马疱疹病毒1型主要毒力基因分析及TK基因缺失载体的构建     

范斌  陈卓  刘建华  鲍子磊  胡月  贾钦瑞  王雪竹  冉多良 《中国畜牧兽医》2018,45(10):2681-2690

为了解新疆马疱疹病毒1型（EHV-1）主要毒力基因遗传进化情况并构建TK基因缺失株，本研究以EHV-1 XJ2015株DNA为模板，对其主要毒力基因TK、gI和gE全长进行克隆、测序及生物信息学分析，并扩增TK基因左右重组臂TKL和TKR，构建质粒pUC-TKLR，将扩增后的增强绿色荧光蛋白（EGFP，含有CMV+polyA）插入pUC-TKLR质粒，构建TK基因缺失打靶质粒。TK、gI和gE基因同源性分析结果显示，XJ2015株与国外EHV-1分离株TK、gI和gE基因同源性均较高，分别为99.8%~100.0%、99.6%~100.0%和99.9%~100.0%；与EHV-3分离株同源性均最低，分别为72.9%、59.4%和62.1%；遗传进化分析显示，3个基因均与国外EHV-1同属于一个遗传进化分支，与EHV-9和EHV-4进化关系较近，但与EHV-3进化关系较远，表明XJ2015毒株与国外EHV-1毒株TK、gI、gE基因核苷酸上差异不明显，没有明显的地域性特征，功能基因保守且进化缓慢，同源基因功能相同或相近；经PCR扩增、酶切、测序及转染鉴定，本试验成功构建了用于TK基因缺失的打靶质粒pUC-TKLR-EGFP。通过对EHV-1主要毒力基因的分析及TK基因缺失打靶载体的构建，为新疆地区马鼻肺炎流行病学调查分析、TK基因缺失株的构建提供理论依据。  相似文献   

Identification of an H1N1 subtype of swine influenza virus and serological analysis     

Fa-chao SUN  Min TAN  Yuan-chao ZHANG  Yu-chao WANG  Sheng-liang CAO  Guo-fei DING  Fang-yuan CONG  Li-hong GUO  Si-dang LIU  Yi-hong XIAO 《农业科学学报》2019,18(7):1436-1442

To investigate the epizootic of swine influenza virus(SIV), 60 nasal swabs were collected from a clinical cases of pig farm in Tai'an City, Shandong Province of China in April 2017. SIV was isolated by inoculating into 10-day-old Special Pathogen Free embryonated eggs and the whole genome was sequenced. An H1N1 subtype SIV was isolated and designated as A/swine/Shandong/TA04/2017(H1N1). Phylogenetic analysis showed that apart from the polymerase A(PA) fragment belonging to the 2009 pandemic H1N1 branch, seven genome segments belonged to avian-like H1N1 influenza virus lineage. The cleavage site sequence of the hemagglutinin(HA) protein was PSIQSR↓G, which is a typical molecular biological characteristic. Five potential N-glycosylation sites(N14, N26, N277, N484 and N543) were found in the HA gene. To further investigate the epidemiology of SIV in this farm, the 995 serum samples were assessed with EAH1N1 2009 pandemic H1N1 and H3 N2 antigens. The results showed that the total positive rate was 65.43%. The positive rates of single virus infection detected by EAH1N1, 2009 pdmH1N1 and H3 N2 for serum HI(Hemagglutination inhibition) were 48.35, 30.85 and 7.47%, respectively. The results showed that SIV in Shandong Province has been reassorted in some segments and the SIV-positive rate was high on the SIV outbreak farm. These data provide evidence of an epizootic of SIV.  相似文献   

A comparison of the effects of carbon dioxide and medical air for abdominal insufflation on respiratory parameters in xylazine-sedated sheep undergoing laparoscopic artificial insemination     

JD Haan  BL Hay Kraus  SR Sathe 《New Zealand veterinary journal》2018,66(4):167-171

AIMS: To determine if abdominal insufflation with medical air will improve oxygenation and ventilation parameters when compared to insufflation with CO₂ in xylazine-sedated sheep undergoing laparoscopic artificial insemination (AI).

METHODS: Forty-seven sheep underwent oestrus synchronisation and were fasted for 24 hours prior to laparoscopic AI. Each animal was randomised to receive either CO₂ or medical air for abdominal insufflation. An auricular arterial catheter was placed and utilised for serial blood sampling. Respiratory rates (RR) and arterial blood samples were collected at baseline, after xylazine (0.1?mg/kg I/V) sedation, 2 minutes after Trendelenburg positioning, 5 minutes after abdominal insufflation, and 10 minutes after being returned to a standing position. Blood samples were collected in heparinised syringes, stored on ice, and analysed for arterial pH, partial pressure of arterial O₂ (PaO₂), and CO₂ (PaCO₂). The number of ewes conceiving to AI was also determined.

RESULTS: Repeated measures ANOVA demonstrated temporal effects on RR, PaO₂, PaCO₂ and arterial pH during the laparoscopic AI procedure (p<0.001), but no difference between insufflation groups (p>0.01). No sheep experienced hypercapnia (PaCO₂>50?mmHg) or acidaemia (pH<7.35). Hypoxaemia (PaO₂<70?mmHg) was diagnosed during the procedure in 14/22 (64%) ewes in the CO₂ group compared with 8/23 (35%) ewes in the medical air group (p=0.053). Overall, 15/20 (75%) ewes in the CO₂ group conceived to AI compared with 16/22 (72.7%) in the medical air group (p=0.867).

CONCLUSIONS AND CLINICAL RELEVANCE: There were no statistical or clinical differences in RR, PaO₂, PaCO₂, pH, or conception to AI when comparing the effects of CO₂ and medical air as abdominal insufflation gases. None of the sheep experienced hypercapnia or acidaemic, yet 42% (19/45) of sheep developed clinical hypoxaemia, with a higher percentage of ewes in the CO₂ group developing hypoxaemia than in the medical air group. Based on the overall analysis, medical air could be utilised as a comparable alternative for abdominal insufflation during laparoscopic AI procedures.  相似文献   

桃蚜电压门控钠离子通道基因cDNA克隆及其生物信息学分析          下载免费PDF全文

王颢  段文波  李芬  李为争  吴少英 《中国生物防治学报》2020,36(3):443-451

克隆获得桃蚜电压门控钠离子通道基因cDNA序列，明确钠离子通道的典型特征，为研究桃蚜抗性分子机理奠定基础。采用实验技术主要有RT-PCR和PCR，克隆桃蚜钠离子通道基因cDNA序列，利用相关软件对其序列进行生物信息学分析。克隆得到两段cDNA序列MpNa_v-1（NCBI登录号：MN124170）和MpNa_v-2（NCBI登录号：MN176136）。MpNa_v-1长度为2945 bp，包括2877 bp的完整开放阅读框，共编码958个氨基酸；MpNa_v-2长度为3546 bp，包括3486 bp的完整开放阅读框，共编码1161个氨基酸。MpNa_v-1和MpNa_v-2共同组成桃蚜的钠离子通道α亚基，MpNa_v-1包含同源结构域Ⅰ和同源结构域Ⅱ，MpNa_v-2包含同源结构域Ⅲ和同源结构域Ⅳ。同源比对发现，桃蚜与豌豆蚜和高粱蚜钠离子通道基因相似度分别高达97.67%和97.65%，所克隆序列包含昆虫钠离子通道α亚基典型特征，具有MFM模块，并含有蚜虫类钠通道特有模块DENS。成功地克隆桃蚜钠离子通道基因，为阐明其对拟除虫菊酯类药剂产生靶标抗性的分子机制奠定基础。  相似文献   

橡胶园酸性、中性土壤交换性钙、镁测定方法研究     

许木果  丁华平  刘忠妹  陈桂良  陈永川  黎小清 《中国农学通报》2020,36(18):73-79

为了获得简单、易操作、准确度高的橡胶园酸性、中性土壤交换性钙、镁测定方法,本试验通过响应面优化试验,建立称样量、提取液体积、提取方式与交换性钙测定结果之间的数学模型。结果表明：在25℃条件下,以1 mol/L的乙酸铵为土壤浸提液,称样量为2 g、提取液体积175 mL、振荡36 min,为橡胶园酸性、中性土壤的最佳提取条件。将上述条件下得到的结果与标准方法条件下得到的结果相对比,两结果的相对误差在1.25~3.04之间,均属合理范围。利用标准样品对优化方法进行试验验证,结果与推荐值相符。改进后的方法分析成本低、操作简便、测定结果准确、稳定性好,可用于橡胶园酸性、中性土壤交换性钙、镁的测定。  相似文献   

Measuring trail users’ perception of crowding in a peri-urban nature park: A best-worst scaling experiment     

《Urban Forestry & Urban Greening》2018

This paper evaluates the perception of crowding, its determinants and relationship with trail users’ experience in a peri-urban nature park. Existing crowding surveys rely heavily on a single-item 9-point crowding scale which was originally developed for backcountry areas. Receiver operating characteristic (ROC) analysis was used to test the validity and reliability of this measure in detecting crowding issues and formulating visitor capacity limits. Low validity and reliability raised concerns about the applicability of this scale in similar frontcountry areas. Inability of this simple crowding measure to detect a visitor threshold brings to the fore its another important limitation – ignorance of the complexity of crowding phenomenon. The perception of crowding varies with various social, psychological and situational factors, and a best-worst scaling (BWS) experiment was designed to simultaneously evaluate different aspects of crowding in the Medvednica Nature Park, a peri-urban nature park near Zagreb, Croatia. The findings of BWS experiment revealed that occupancy of mountain huts, number of other hikers on the trail and amount of litter along the trail on average contributed most to the trail users’ sense of crowding. A latent class analysis identified a large heterogeneity in the perception of crowding; while one group of trail users (63%) was more disturbed by the direct social impacts, the others (37%) were more disturbed by the secondary impacts of other visitors, especially by the negative externalities from road traffic in the park. Older visitors had a greater tendency to primarily associate crowding with the environmental conditions. A higher impact of trail use level in the perception of crowding, younger age and more frequent use were associated with a more negative evaluation of crowding. The park management should closely monitor road traffic in the park and visitor activity on highly used trails, especially on those popular among younger trail users.  相似文献   

秀珍菇原基形成相关基因PpFBD1的克隆与表达研究     

王伟科  宋吉玲  陆娜  袁卫东  闫静  陈观平 《浙江农业学报》2020,32(1):93

前期研究中通过对秀珍菇经低温诱导后不同发育阶段样本进行转录组测序及差异表达基因分析发现,ID为Cluster-6377.64510的基因(命名为PpFBD1)在原基形成前期的样本中高表达。为进一步了解分析其表达特性及功能,根据转录组测序结果设计特异性引物,利用RT-PCR技术从秀珍菇中克隆获得了该基因的cDNA全长。该基因由342个核苷酸组成,编码一个由113个氨基酸组成的蛋白,蛋白相对分子质量约11 ku。进化树分析显示,PpFBD1编码蛋白与糙皮侧耳hydrophobin1编码的疏水蛋白亲缘关系最近。Gene Ontology功能分析表明,PpFBD1主要参与真菌类细胞壁的合成。荧光定量RT-PCR检测显示,该基因在菌丝体经低温诱导后恢复至室温阶段(原基形成前期)表达量最高,这表明其可能参与调控秀珍菇原基形成过程。本研究结果为阐明秀珍菇原基形成机制提供参考。  相似文献   

狂犬病病毒SRV9克隆株核蛋白基因的克隆、表达与特性分析   总被引：4，自引：0，他引：4  

袁慧君  扈荣良  张守峰  张茂林  涂长春 《中国预防兽医学报》2003,25(1):5-8

根据已发表的狂犬病病毒核蛋白基因序列，设计并合成了一对引物，从SAD株驯化的SRV9。蚀斑株中提取病毒RNA，通过RT-PCR扩增出核蛋白的全长cDNA序列，测序结果显示，其序列与国外报道的SAD母源株序列一致。将核蛋白的cDNA克隆至原核表达载体pET-28b( )中，转化大肠杆菌BL21（DE3）plyss，于30℃1mmol/LIPTG条件下诱导表达，大肠杆菌菌体裂解产物经SDS-PAGE分析，在分子量约为56kDa处出现一新的蛋白带。和预期的目的蛋白分子量相符，Western-blotting检测表明，表达产物能与狂犬病病毒阳性血清发生特异性反应，出现单一反应带，扫描分析显示，表达产物占菌体总蛋白的23%，包涵体分离，纯化后，纯度达89%，上述结果为核蛋白在狂犬病基因免疫和免疫检测中的进一步应用奠定了基础。  相似文献   

猎豹与虎猫杯状病毒的分离及其超变区基因比较研究   总被引：1，自引：0，他引：1  

高玉伟  夏咸柱  扈荣良  黄耕  徐春忠  王铁东 《中国预防兽医学报》2003,25(3):179-182

用F81细胞从上海某动物园患口腔溃疡的猎豹和虎的唾液病料中分离获得两株杯状病毒，经形态学、理化学、生物学鉴定和病毒核酸超变区基因RT-PCR扩增与序列测定证明两株病毒均为猫杯状病毒(FCV)，分别命名为FCV/cheetah/Shanghai/02/2002与FCV／tiger/Shanghai／03/2002。人工感染猫可引起体温升高，口腔溃疡，食欲下降等症状。两株病毒的超变区序列520bp长片段问的同源性为99.2％，与国内桂林虎分离株(TFCV9710)的同源性为74．0％，与国外分离株的总体同源性为58．1％，说明不同宿主或同种不同个体间杯状病毒分离株超变区核酸差异十分显著，符合猫杯状病毒的分子生物学特征。  相似文献   

6株牛传染性鼻气管炎病毒BICP4基因序列的同源性分析   总被引：1，自引：0，他引：1  

李继昌  童光志  仇华吉  薛强  周艳君 《中国预防兽医学报》2003,25(2):92-95

牛传染性鼻气管炎病毒的立即早期启动基因IER4.2编码感染细胞蛋白BICP4基因。各种疱疹病毒的BICP4基因的Ⅰ，Ⅲ，Ⅴ区变异性较大，并为其特征区。为了比较分离自不同牛种，不同部位的5种病毒株BICP4蛋白I区基因的差异性，本文根据已发表的IBBV K22毒株重复序列IRs中BICP4的基因序列设计了一特异性引物，对各毒株进行PCR扩增，均得到约540bp的片段，将其克隆，测序并连同K22株进行同源性比较。结果表明，6株病毒的核苷酸序列及氨基酸序列同源性均在98%以上，说明这些毒株重复序列中的早期基因高度保守，在一定程度上也显示出IBRV的高度保守性。  相似文献