1株奶牛源松鼠葡萄球菌的分离鉴定及耐药性分析

为确定奶牛垫料中是否含有病原菌并深入了解垫料中优势生长菌的类型、耐药性及致病性等情况,本试验进行了细菌分离培养、革兰染色镜检、生化试验鉴定、16S rDNA序列分析及同源性比对、药敏试验、小鼠致病性试验。结果显示,分离菌在普通营养琼脂平板上形成圆形、表面光滑的白色菌落,血琼脂平板上形成黏稠、较大的白色菌落。革兰染色镜检结果显示,分离菌为革兰阳性,球型,呈葡萄串状排列,或单个散落。生化试验结果显示,分离菌对木糖、葡萄糖、麦芽糖、果糖、乳糖、硝酸盐还原等呈阳性反应,而蜜二糖、木糖醇、山梨醇、尿素、V-P反应等呈阴性反应。16S rDNA序列分析结果显示,扩增的16S rDNA序列长度为1 298 bp,与松鼠葡萄球菌的核苷酸同源性达99.85%~100%;系统进化树结果显示,分离菌与松鼠葡萄球菌处于同一分支。动物试验结果表明,分离菌株对试验小鼠有较强的致病性,以0.2 mL/只（7.9×10⁸ CFU/mL）菌液的剂量接种小鼠,在48 h内死亡率为60%（3/5）。药敏特性分析结果显示,分离菌株对复方新诺明、氨苄西林等7种药物敏感,对克林霉素、头孢噻肟和头孢呋辛中度敏感,对青霉素、红霉素和林可霉素耐药。本研究为奶牛源松鼠葡萄球菌的分离鉴定及防控提供了参考依据。     

气肿疽梭菌CctA基因的原核表达及其间接ELISA检测方法的建立

研究旨在原核表达气肿疽梭菌细胞毒素A （CctA）基因,用纯化的重组蛋白建立其间接ELISA检测方法,a。利用大肠杆菌密码子的偏爱性优化CctA基因序列,克隆至原核表达载体pET-28a （+）,双酶切鉴定原核表达质粒pET28a-CctA并测序。将重组质粒转入大肠杆菌,经IPTG诱导得到高表达的重组CctA包涵体蛋白。包涵体蛋白变性后经镍（Ni）柱纯化,SDS-PAGE检测其纯化效果。以豚鼠抗气肿疽梭菌抗血清为一抗,用Western blotting方法检测重组CctA蛋白的反应原性。用棋盘滴定法建立间接ELISA检测方法,以复性后的重组CctA蛋白作为检测抗原,摸索抗原包被浓度、封闭液的种类及浓度、抗体的最适稀释度和反应条件等。选用3批气肿疽灭活疫苗免疫豚鼠后采集血清,同时用攻毒和间接ELISA两种方法验证疫苗的免疫效力。质粒双酶切结果显示,得到大小约853 bp的条带,与预期相符,且测序结果正确。SDS-PAGE结果表明,成功表达并纯化大小为35 ku的重组CctA蛋白。Western blotting结果显示,豚鼠抗气肿疽梭菌抗血清与重组CctA蛋白具有良好的反应原性。建立的间接ELISA法的最适条件为：抗原的包被浓度为0.5 μg/mL,于4 ℃包被过夜;封闭液选择10%胎牛血清,37 ℃孵育2 h;二抗的稀释度为1：8 000;室温避光显色10 min后终止反应,测定D_{450 nm}值。当P/N>4.6时,间接ELISA法检测结果与豚鼠攻毒试验结果拟合度较好。本研究成功表达CctA基因并纯化了重组CctA蛋白,建立的以重组CctA蛋白为检测抗原的间接ELISA检测方法,有望成为气肿疽灭活疫苗免疫效果验证的替代方法。     

MSTN基因编辑牛肌肉转录组测序及生物信息学分析

为探究肌生长抑制素（myostatin,MSTN）基因对牛肌肉发育的具体调控机制,本研究选取同一牛场健康鲁西黄牛10头,其中通过转基因技术得到的基因编辑牛MSTN^-/-和同种非转基因野生型牛各5头。分别采集两组牛腿臀肌肉样品,利用IIlumina HiSeq高通量测序技术进行转录组测序分析,通过生物信息学方法比较两组样本间的差异表达基因,并进行GO和KEGG富集分析,最后利用实时荧光定量PCR验证转录组测序数据。结果显示,基因编辑型牛和野生型牛之间共检测到18 071个基因。在log₂|FoldChange|≥ 1.48条件下,筛选出406个差异表达基因,其中347个显著上调,59个显著下调。GO功能富集分析显示,MSTN基因编辑后显著影响915个功能类别（P<0.05）,差异基因主要参与结合、生物系统调节、免疫系统等相关功能。KEGG通路富集分析结果共涉及211个通路,差异基因主要富集在细胞黏附分子、趋化因子信号通路、细胞因子互作等信号通路上,进一步从中筛选出可能参与细胞生长、肌肉发育的差异基因（CD14、KIT、CSF1R、FBP1、DUSP4、ULBP21、PRKCB、SPN、CHAD、SRC）。实时荧光定量PCR检测结果显示,所选差异基因表达水平与转录组表达水平一致,证明测序结果的可靠性。本研究结果表明,MSTN基因发挥作用后可以介导多个下游基因表达,从而影响相关信号通路及生物学过程;同时,所筛选出的差异表达基因可作为进一步研究骨骼肌调控机制的候选靶标。     

MSTN基因对牛肌动蛋白细胞骨架调节通路的影响

为探究肌肉生长抑制素（myostatin,MSTN）对牛骨骼肌生长发育的作用机制,本研究前期利用定量蛋白质组学与磷酸化蛋白质组学分析野生型蒙古牛（MG.WT）和MSTN^+/-蒙古牛（MG.MSTN^+/-）腿臀肌肌肉组织中蛋白质水平和磷酸化修饰水平的差异变化,使用已建立的牛骨骼肌卫星细胞体外诱导成肌分化模型,检测设计合成的MSTN siRNA （si-MSTN）干扰效果;采用实时荧光定量PCR和Western blotting方法检测转染si-MSTN的增殖期（GM）和分化第3天（DM3）牛骨骼肌卫星细胞中肌动蛋白细胞骨架调节通路相关基因的mRNA和蛋白水平的表达变化,研究敲低MSTN表达对肌动蛋白细胞骨架调节通路的影响。结果显示,在MSTN^+/-蒙古牛肌肉组织中共鉴定到16个肌动蛋白细胞骨架调节通路相关基因表达丰度上调;转染si-MSTN细胞中的MSTN表达水平极显著降低（P<0.01）;在转染si-MSTN的GM期牛骨骼肌卫星细胞中,肌动蛋白细胞骨架调节通路相关基因ENAH、ACTN4和Cdc42的mRNA水平均显著升高（P<0.05）,PFN1、RhoA和ACTN4的蛋白水平均显著或极显著升高（P<0.05;P<0.01）;在转染si-MSTN的DM3牛骨骼肌卫星细胞中,ENAH、CFL1、SCIN和Cdc42基因mRNA水平均显著升高（P<0.05）,RhoA基因mRNA水平极显著升高（P<0.01）,PFN1和ACTN4的蛋白水平均显著升高（P<0.05）。结果表明,干扰MSTN可以促进肌动蛋白细胞骨架调节通路相关基因的表达,探明了MSTN可能通过介导肌动蛋白细胞骨架调节通路影响牛骨骼肌卫星细胞增殖和成肌分化的分子机制,为进一步研究MSTN对牛成肌分化的调控机制提供参考。     

母猪难产的病因病机与预防诊治

难产又称异常分娩,轻则胎儿窒息死亡,重则母猪和仔猪全部死亡。如果难产过程中对生殖器官造成损伤,会影响母猪分娩后身体的恢复,易发生子宫炎、乳房炎和无乳症等疾病。母猪难产,不仅仅影响当前胎次生产性能,还可能持续影响之后的繁殖性能,甚至发生过难产的母猪失去种用价值,不得不被提前淘汰。     

母猪不食的原因分析及综合防控措施

母猪通过采食饲料,获得营养物质,一旦不食,养分没有来源,必然不能维持生产和基本的健康。无论是妊娠期还是分娩期不食,都影响母猪和仔猪的健康及生产性能,从而增加生产成本、降低养殖效益。母猪产后不食,生成乳汁的营养来源不足,仔猪因不能摄入足够的母乳,引发腹泻、生长受阻,甚至死亡。营养摄入不足,也影响母猪产后机体恢复,母猪表现出身体虚弱、生理功能紊乱,如不及时治疗好,可能会导致母猪不发情被淘汰。     

Protein and energy concentrations of meat meal and meat and bone meal fed to pigs based on in vitro assays

The objective of this study was to develop equations for estimating ileal digestible crude protein (CP) and metabolizable energy (ME) contents of meat meal (MM) and meat and bone meal (MBM) as feed ingredients for pigs based on in vitro assays. Test ingredients were 4 sources of MM and 3 sources of MBM. Ash and CP contents of the ingredients ranged from 3.8% to 33.1% and 46.8% to 82.9% (as-is basis), respectively. In vitro ileal disappearance (IVID) of CP was determined and ileal digestible CP content was calculated by multiplying CP content by IVID of CP. In vitro total tract disappearance (IVTTD) of dry matter (DM) was determined and ME was calculated using gross energy, CP contents, and IVTTD of DM. The IVID of CP and IVTTD of DM ranged from 77.2% to 88.7% and from 82.7% to 92.4%, respectively. Calculated ileal digestible CP and ME contents ranged from 37.8% to 73.5% DM and 2,405 to 3,905 kcal/kg DM, respectively. Ash contents were negatively correlated (P < 0.001) with CP (r = −0.99), in vitro ileal digestible CP (r = −0.97), gross energy (r = −1.00), in vitro digestible energy (r = −0.97), and adjusted ME (r = −0.97). The most fitting equations for ileal digestible CP and adjusted ME were: ileal digestible CP (% DM) = 11.91 − 0.90 × Ash (% DM) + 0.74 × IVID of CP (%) (R² = 0.99) and adjusted ME (kcal/kg DM) = 130.85 − 50.90 × ash (% DM) + 47.06 × IVTTD of DM (%) (R² = 0.99). To validate the accuracy of the prediction equations for ME, mean bias and linear bias were determined using a regression analysis. Calculated ME values of MM and MBM were in a good agreement with data obtained from animal experiments based on a statistically insignificant bias in the models. In conclusion, ME concentrations of MM and MBM as swine feed ingredients can be calculated using ash concentration and in vitro disappearance of dry matter.     

野生二粒小麦与野生燕麦族间杂交研究——Ⅱ、杂种 F_2成株期形态学和细胞学研究

野生二粒小麦与野生燕麦杂交F_2共61株,其中抽穗前(?)亡和黄化未能抽穗的共21株。分离出野生二粒小麦型、硬粒小麦型、斯卑尔脱小麦型、燕麦型和普通小麦型。野生二粒小麦型结实率11.2～63.5%,PMC染色体数为27～36。硬粒小麦型结实正常,PMC染色体数为2n=14″。斯卑尔脱小麦型结实率2.7～43.4%,PMC染色体数为37～50。燕麦型结实基本正常,PMC染色体数为41～43,出现1～2个单价体。普通小麦型的护颖包裹籽粒较松,不断穗节,容易脱粒,染色体数为39～45,结实率1.2～68.2%。杂种F_2不同类型植株的PMC染色体数为27～50,每株能抽穗扬花的植株都能结实,结实率1.2～92.3%。杂种F_2的小麦型中有一部分植株的芒长在外颖背上,表现出燕麦族的分类特征性状。     

Fortification of Wheat Dough with Calcium and Magnesium Ions Affects Empirical Rheological Properties

This study evaluated the influence of calcium and magnesium ions on the empirical rheological properties of wheat flour to verify possible effects of these ions on processing because, in addition to their nutritional importance, they are also responsible for water hardness. Calcium (0–1.30 g/100 g) and magnesium (0–0.34 g/100 g) ions from sulfate salts were added to wheat flour, according to a central composite rotatable design. The farinograph and extensigraph properties of wheat flour and its mixtures were evaluated. The results were analyzed by response surface methodology. Calcium ions stood out for increasing water absorption, decreasing mixing stability, and producing a delayed effect on dough extensibility (reduced at 135 min). Magnesium ions influenced most flour rheological properties in a similar manner to oxidizing agents (increased dough stability, increased resistance to extension, and reduced extensibility), thus proving to be a possible replacement agent for these additives. An interaction effect of the combined calcium and magnesium ions was observed on dough development time. The results showed that effects on processing can occur when wheat flour fortification is made, and adaptations on wheat flour specifications, product formulation, and processing parameters may be required.     

Analysis on Differential Expression Genes in Porcine Aortic Vascular Endothelial Cells Induced by Apolipoprotein C Ⅲ

The aim of this study was to investigate the differential expression genes induced by ApoCⅢ,and study the function of ApoCⅢ.Porcine aortic vascular endothelial cells were successfully isolated using enzyme digestion,and then screened the differential expression genes induced by ApoCⅢ using the Solexa high-throughput sequencing technology.The results showed 647 differential expression genes,including 390 up-regulated genes and 257 down-regulated genes.The qRT-PCR results verified that the gene expression results from Solexa sequencing data were reliable.GO and Pathway analysis showed that the function of differential expression genes were related to immune response,cell apoptosis and death.These findings suggested that ApoCⅢ affected the physiological function of porcine aortic endothelial cells by the molecular pathways of inflammation,cell adhesion and apoptosis,which provided a theoretical basis for further understanding the molecular mechanisms of atherosclerosis caused by ApoCⅢ.