鸭源呼肠孤病毒DRV-GZ株S2基因的序列分析及其表达

采用RT-PCR方法从鸭源呼肠孤病毒DRV—GZ株中扩增出了S2基因片段,将其克隆到表达载体pET-28a（＋）中,测序验证后转化入表达宿主菌Rosetta^TM2（DE3）plysS,进行IPTG诱导表达。结果表明,重组菌可表达出相对分子质量约为50000的重组融合蛋白,在浓度为0．6mmol／L的IPTG诱导4h的情况下表达效果最好。表达的蛋白以包涵体的形式存在于菌体中。表达产物经Ni柱纯化后可得到纯度较高的目的蛋白。经Western-blot分析,所纯化的蛋白能与抗呼肠孤病毒DRV—GZ株阳性血清进行特异性的免疫印迹反应,证实表达的蛋白具有较好的反应原性。     

地毯草耐盐浓度梯度筛选与临界盐浓度研究

以地毯草(Axonopus compressus)为对象,利用水培法进行耐盐鉴定,初步研究不同盐浓度对地毯草坪用品质、叶色和枯黄率的影响,并对地毯草耐盐性进行了评价。结果表明,不同的处理之间存在显著或极显著性差异,在低浓度处理下（0～120 mmol·L-1）,叶色和坪用品质得分6分以上;而在高浓度处理下（140～180 mmol·L-1）,景观价值低于6分。叶片枯黄率与之正好相反,在低浓度下（0～140 mmol·L-1）,枯黄率较低;而在高浓度下（160～180 mmol·L-1）,枯黄率超过50%,从而导致地毯草草坪观赏价值过低。通过建立回归方程,以枯黄率50%作为确定存活的临界值指标,求得地毯草具有盐半致死临界浓度为141 mmol·L-1。     

Q热贝纳柯克斯体TaqMan荧光定量PCR检测方法的建立

根据Q热贝纳柯克斯体(Coxiella burnetii)插入IS1111序列设计引物和探针,建立快速检测Q热的TaqMan实时荧光定量PCR方法。以梯度稀释含有目的扩增片段的重组质粒作为标准品,进行定量PCR反应。结果显示,该方法能够检测出10个拷贝数的阳性质粒;标准曲线相关系数为0.995,扩增效率为103%;结核分枝杆菌(M.tuberculosis)、衣原体(C.psittaci)、布鲁氏菌(Brucella.spp)及牛血液的核酸样本特异性检测结果均为阴性。本研究建立的TaqMan荧光定量PCR法灵敏度高、特异性好,对Q热的检测与鉴定中具有良好的应用前景。     

广西猪瘟病毒E0和E2基因的克隆及序列分析

通过分析目前广西猪瘟病毒(CSFV)的E0和E2基因特征,为了解广西地区CSFV的分子流行病学、遗传变异及综合防控提供科学依据。试验采用RT-PCR方法,对阳性猪瘟样品进行CSFV的E0及E2基因的扩增,经克隆、测序后,利用DNAStar软件对序列进行比对分析,同时绘制系统遗传进化树。结果表明,从阳性猪瘟样品中成功扩增CSFV的E0及E2基因。序列比对分析发现,GX2毒株与参考毒株的E0基因核苷酸同源性在83.1%~94.1%,其推导氨基酸同源性在85.9%~99.6%;与参考毒株的E2基因核苷酸同源性为81.7%~93.7%,其推导氨基酸同源性为89.0%~97.0%;E0与E2基因均属于基因Ⅱ群。氨基酸变异位点分析表明,E0蛋白的RNase活性区域氨基酸基序位点没有发生变异;E2蛋白中15个位点上的半胱氨酸均未发生变异,但单抗识别位点S734R发生变异。遗传进化分析显示测定的GX2毒株与近年来广西CSFV流行毒株的变异趋势相似,与中国传统疫苗株HCLV、经典强毒株Shimen的同源性较低,亲缘关系较远,与广西近年来的流行毒株GXWZ02株的同源性较高,亲缘关系较近。     

种蛋孵化死胚及孵化厅环境样本中铜绿假单胞菌的分离鉴定及其耐药性分析

本研究旨在了解种鸡场中种蛋孵化死胚和周围环境中感染或污染铜绿假单胞菌(Pseudomonas aerugino-sa,PA)的比率及分离株的耐药性.从广东省的5个规模化种鸡场采集孵化厅死胚及其周围环境样本进行PA的分离鉴定,采用K-B纸片扩散法分析其抗菌药物敏感性,并通过常规PCR检测对应的耐药基因.结果 显示,在采集...     

一例猫卵巢恶性颗粒细胞瘤的病理学分析

本文报道猫伴发腹腔种植性转移的卵巢恶性颗粒细胞瘤1例,较为罕见,国内外文献未见报道。1例15岁雌性家猫,因不食和腹围增大就诊。超声检查显示,左肾尾侧可见一大小约4.27 cm×2.63 cm的肿物。开腹探查,可见大网膜、肠壁及胃壁弥散性分布大量大小不等的球形肿物,左侧卵巢明显增大,表面形成坚实或柔软的结节。对肿物进行组织病理学检查,诊断为卵巢恶性颗粒细胞瘤。本文提供该病例临床和组织病理学资料,并回顾相关文献资料,希望对该肿瘤的诊疗和研究提供帮助。     

奶牛源MRSA菌株的分离鉴定和耐药性分析

本研究对部分奶牛场采集的502份隐性乳房炎试验（california mastitis test,CMT）检测呈阳性的奶样进行细菌分离、鉴定,运用多重PCR方法筛选出耐甲氧西林金黄色葡萄球菌（methicillin-resistant Staphylococcus aureus,MRSA）,并对分离出的MRSA用纸片扩散法进行药敏试验,检测其对19种抗菌药物的敏感性。结果显示,分离到细菌的奶样有452份,其中检出葡萄球菌338株,MRSA 89株;MRSA分离株主要对万古霉素、磷霉素、氯霉素和克拉霉素敏感,对青霉素、多黏菌素、大观霉素及头孢他啶具有较强的耐药性。本研究为指导奶牛乳房炎用药、MRSA菌株的快速鉴定及制定MRSA感染的防控措施提供参考。     

Analysis on Target Genes with Mutation in microRNA of Blue-shelled Chicken and White Leghorn

The study was conducted to explore the potential different characters between Blue-shelled chicken and White leghorn.Global genome microRNA was combined the identified microRNA with complementary lab-predicted microRNA.Then the two breed chicken's SNP data got by GGRS were mapped to the microRNA and focused on SNP that deliberately located in mature-microRNA.Bioinformatics method was adopted for target prediction on microRNA which had SNPs.By further gene enrichment analysis,the study found these genes enriched in 22 GO terms,10 KEGG pathways,and 3 IPA important networks.And they enriched in traits which associated with growth,such as mTOR signaling pathways,Wnt signaling pathways,growth hormone receptor networks and insulin-like growth factor Ⅰ receptor networks.And they also enriched in some laying traits,such as oocyte meiotic signaling pathways and progesterone mature oocytes signaling pathways.The methods and the results might provide references for further studies.     

Cloning and Expression of CMKLR1 Gene and Its Correlation with Intramuscular Fat in Goat (Capra hirus)

In order to enrich basic date in goat (Capra hirus) CMKLR1 gene and investigate the correlation between CMKLR1 gene expression and intramuscular fat (IMF) content in muscles, the CMKLR1 gene was cloned from the goat of subcutaneous adipose tissue by RT-PCR, characterized by bioinformatics methods. The expression profiles of CMKLR1 gene of goat in various tissues were constructed liver was benchmark, the GAPDH (GenBank accession No.: AJ431207.1) as a reference gene.Then, the correlation between CMKLR1 mRNA expression and IMF content in muscles was analyzed. The results showed that the CMKLR1 gene CDS of goat (GenBank accession No.: KT165374) was 1 089 bp. Real-time PCR indicated that CMKLR1 was widely expressed in various tissues, including heart, liver, spleen, lung, kidney, adipose, longissimus dorsi, biceps femoris and triceps brachii, and was the highest in lung (P< 0.05). Similar expression variation trend of CMKLR1 was observed between the muscles from 1 to 3 and 24 months old goat, and was the highest in biceps femoris. Reversely, in 8 to 10 months old CMKLR1 was the highest in triceps brachii. The IMF of longissimus dorsi from 24 months old was the highest. Correlation analysis demonstrated that different correlations were observed between expression of CMKLR1 mRNA and IMF content in longissimus dorsi, biceps femoris, and triceps brachii in goat. The CMKLR1 gene did not participate in the deposition of IMF in goats. The research built the theoretical basis for further studies about the CMKLR1 gene.     

地衣芽孢杆菌CP-16脂类水解酶的研究

本试验旨在通过异源表达获得地衣芽孢杆菌(Bacillus licheniformis)CP-16的脂类水解酶,并探究其在羽毛降解过程中的作用。试验以地衣芽孢杆菌CP-16基因组DNA为模板,扩增脂类水解酶基因,转化入大肠杆菌中表达,获得重组酶L-4。研究重组酶L-4的适宜p H、p H稳定性、适宜温度、温度稳定性以及有机溶剂和金属离子对其相对活性的影响,同时探究其对角蛋白酶K水解天然羽毛角蛋白的作用。结果显示,获得的脂类水解酶基因大小为747 bp,编码248个氨基酸,在大肠杆菌中成功表达出重组酶L-4,其分子质量约为28.3 ku,酯酶活性为0.42 U/m L,适宜p H为6.5,适宜温度为50℃;在p H 6.5~9.5条件下处理30 min相对活性保持80%以上,在低于50℃温度条件下处理30 min相对活性保持70%以上。二价铁离子(Fe~(2+))、钠离子(Na~+)、锰离子(Mn~(2+))、钙离子(Ca~(2+))对重组酶L-4相对活性具有激发作用,钡离子(Ba~(2+))、锌离子(Zn~(2+))、铜离子(Cu~(2+))、镍离子(Ni~(2+))对重组酶L-4相对活性具有抑制作用。当有机溶剂浓度为30%时,重组酶L-4在二甲基亚砜(DMSO)和甲醇中保存97%和85%的相对活性,在丙酮、乙醇中保存45%以上的相对活性,在异丙醇中保存不到20%的相对活性,而在乙腈中相对活性基本完全丧失。用重组酶L-4预处理天然羽毛底物,可提高角蛋白酶K对底物的水解效率,促进率为4.32%。由此可见,脂类水解酶可降解羽毛表层脂质,可在促进角蛋白酶水解羽毛角蛋白中发挥作用。