细胞中CSFV E0基因的变异性。结果显示,接种病毒第10代的病毒E0基因在630核苷酸位点出现变异,第15代病毒在632核苷酸位点出现变异。本研究通过分子生物学方法发现CSFV Shimen株在ST细胞连续传代过程中E0基因能保持遗传的稳定性。     

ST贴壁细胞悬浮驯化及应用

为将ST贴壁细胞通过自主驯化,使其能在ST-S细胞无血清培养基中悬浮生长且能稳定传代,在摇瓶中实现高密度生长,并应用于猪伪狂犬病毒(PRV)悬浮培养,经ST-A低血清培养基适应培养,对一株贴壁的ST细胞进行了无血清的全悬浮驯化,并将PRV在悬浮细胞中连续盲传培养。结果显示:ST悬浮细胞能在无血清培养基中传代,培养48 h至第3代后,所能达到的最终细胞密度为3.00×10^(6 )cells/mL以上;PRV连续培养至第5代,毒价可达到109.0 TCID50/mL。结果表明,用专用培养基可使ST贴壁细胞实现悬浮驯化,并可应用于PRV悬浮培养。本研究为获得高密度ST悬浮细胞和提高PRV增殖效率奠定了技术基础。     

猪源ST9型耐甲氧西林金黄色葡萄球菌中前噬菌体的流行状况与转导分析

本研究旨在分析猪源ST9型耐甲氧西林金黄色葡萄球菌(Staphylococcus aureus,MRSA)中前噬菌体的流行情况、结构特点和转导能力,探究前噬菌体在猪源MRSA流行克隆形成中的作用。基于全基因组信息,分析了近年来从我国多省分离的131株ST9型MRSA中前噬菌体的流行率、分型、亲缘关系和结构特征;选取含不同分型前噬菌体的菌株进行诱导,对诱导获得的噬菌体颗粒进行转导,测定转导子的耐药表型及体外适应性。研究结果显示：猪源ST9型MRSA的前噬菌体携带率为78.6%(103/131株),其中,63株携带完整前噬菌体序列,所有前噬菌体序列均不含耐药基因,仅2.9%(3/103株)的前噬菌体序列含毒力基因;前噬菌体谱型丰富,其整合酶分型主要为Sa2int和Sa4int;各型别前噬菌体结构同源性较高,完整前噬菌体可被诱导为长尾噬菌体;噬菌体颗粒可包装供体菌的aadD、tet(L)耐药基因并转导至受体菌中;转导子可获得卡那霉素、四环素耐药表型,体外生长能力与受体菌株无明显差异(P>0.05)。研究结果表明：猪源ST9型MRSA的前噬菌体携带率较高,谱型丰富,不携带耐药基因,部分噬菌体可包装供体菌的耐药基因转导至受体菌,产生的适应性代价小。     

大豆GmFTL3和GmFTL5启动子的组织特异性表达分析

大豆（Glycine max (L.) Merr.）GmFTL3和GmFTL5基因具有很高的序列相似性,均参与大豆开花调节,但表达模式并不相同,本研究以启动子为突破口,分析这两个基因之间的表达差异。从大豆品种天隆1号中克隆GmFTL3和GmFTL5的启动子,构建载体pGmFTL3pro::GUS和pGmFTL5pro::GUS,转化拟南芥进行GUS染色分析。结果显示：无论长日条件还是短日条件,苗期和花器官中GmFTL3启动子均不驱动GUS基因在拟南芥中表达,而GmFTL5启动子则驱动GUS基因在拟南芥中表达,且在苗期的不同阶段呈现组织表达特异性。在花器官中,GmFTL5启动子驱动GUS基因主要在花萼、花瓣、花药和花粉粒中表达。实时定量结果显示,在大豆中,GmFTL3和GmFTL5基因均有表达。综上所述,大豆GmFTL3启动子在大豆中有活性但在拟南芥中无活性;GmFTL5启动子在大豆和拟南芥中均有活性,但表达可能存在差异; GmFTL5启动子在花器官中表达可能暗示GmFTL5基因与花器官发育有关。     

猪细小病毒CG-05株在ST细胞上的增殖规律研究

通过将猪细小病毒CG-05株同步接种于ST细胞,测定不同时间收获的病毒液的TCID50和HA,研究了CG-05株病毒在ST细胞上的增殖规律。病毒接种后镜检观察接毒细胞,在接种病毒后24 h,即可在细胞较少的区域看到非常轻微的病变。测定病毒TCID50,检测到接毒后培养17h病毒已经明显增殖;当培养到40h左右,其TCID50即接近高峰,达到10-7.2/0.1mL以上,并进入平台期;继续培养,TCID50最高可达到10-8.5/0.1mL,且直到122 h也不见明显降低。病毒HA价也出现同样的平台期,但比TCID50的平台期出现得晚,到50 h才进入平台期。结果表明,用ST细胞培养CG-05株病毒,接种病毒培养56～96 h后,如病变达到80%以上,且细胞脱落已形成20%以上空斑,此时收获病毒可获得高效价的病毒液。该研究可为制备高效价的PPV病毒抗原提供数据资料。     

Effect of size reduction by freeze-milling on processing properties of beta-glucan oat bran

An effect of freeze-milling on processing properties of beta-glucan oat bran has been evaluated. A comparison with existing, patented methods of high molecular weight oat beta-glucan has been carried out. The new method employs raw material pre-treatment with freezing and milling in a hammer mill, resulting in significant reduction of particle size – 89% was between 80 and 50 μm in comparison with initial material when 79% were between 250 and 200 μm. Reduction of particle size also improved some process parameters of extraction technology – 30% improvement in fat removal during first stage of beta-glucan recovery was observed. The achieved pilot plant yield of product containing beta-glucan extraction was 64,03%, purity (ie. beta-glucan content), of achieved product was 84,4% and average molecular weight was about 69,500 g/mol.     

犬低温体外循环手术及其灌注管理的试验研究

试验应用现代体外循环技术，实施完全心肺转流的低温体外循环，首次在我国兽医临床建立了２８只犬低温体外循环实验模型。探索适用于小动物心脏直视手术的体外循环手术操作、心脏停搏与复苏、体外循环灌注管理的技术方法。研究表明，选择右侧第４肋间进胸，从右心房施行前后腔静脉插管及升主动脉根部置供血管是简便易行的手术通路。采用部分晶体液与全血作预冲液，体外转流维持中度血液稀释（平均２３６８±３２７％），最低温度控制２６℃左右（平均２６１４±０７７℃），血流复温至３６℃左右（平均３５９７±０６８）开放升主动脉，转流中灌注压维持５０～９０ｍｍＨｇ（平均７２±１３ｍｍＨｇ），灌注流量８０～１００ｍＬ·ｋｇ－１·ｍｉｎ范围，是低温体外循环下开展犬心内直视手术成功的基本保障。采用冷晶与冷温血停搏液不同灌注。结果显示，冷温血停搏液联合灌注组心脏自动复跳率最高，并且复跳迅速，心搏有力，正性肌力药应用剂量小，复跳后辅助循环时间明显低于冷晶停搏液灌注组（Ｐ＜００５）。     

Population structure of poplar rust Melampsora larici-populina in the UK inferred from AFLP

AFLP profiles were generated from poplar leaf rust ( Melampsora larici-populina ) collected from Populus trichocarpa cv. Trichobel at six sites in the UK in 2001. Of a total of 145 isolates, 121 were unique AFLP phenotypes. On one occasion, the same AFLP phenotype was found at two sites. The analysis indicated that 81·88% of the total variation was attributable to differences among individuals within replicate plots, 3·47% to differences among plots within sites and 14·65% to differences among sites. Weir & Cockerham's θ was calculated as 0·138 and θ ^B , the Bayesian analogue of F _ST, as 0·146. Overall, there was sufficient gene flow among different sites to prevent genetic drift, as Nm was calculated as 1·562 using θ and as 1·462 using θ ^B . Nei's genetic distance and population-assignment test indicated that the rust from a site in north-east England was differentiated from those from other sites. There was no significant correlation between genetic and geographical distances. The results indicate that sexual reproduction has a major influence on the relative lack of population structure of M. larici-populina in the UK.