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旨在制备毒害艾美耳球虫(Eimeria necatrix)配子体抗原EnGAM22单克隆抗体,用Ni-NTA亲和层析纯化的重组抗原rEnGAM22免疫BALB/c小鼠,免疫3次后,取脾细胞与骨髓瘤细胞SP2/0进行融合,用预先建立的ELISA方法筛选阳性杂交瘤细胞,经有限稀释法对阳性杂交瘤细胞进行4次亚克隆后,获得2株能稳定分泌特异性抗体的杂交瘤细胞株并命名为2F3和3D3;对单抗亚类和特异性进行鉴定,应用单抗识别天然蛋白和虫体定位。结果显示,单克隆抗体2F3和3D3亚类分别为IgG2a、IgG2b,纯化后腹水效价分别为1:256 000、1:64 000,能特异性识别重组抗原rEnGAM22和毒害艾美耳球虫天然配子体蛋白;免疫荧光定位显示单克隆抗体2F3和3D3能特异性识别大配子体的成壁体和卵囊壁,表明EnGAM22抗原参与卵囊壁的形成。制备的单克隆抗体为研究球虫卵囊壁形成的分子机制奠定了基础。 相似文献
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利用聚丙烯酰胺凝胶电泳技术(SDS-PAGE)分析比较了11株巨型艾美耳球虫(Eimeria maxima)孢子化卵囊可溶性蛋白。用上海株、扬州株和美国株的阳性血清,分别对上海株、扬州株和美国株的孢子化卵囊可溶性蛋白进行Western blotting分析,比较这3株球虫孢子化卵囊可溶性蛋白的抗原性。结果显示,11株巨型艾美耳球虫孢子化卵囊可溶性蛋白的SDS-PAGE电泳图谱相似,至少有18条电泳条带,其中有7条相对明显的主带,相对分子质量分别为100 2007、7 000、63 2004、8 3004、1 200、36 300和30 000。Western blotting分析显示,在同源株间与异源株间的印迹图谱未见差异,均有4条免疫印迹条带,相对分子质量分别为103 700、64 200、43 400和37 100。 相似文献
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旨在探析毒害艾美耳球虫氧化还原酶EnOXIO1的功能。提取毒害艾美耳球虫扬州株配子体总RNA,应用RT-PCR获取EnOXIO1编码基因,构建原核表达载体pET-28a(+)-EnOXIO1,转化至大肠杆菌BL21(DE3),体外诱导表达,对重组蛋白进行抗原性分析,应用制备的多抗进行激光共聚焦免疫荧光定位。结果显示,获得的EnOXIO1基因全长为2 535 bp,体外重组表达的蛋白大小约100 ku,主要以包涵体的形式存在,表达量约为0.5 mg·mL-1。Western blot分析结果显示,该重组蛋白能被6×HIS标签单克隆抗体识别,同时能被制备的鼠多抗以及毒害艾美耳球虫、堆型艾美耳球虫和柔嫩艾美耳球虫病鸡康复血清所识别,表明该重组蛋白具有较好的反应原性和交叉反应原性。激光共聚焦免疫荧光定位结果显示,EnOXIO1基因表达产物主要存在于配子体内的成壁体上,参与了卵囊壁的形成。本研究成功克隆和表达了毒害艾美耳球虫EnOXIO1蛋白,并将其定位于配子体和卵囊壁上,为进一步解析EnOXIO1蛋白参与卵囊壁形成的分子机制奠定基础,为研制新型免疫阻断型球虫亚单位疫苗提供重要靶抗原。 相似文献
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《畜牧与兽医》2020,(11)
提取柔嫩艾美耳球虫(扬州株)配子体基因组DNA,PCR扩增配子体基因Etgam56,经克隆和序列分析后,优化密码子,连接至pET-28a(+),构建原核表达载体pET-28a(+)-Etgam56,优化条件进行体外诱导表达,Western blot分析其抗原性。结果显示,Etgam56基因全长1 425 bp,为一个完整开放阅读框,共编码474个氨基酸;体外表达重组蛋白的大小约为56 ku,IPTG终浓度为0.10 mmol/L,37℃诱导3 h时的表达量最高,且主要以包涵体形式存在。Western blot结果显示,该重组蛋白能被6×HIS标签单克隆抗体、柔嫩艾美耳球虫鸡康复血清和毒害艾美耳球虫鸡康复血清识别,表明该重组蛋白为特异性表达的蛋白,具有很好的抗原性和交叉反应性。本研究将为进一步探析柔嫩艾美耳球虫配子体蛋白的功能与研制鸡球虫基因工程疫苗奠定基础。 相似文献
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《中国兽医学报》2017,(5):844-849
本研究将巨型艾美耳球虫Emgam56基因去除信号肽后的ORF序列连接至pET-28a(+)载体,构建表达载体pET-28-Emgam56,转化表达宿主菌大肠杆菌BL21(DE3),经IPTG诱导表达;分析表达产物的可溶性和重组蛋白的分子相对质量,并进一步鉴定重组配子体蛋白rEmGAM56的抗原性。SDS-PAGE分析表明,表达载体能表达56000左右蛋白质,主要以可溶性蛋白的形式存在;Western blot检测表明,rEmGAM56蛋白能被鼠抗rEmGam56多克隆抗体和巨型艾美耳球虫感染鸡的康复血清特异性识别,显示重组蛋白具有良好的抗原性。本研究结果为研制重组配子体抗原亚单位疫苗奠定了基础。 相似文献
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将毒害艾美耳球虫重组蛋白rEnApiAP2单独免疫(单免)或分别与柔嫩艾美耳球虫重组蛋白rEtGAM56或rEtGAM59联合免疫(联免)雏鸡,同时设rEtGAM56单免、rEtGAM59单免、未免疫攻虫和空白组为对照,分别攻毒害艾美耳球虫或柔嫩艾美耳球虫,以成活率、排出血便数、平均增重、相对增重率、病变记分、卵囊减少率、抗球虫指数(ACI)和血清抗体水平为指标,评价rEnApiAP2单免及与rEtGAM联免的免疫保护效果。结果显示:各试验组的成活率均为100%;与未免疫攻虫组比较,各免疫组鸡排血便堆数减少、平均增重增加。在rEnApiAP2单免组间,低剂量组的相对增重率(82.36%)、卵囊减少率(73.88%)和抗球虫指数(155.26)均为最高,平均病变记分最低(1.71)。攻毒害艾美耳球虫后,联免组的ACI值均大于相应的单免组,其中rEnApiAP2与rEtGAM56联免组的ACI值(169.83)最高。攻柔嫩艾美耳球虫后,rEnApiAP2单免组的ACI值(128.37)低于rEtGAM56、rEtGAM59单免组(130.12、151.88),rEnApiAP2与rEtGA... 相似文献
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ELISA检测DLV虫苗免疫鸡抗体消长规律的研究 总被引:5,自引:0,他引:5
将柔嫩、毒害、巨型艾美耳球虫的孢子化卵囊制成可溶性抗原,用酶联免疫吸附试验检测鸡球虫双重致弱多凤苗虫苗、为柔嫩、毒害、巨型艾美耳球虫三价苗免疫鸡血清抗球虫抗体的消长规律。 相似文献
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利用本实验室前期获得的柔嫩艾美耳球虫(Eimeria tenella)孢子化卵囊和未孢子化卵囊差异表达ESTs序列,选取编号为BW4-C03的孢子化卵囊,采用RACE技术,获得该基因全长序列。经BLAST分析,该序列与柔嫩艾美耳球虫表面抗原有72%以上的同源性,命名为EtSAG。利用荧光定量PCR(Real-time PCR)检测发现该基因在孢子化卵囊的转录拷贝数最高,且随着孢子化时间的延长,转录拷贝数逐渐增加。采用原核表达载体pET-28C表达该基因,得到的融合蛋白大小约为36 kDa,符合预期大小。经Western blot分析,该重组蛋白可被兔抗柔嫩艾美耳球虫的多克隆抗血清识别,表明该蛋白具有较好的反应原性。本研究结果为进一步研究该基因的生物学功能奠定了基础。 相似文献
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魏静 《四川畜牧兽医学院学报》2009,(4):28-32
在现代法律秩序中,商会自治规范是制定法的基础和必要的补充,甚至在某些方面替代了制定法;商会自治规范主要包括商会组织规范、行为规范、惩罚规范以及争端解决规范等;其效力仅及于其内部成员;商会自治规范和制定法之间存在冲突,但也存在整合的基础。 相似文献
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本文概述了猪的毛色类型、猪的毛色遗传模式,着重综述了猪毛色基因分子基础的研究进展,指出存在问题并就未来发展方向做了思考。 相似文献
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REASONS FOR PERFORMING STUDY: Centesis of the bicipital bursa using an 8.9 cm long spinal needle has been reported but the alternative of employing a 3.8 cm long hypodermic needle requires validation. OBJECTIVE: To compare the efficacy of 2 different methods of centesis of the bicipital bursa and to evaluate the usefulness of ultrasonographic imaging to determine the location of solution administered when centesis of the bursa is attempted. METHODS: For Trial 1, 6 clinicians, who had no previous experience of centesis of the bicipital bursa, attempted to inject a solution composed of an aqueous radiopaque contrast medium and physiological saline solution (PSS) into the bicipital bursae of 2/12 horses using the previously described distal approach to inject one bursa and a proximal approach to inject the contralateral bursa. The bicipital tendon and bursa were examined ultrasonographically before and after injection; and both shoulders were examined radiographically to identify the location of the medium. In Trial 2, another 6 clinicians, also with no previous experience of centesis, repeated Trial 1, using 6 horses, but the radiopaque contrast medium was mixed with air instead of PSS. RESULTS: Accuracy of centesis using the proximal approach was 39% and that of the distal approach 28%. Ultrasonographic examination of the shoulder allowed the location of solution and air to be accurately predicted in all 12 shoulders examined. CONCLUSIONS: Clinicians who have had no previous experience performing centesis of the bicipital bursa are unlikely to be successful in centesis using either approach. Radiographic examination after injecting a radiopaque contrast medium may be necessary to assess the success of centesis especially if bursal fluid is not obtained during centesis. Injecting air along with the radiopaque contrast medium provides more accurate ultrasonographic confirmation of centesis and better radiographic definition than does injection without air. 相似文献
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Effects of size of ingestively masticated fragments of plant tissues on kinetics of digestion of NDF
Ingestively masticated fragments were collected and sized via sieving. Different sizes of esophageal masticate and ruminal digesta fragments, and ground fragments of larger masticated pieces were incubated in vitro, and undigested NDF remaining at intervals of up to 168 h of incubation was determined. The ruminal age-dependent time delay (tau) for onset of digestion of NDF was positively correlated (P < 0.004) with the mean sieve aperture estimated to retain 50% of the fragments between successive sieve apertures (MRA). Degradation rate of potentially degradable NDF (PDF) and level of indigestible NDF were not related (P > 0.10) to MRA of masticated and ground fragments. Estimates of tau were positively related to MRA, with slopes of bermudagrass < corn silage < ruminal fragments of corn silage. It was concluded that fragment size-, and consequently, ruminal age-dependent onset of PDF degradation of a mixture of different fragment sizes results in an age-dependent rate of degradation of the more rapidly degrading of two subentities of PDF. Models are proposed that assume a tau before onset of simultaneous degradation of PDF from two pools characterized as having gamma-modeled age-dependency and age-constant rates. The ruminal age-dependent pool seems to be associated with the faster-degrading pool, and its rate parameter increases with range in MRA in the population of fragments. Conceptually, the ruminal age-dependent rate parameter for PDF degradation seems to represent a composite of several effects: 1) effects of the size-dependent tau; 2) range in MRA of the population of ingestively masticated fragments; and 3) subentities of PDF that degrade via more rapid age-dependent rates compared with subentities of PDF that degrade via age-constant rates. The estimated fractional rates of ruminative comminution of ingestively masticated fragments (0.060 to 0.075/h) were of a magnitude similar to the mean fractional rates of PDF digestion (0.030 to 0.085/h), which implies that ruminative comminution may be first-limiting to fractional rate of PDF digestion. The in vivo roles of ingestive and ruminative mastication of fragments on PDF degradation must be considered in any kinetic system for estimating PDF digestion in the rumen. These results and others in the literature suggest that the rate of surface area exposure rather than intrinsic chemical attributes of PDF may be first-limiting to degradation rate of PDF in vivo. 相似文献
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以国际标准强毒R株人工感染非免疫产蛋鸡,定时扑杀,分别从鼻窦、眶下孔、气管、肺、气囊、卵巢和输卵管分离MG,并收集感染鸡所产蛋分离MG。结果表明,人工感染48小时后上、下呼吸道及肺已被全面感染,96小时气囊已被感染,120小时输卵管已能分离到MG,卵巢始终分离不到MG。人工感染鸡自144小时便能在其所产蛋中分离出MG。药物治疗能在72小时内消除感染,油乳剂苗则需24天后逐渐降低蛋内MG分离率,药物卵内注射、种蛋药浴、高温处理均能杀死卵内MG,但以研制的种蛋浸泡剂药浴效果为最好。 相似文献