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堆型艾美耳球虫单链抗体-PE40重组免疫毒素的构建 总被引：1，自引：0，他引：1
用HindⅢ对已构建的质粒pT-PE40和原核表达载体pET22-ScFv进行单酶切,回收纯化1 101 bp的PE40基因片段并将其亚克隆至pET22-ScFv载体中,构建重组免疫毒素表达质粒pET22-ScFv-PE40。将该质粒转化入感受态大肠杆菌J M109中增殖,提取质粒后用SalⅠ和NotⅠ进行双酶切鉴定,得到了1 114 bp和6 181 bp目的基因片段。测序鉴定PE40的插入方向,选取以PE40基因5′端与ScFv基因3′端连接的重组质粒,构建了抗堆型艾美耳球虫重组免疫毒素质粒pET22-ScFv-PE40。抗堆型艾美耳球虫重组质粒pET22-ScFv-PE40经1 mmol/LIPTG诱导,在大肠杆菌中表达约68 000的目的蛋白。使用抗PE多克隆抗体对目的蛋白进行蛋白印迹分析,结果表达产物与抗PE多克隆抗体发生抗原抗体反应,证明融合基因得到表达。 相似文献
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通过电子克隆手段,克隆到大小为959 bp的cDNA序列.然后从鸡的脾脏提取RNA,用RT-PCR方法扩增出目的片段.将该片段连接到原核表达栽体pET-32a和真核表达载体pEGFP-C1中,采用SDS-PAGE检测原核表达载体pET-BCL10在大肠杆菌中的表达,脂质体转染法将真核表达载体pEGFP-BCL10转入vero细胞,Western blotting检测其在细胞中的表达.结果显示,PCR扩增到735 bp的DNA片段,原核表达载体和真核表达栽体经双酶切和核酸测序表明载体构建成功.SDS-PAGE电泳检测到pET-BCL10重组质粒在大肠杆菌中表达.western blotting检测到pEGFP-BCL10重组质粒在veto细胞中表达.结论表明,BCLl0基因在鸡体内存在,该基因的重组质粒能在大肠杆菌和vero细胞中表达. 相似文献
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构建新城疫病毒(Newcastle disease virus, NDV)核衣壳蛋白(nucleocapsid protein, NP)基因的原核表达载体,并将其在宿主菌E.coli BL21(DE3)感受态细胞中表达。以NDV La Sota株NP基因序列为模板,设计特异性引物,通过PCR扩增获得NP蛋白全长基因片段,定向插入原核表达载体pET-30a,构建重组质粒pET-NDV NP;将构建的重组质粒转化宿主菌E.coli BL21(DE3)感受态细胞,经IPTG诱导,表达出目的蛋白,并采用亲和层析的方法纯化表达蛋白;表达蛋白采用Western blotting方法检测其反应原性。结果显示,成功克隆了新城疫病毒NP基因全长,片段序列1470 bp;构建的pET-NDV NP载体经PCR、双酶切、测序鉴定均无误;转化表达宿主菌后经SDS-PAGE检测结果显示目的基因得到成功表达,且表达蛋白经Ni-NTA镍离子亲和层析法被成功纯化,蛋白质浓度为3 mg/mL;Western blotting检测结果显示表达蛋白具有良好的反应原性。试验结果表明,成功构建了含有新城疫病毒核衣壳蛋白基因的原核表达载体,成功表达、纯化得到了NDV NP蛋白。 相似文献
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为了克隆巴什拜羊SPLUNC1基因并表达该蛋白,本试验从巴什拜羊肺脏组织中提取总RNA,利用RT-PCR方法扩增出SPLUNC1基因开放阅读框序列,将该基因片段插入到真核表达质粒pPIC9K中构建pPIC9K-SPLUNC1重组质粒,然后将pPIC9K-SPLUNC1重组质粒线性化并电击转化入毕赤酵母GS115中,经甲醇诱导蛋白表达后用SDS-PAGE和Western blotting分析鉴定目的蛋白。结果表明,经RT-PCR扩增成功获得大小为748 bp的SPLUNC1基因;构建的pPIC9K-SPLUNC1重组质粒经PCR、酶切及测序鉴定与预期结果一致;SDS-PAGE及Western blotting检测鉴定结果表明获得大小为25.53 ku的SPLUNC1蛋白。本试验结果为进一步研究巴什拜羊SPLUNC1蛋白的生物学活性奠定基础。 相似文献
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试验旨在构建微小隐孢子虫表面抗原CP15基因原核表达载体并获得重组CP15蛋白。试验以已知重组质粒pMD-CP15为模板,PCR方法扩增出CP15基因片段,并亚克隆到pGEX4T-3,构建了在E.coli BL21中GST融合表达载体pGEX-CP15;经1 mmol/L IPTG进行诱导表达获得目的蛋白,其大小采用SDS-PAGE电泳和Western blotting进行鉴定。结果表明,扩增出约390 bp的微小隐孢子虫表面抗原CP15基因片段并成功构建pGEX-CP15质粒,表达出分子质量为42 ku的融合蛋白,与推导的理论值相符。Western blotting显示该蛋白能被GST血清识别。牛微小隐孢子虫表面抗原CP15基因在大肠杆菌中得到了高效表达。 相似文献
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为了克隆巴什拜羊SPLUNC1基因并表达该蛋白,本试验从巴什拜羊肺脏组织中提取总RNA,利用RTPCR方法扩增出SPLUNC1基因开放阅读框序列,将该基因片段插入到真核表达质粒pPIC9K中构建pPIC9K-SPLUNC1重组质粒,然后将pPIC9K-SPLUNC1重组质粒线性化并电击转化入毕赤酵母GS115中,经甲醇诱导蛋白表达后用SDS-PAGE和Western blotting分析鉴定目的蛋白。结果表明,经RT-PCR扩增成功获得大小为748bp的SPLUNC1基因;构建的pPIC9K-SPLUNC1重组质粒经PCR、酶切及测序鉴定与预期结果一致;SDS-PAGE及Western blotting检测鉴定结果表明获得大小为25.53ku的SPLUNC1蛋白。本试验结果为进一步研究巴什拜羊SPLUNC1蛋白的生物学活性奠定基础。 相似文献
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为了成功克隆外膜蛋白16(outer membrane proteins 16, Omp16)基因并对其进行原核表达,试验根据GenBank中羊布鲁氏菌M5-90株外膜蛋白Omp16基因序列(登录号:JF918760.1)设计1对引物,从布鲁氏菌基因组中扩增出大小约为507 bp的目的基因片段,凝胶回收纯化目的片段,连接入pMD20-T质粒,转化E.coli DH5α并测序,测序正确后再亚克隆入pET-28a(+)表达载体,构建重组质粒pET-Omp16,转化入E.coli BL21(DE3),经IPTG诱导其表达,最后用Western blotting分析方法鉴定诱导得到的蛋白。结果表明,成功构建了pET-Omp16原核表达载体,并在E.coli BL21中表达了Omp16基因,诱导得到的蛋白经鉴定与目的蛋白大小一致,证明成功表达了目的基因。 相似文献
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新城疫病毒F基因在大肠埃希菌中的表达及其抗原性分析 总被引：2，自引：0，他引：2
构建新城疫病毒融合蛋白F基因的原核表达载体,并将其在宿主菌BL21(DE3)感受细胞中表达.以含有融合蛋白F基因的重组质粒pMD19T-F为模板,设计特异性引物,应用PCR技术扩增获得F基因的F1片段,定向插入原核表达载体pET-28a,构建重组质粒pET-F1,将构建成功的重组质粒pET-F1转化宿主菌BL21(DE3)感受态细胞经IPTG诱导,将其在宿主菌细胞中表达,表达产物用SDS-PAGE和Western blot方法检测.结果显示,成功克隆出了新城疫病毒F1基因片段序列852 bp.构建的pET-F1载体经PCR、双酶切、测序鉴定均无误,转化表达宿主菌后经SDS-PAGE和Western blot检测结果显示,目的基因被成功表达,并具有良好的反应原性.成功构建了新城疫病毒的融合蛋白F基因原核表达载体,转化宿主细胞后成功表达了融合蛋白F1重组蛋白片段. 相似文献
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目的克隆边缘无浆体MSP5基因,构建重组质粒,并进行表达与鉴定。方法根据GenBank公布的边缘无浆体MSP 5基因序列(AY714547)设计一对引物,无菌颈静脉采集边缘无浆体阳性的牛血,用PCR方法从4血的DNA模板中扩增MSP 5基因582bp的片断。将该片段克隆到原核表达载体pGEX-KG中,构建原核表达载体KG-MSP5,转化BL21,经IPTG的诱导表达蛋白。利用BugBuster GST Bind Purification Kit将其纯化,经Western blotting进行分析。结果重组质粒KG-MSP5,转化BL21,在IPTG的诱导下表达大小约46 kPa蛋白。western blotting分析表明其具有很好的免疫原性。结论本研究为边缘无浆体的血清学诊断试剂盒的研制奠定了基础。 相似文献
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【目的】探讨禽致病性大肠杆菌(avian pathogenic Escherichia coli,APEC)外膜蛋白OmpA对DF-1细胞自噬的影响,为研究OmpA是否协助APEC逃逸宿主细胞自噬介导的清除作用提供依据。【方法】以APEC E058株基因组DNA为模版,通过PCR扩增回收ompA基因片段,经EcoRⅠ和XhoⅠ双酶切后克隆至真核表达载体pEGFP-N1,通过酶切及测序鉴定筛选阳性的重组表达质粒pEGFP-N1-ompA;随后将质粒转染至鸡胚成纤维细胞DF-1并通过Western blotting和免疫荧光试验检测OmpA蛋白的表达情况;通过透射电子显微镜、免疫荧光试验及Western blotting方法检测OmpA对DF-1细胞自噬的影响。【结果】测序鉴定正确的重组质粒pEGFP-N1-ompA经EcoRⅠ和XhoⅠ双酶切,获得大小分别为1 038和4 700 bp的条带,大小与载体和目的片段相符,表明真核表达质粒pEGFP-N1-ompA构建成功。将重组质粒经脂质体转染DF-1细胞后,通过免疫荧光试验可观察到大量绿色荧光,Western blotting检测到大小为... 相似文献
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Beran GW 《Preventive veterinary medicine》2008,86(3-4):198-207
When early people made their appearance, zoonotic infectious diseases were already waiting, but epidemic diseases did not appear in human history until people began to live in large numbers under conditions of close contact, mainly during the last 10,000 years. Disease has decimated urban populations, conquered armies, and disrupted society. The focus here is on (1) the plague of Athens and the Black Death; (2) smallpox, influenza, and rabies; (3) avian influenza prion diseases, and foot & mouth disease; and (4) emerging and re-emerging diseases. All have veterinary public health associations. In Athens, Greece, in 430 BC, when the Spartans ravaged the countryside, hordes crowded into Athens so that orderly movements, space in which to live, and adequate supplies of food became impossible. Crowding of any population fosters disease transmission; chaos and disorder enhance it all the more. Out of northern Egypt came a terrible plague from across the Mediterranean Sea. The identity of the plague of Athens remains unsure, but the well-considered conclusion is Rift Valley Fever, a mosquito borne, viral zoonosis. The Black Death, also called the Plague, raged in Asia for centuries. In 1347, the Black Death was brought by a ship out of Asia to Sicily. The scenes of devastation were repeated throughout Europe, with 90% or more of the people dying in city after city. Influenza, too, has been a cause of periodic human epidemics, but the great pandemic of influenza occurred in the last months of World War I. In the years of highest occurrence, more than half the world's population became clinically infected. If veterinary public health had been born earlier, it could have led to elucidating the epidemiology of influenza and the plagues of Athens, Europe, and Asia. In turn, smallpox had also caused continual tragedy. In 1796, Edward Jenner began to harvest pustules of cowpox from children or infected cows and inject them into susceptible children. In 1980, the World Health Organization declared that smallpox had been eliminated from the world. Rabies, though, still strikes terror. A number of animal diseases, broadly termed emerging and re-emerging diseases, need surveillance because they have the potential to impact human health. From late in 2003 to 2007, the highly pathogenic H5N1 influenza virus in poultry infected at least 121 people and caused 62 deaths in four countries. The prion diseases, too, all have very high numbers in concentrated contacts. To control these diseases, veterinary public health is essential, with diagnosis, epidemiological surveillance, clinical manifestations, and prevention as primary measures. 相似文献
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在动物的异常行为中以刻板行为最为常见,而咽气癖又是马最常见的口部刻板行为之一。作者就咽气癖在生理方面和心智方面对马体产生的影响、行为基础、诱因的研究进展及常见的防治措施进行综述。 相似文献
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Dekker A Dercksen D Snoep J van Wuyjckhuise L 《Tijdschrift voor diergeneeskunde》2007,132(18):695-701
On 26th of july 2007 a new case of bluetongue was notified in the Netherlands and on 2nd of august 2007 foot-and-mouth disease was diagnosed in Surrey, England, which raised the threat of having both infections simultaniously in one area. Bluetongue and foot-and-mouth disease have a different pathogenesis, but symptoms may resemble each other at a later stage of infection. The pathogenesis and possible clinical symptoms of both infections are discussed and illustrated with pictures. 相似文献
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近几年,世界饲用添加剂市场增长较快,预计今后将进一步增长,据世界粮农组织(FAO)首领会议的倡议,到2015年,将使世界营养不良的人口削减一半,其间,肉制品将会以每年2％的速度增长,特别是加快猪肉和禽肉增长。这将促进世界添加剂,尤其是蛋氨酸和赖氨酸需求量的增加。 相似文献
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