猪瘟病毒E2基因的克隆及分子特性分析

为研究四川地区猪瘟病毒（classical swine fever virus,CSFV）及E2基因的遗传变异情况,试验对CSFV阳性病料抽提RNA,通过RT-PCR扩增CSFV E2基因并将其克隆到pMD18-T载体,经菌落PCR、双酶切、序列测定后利用生物信息学软件分析了E2基因的分子特性。结果显示,扩增的E2基因大小为1 125 bp,编码375个氨基酸,与GenBank中CSFV GXWZ02、CSFV/2.1、HEBZ、YC11WB、SXYL2006、0406/CH/01/TWN亲缘关系较近,核苷酸同源性分别为98.3%、96.5%、94.7%、92.4%、92.1%和90.9%,而与疫苗株HCLV E2的同源性较低,为87.6%。密码子偏向性分析显示,E2基因的ENc值为53.161,密码子使用频率与大肠杆菌、酵母菌和人差异较大的分别有33、25和25个;此外,E2基因共有34个稀有密码子,且有多个稀有密码子多次串联成簇的情况出现。结果表明,E2基因在密码子使用中整体上不存在较明显的偏向性,且密码子使用模式更接近真核生物,本试验克隆的E2基因与疫苗株相比,部分核苷酸出现了变异,但主要抗原域氨基酸未发生改变,该结果为进一步开展CSFV E2功能研究及后期基因工程苗的研制提供了基础数据。     

猪繁殖与呼吸综合征病毒双基因多组合DNA疫苗的构建

本实验根据PRRSV结构蛋白的特性和IFNγ及IL-2这两种细胞因子的特性,构建表达PRRSVGP4-GP5、GP5-M、GP5-N、GP5-IFNγ、GP5-IL-2、N-IFNγ、N-IL-2的重组质粒。这些重组质粒在真核细胞中得到了表达。用这些真核表达质粒可以免疫小猪,通过检测其对猪体免疫应答的诱导能力和接种后动物对PRRSV的抵抗力来对基因疫苗的效能进行研究,以期筛选到有效的基因疫苗并为今后进一步的研究提供思路和理论基础。     

日本乙誓脑炎病毒NS1基因及其疫苗的研究进展

日本乙型脑炎是由日本乙型脑炎病毒引起的严重的人畜共患传染病。乙型脑炎病毒的三种糖基化蛋白PrM、E和NS1是其主要免疫保护性抗原,其中PrM和E蛋白是日本乙型脑炎病毒的结构蛋白,其结构和相关疫苗研究的报道较多。NS1蛋白是日本乙型脑炎病毒的非结构蛋白,其主要参与病毒复制的早期阶段,推测可能参与病毒组装和释放。可诱导补体依赖性溶细胞反应,不能产生中和抗体,能诱发机体在非中和性抗体存在的情况下产生保护性免疫。在接种乙脑病毒的细胞的细胞内、细胞表面及上清液中均含有大量的NS1蛋白。对小鼠接种NS1蛋白或NS1蛋白特异性抗体,均能让小鼠产生对乙脑病毒感染的保护性免疫作用。本文主要就乙型脑炎病毒非结构基因NS1及其免疫疫苗的研究进展进行综述,为其更进一步深入研究IEV NS1基因及其相关疫苗奠定基础。     

复方金银花对鸡体内NDV分布的影响

选用1日龄雏鸡,无污染环境饲养21 d后,随机分成感染组、中药治疗组、空白组。中药治疗组饮水给予复方金银花制剂。以单克隆抗体介导的免疫组化（IHC）法检测攻毒鸡体内NDV抗原的分布及定位,以确定复方金银花对新城疫病毒感染鸡的组织嗜性的影响。结果显示,感染组和中药治疗组鸡的16个组织器官中均能检测到NDV抗原,病毒以气管、肺脏、十二指肠、空肠等器官中分布为最高。复方金银花对新城疫的组织损伤有一定的保护作用,可缩短组织修复的时间。     

Pathogenicity of emerging Japanese type 1 porcine reproductive and respiratory syndrome virus in experimentally infected pigs

To clarify the pathogenicity of Japanese type 1 porcine reproductive and respiratory syndrome virus (PRRSV) isolate in experimentally infected pigs, we evaluated clinical signs and monitored viremia for 21 days post-inoculation (dpi). Lungs were mottled, tanned and reddish in appearance; had lesions predominantly in the cranial, middle and accessory lobes; and failed to collapse at 10 dpi. Although microscopic lesions of lungs were reproduced using the Japanese emerging type 1 PRRSV isolate under experimental conditions, no significant differences were noted between the challenge and control groups regarding mean rectal temperature and daily weight gain. These results provide useful insights into the limited pathogenicity of single infection with the Japanese type 1 PRRSV isolate in piglets, which differ from findings in reported field cases.     

表达外源基因的猪繁殖与呼吸综合征病毒(PRRSV)感染性克隆的构建及病毒拯救

为建立表达标记基因的重组猪繁殖与呼吸综合征病毒(PRRSV)反向遗传操作平台,本研究在PRRSV HuN4-F112疫苗株感染性分子克隆的基础上,采用突变PCR技术将新城疫病毒(NDV)NP蛋白末端优势抗原区插入PRRSV NSP2蛋白的复制非必须区,经基因片段的克隆、拼接,构建了含有外源标记基因的感染性的PRRSV cDNA克隆psk-HuN4-F112-Δ52+NP49。用限制性内切酶SwaΙ将psk-HuN4-F112-Δ52+NP49线性化后通过细胞外转录获得病毒RNA,用脂质体法将病毒RNA转染叙利亚仓鼠肾细胞(BHK-21)包装出病毒粒子,再转接到猴胚胎肾上皮细胞(MACR-145)传代拯救病毒。对拯救的病毒进行RT-PCR扩增、酶切和序列分析鉴定。结果表明,拯救病毒含有不同于亲本病毒的分子标记(病毒基因组14667位人工产生的MluⅠ酶切位点)和插入的标记基因序列。间接免疫荧光试验表明,外源基因NP49在拯救的PRRSV中表达,且能够在PRRSV传代过程中稳定遗传。病毒生长特性比较显示,拯救病毒与亲本病毒在MARC-145细胞上具有相似的增殖特性。本研究构建了含有标记基因PRRSV的感染性克隆并获得了拯救病毒,为进一步开发新型PRRS疫苗提供了有效的反向遗传操作平台。     

Contagious caprine pleuropneumonia – a comprehensive review

Contagious caprine pleuropneumonia (CCPP) is a serious disease of goats, occasionally sheep and wild ruminants, caused by Mycoplasma capricolum subspecies capripneumoniae (Mccp). The disease is characterized by severe serofibrinous pleuropneumonia, very high morbidity (～100%), and mortality (80–100%). CCPP affects goats in more than 40 countries of the world thereby posing a serious threat to goat farming around the globe. The characteristic clinical signs of CCPP are severe respiratory distress associated with sero-mucoid nasal discharge, coughing, dyspnea, pyrexia, pleurodynia, and general malaise. In later stages, severe lobar fibrinous pleuropneumonia, profuse fluid accumulation in pleural cavity, severe congestion of lungs and adhesion formation is observed. Mycoplasmal antigen interactions with host immune system and its role in CCPP pathogenesis are not clearly understood. CCPP is not a zoonotic disease. Diagnosis has overcome cumbersome and lengthy conventional tests involving culture, isolation, and identification by advanced serological (LAT, cELISA) or gene-based amplification of DNA (PCR, RFLP, and hybridization) and sequencing. The latex agglutination test (LAT) is rapid, simple, and better test for field and real-time diagnosis applicable to whole blood or serum and is more sensitive than the CFT and easier than the cELISA. Moreover, the studies on antibiotic sensitivity and exploration of novel antibiotics (fluoroquinolones, macrolides) can help in better therapeutic management besides preventing menace of antibiotic resistance. Re-visiting conventional prophylactic measures focussing on developing novel strain-based or recombinant vaccines using specific antigens (capsular or cellular) should be the most important strategy for controlling the disease worldwide.     

甘蓝型油菜品质性状间动态关系的分析

本文采用时间序列分析等方法,研究了油菜种子成熟过程中,蛋白质、油分、纤维素、可溶性糖、ATP 和生长量变化过程中的量化规律和动态相关规律。初步认为 ATP 在各发育阶段对有机物质合成有一定影响,其他物质变化过程表现一定程度的显著优势的周期函数特征。说明种子成熟过程中可能有某种代谢调节的共同系统。为了控制该系统以     

山羊痘病毒SS株5个ANK基因缺失的重组病毒构建

为研究锚定蛋白基因(Ankyrin,ANK)对山羊痘病毒的影响,试验采用融合PCR和Overlap PCR技术扩增山羊痘病毒SS株5个ANK基因(ANK010、ANK138、ANK 140、ANK141.2和ANK145)两端侧翼序列和绿色荧光蛋白(GFP)基因,并将其产物连接Trans1-T 1载体构建ANK缺失的转...