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噬菌体抗体是表面表达有单链抗体(Singlechain Fv,ScFv)或抗体Fab段的噬菌体,用基因克隆技术将B细胞全套可变区基因克隆出来,组装到表达载体内并表达到噬菌体表面成为噬菌体抗体的群体,即为噬菌体抗体库.该技术是丝状噬菌体展示(phage display)与抗体组合文库技术相结合而成.其基本路线为用PCR方法扩增抗体全套可变区基因,重组到噬菌体载体中,并通过与丝状噬菌体的外壳蛋白形成融合蛋白,把Fab段或ScFv表达到噬菌体表面.通过"吸附-洗脱-扩增"的富集过程,从中筛选出特异性抗体的可变区基因. 相似文献
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抗非洲猪瘟病毒单链抗体的构建、表达及活性鉴定 总被引:1,自引:0,他引:1
制备抗非洲猪瘟病毒(ASFV)猪源单链抗体(ScFv),并对其生物学活性进行鉴定,筛选出具有ASFV反应活性的猪源单链抗体(ScFv),为ASFV的诊断提供新的材料。采集ASFV感染康复猪的外周血,分离淋巴细胞,提取淋巴细胞总RNA,以此为模板通过PCR扩增得到猪源IgG重链可变区与轻链可变区基因,利用SOE-PCR技术扩增拼接得到猪源ScFv基因;构建pET-30a-ScFv表达载体,进行蛋白的表达与纯化,用ELISA和IFA鉴定ScFv抗体的反应活性。结果显示,成功扩增出猪源ScFv(VH-VLκ、VH-VLλ)基因,鉴定到1株与ASFV存在反应活性的ScFv(VH-VLλ11)抗体。结果表明,成功筛选到1株与ASFV存在反应活性的ScFv(VH-VLλ11)抗体,为ASFV诊断和防控提供了新型原材料。 相似文献
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首先对猪囊尾蚴头节特异性单链抗体重链可变区基因(Vh)和轻链可变区基因(Vl)进行酶切鉴定,PCR鉴定以及测序分析,然后采用重叠延伸PCR(SOE PCR)技术,将重链可变区基因和轻链可变区基因,通过linker链连接成Vh-linker-Vl单链抗体基因(ScFv)。将此基因与pMD18-T载体连接,转化感受态大肠杆菌J M109,从而进一步克隆并对重组载体pMD-ScFv进行鉴定。结果:成功构建并克隆了抗猪囊尾蚴头节单链抗体,为重组免疫毒素ScFv-PE40的构建?表达及活性测定奠定了良好的基础。 相似文献
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《中国饲料》2015,(9)
以纯化的重组大肠杆菌外膜蛋白Omp C免疫BALB/c小鼠,提取骨髓浆细胞总RNA,逆转录获得c DNA,作为模板通过PCR扩增获得重链和轻链可变区序列。由(Gly4Ser)3 linker连接两片段,获得单链抗体可变区片段基因库。重叠延伸PCR添加T7启动子、核糖体结合位点、gⅢtether和两端茎环结构等元件,成功构建对外膜蛋白C具有特异性亲和力的单链抗体可变区的核糖体展示文库,为特异性强、亲和力高的单链抗体可变区筛选提供了技术和材料基础。构建能与革兰氏阴性菌外膜蛋白C特异性结合的鼠源单链抗体核糖体展示文库,是奠定构建饲料革兰氏阴性腐败菌分子预警和靶向抗菌剂的靶向定位区的高亲和力单链抗体可变区片段的重要基础,对建立有害革兰氏阴性菌分子检测方法和靶向抗革兰氏阴性菌菌剂设计方法具有借鉴作用。 相似文献
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《中国预防兽医学报》2015,(5)
为构建抗1型鸭肝炎病毒(DHV-1)RdRp蛋白的单链抗体(scFv)库,并淘选特异性scFv,本研究采用RT-PCR方法从DHV-1基因组中扩增Rd Rp基因,将其克隆于pET-32a载体中进行重组RdRp蛋白原核表达。将纯化的重组蛋白免疫小鼠,以提取的免疫鼠脾脏总RNA为模板,利用抗体轻链可变区(VL)和重链可变区(VH)简并引物,经RT-PCR扩增抗体的VL和VH编码序列,由Linker序列连接,通过重叠延伸PCR(SOE-PCR)扩增完整的sc Fv基因(VL-Linker-VH)。将扩增的scFv基因克隆于pCANTAB5E载体中构建噬菌体scFv文库,利用纯化的重组RdRp蛋白开展4轮吸附-洗脱-富集以淘选抗RdRp的ScFv;最后对淘选到的阳性ScFv进行可溶性表达,以ELISA方法检测其免疫结合活性。结果显示,获得了7株具有良好抗原结合活性的ScFv。淘选到的DHV-1 RdRp特异的基因工程ScFv,为鸭病毒性肝炎防制新策略研究奠定了基础。 相似文献
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利用在体外构建的三肽囊素抗独特型抗体可变区VH和VL基因,通过重叠延伸反应(SOE),以(Gly4Ser)3为连接肽,将VH和VL基因连接成为VH-Linker-VL ScFv,且ScFv DNA与噬菌粒载体pHENI的连接产物转化于大肠杆菌TGl,经辅助噬菌体M13K07感染后,获得重组的鸡源抗三肽囊素抗独特型抗体全套单链噬菌体抗体库,并将该抗体库展示在噬菌体表面,以利用噬菌体展示技术的强大筛选能力筛选出与三肽囊素同功能单链抗体(Bursin-ScFv)。 相似文献
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《中国兽医学报》2017,(2):243-249
选取经产气荚膜梭菌ι毒素免疫且抗体效价较高的小鼠,提取脾脏mRNA,通过PT-PC技术,分别获得340,325bp的重链可变区基因(VH)和轻链可变区基因(VL)。利用重叠延伸PCR(SOE-PCR)获得大小为750bp的VH-Linker-VL单链抗体基因(ScFv)。利用One Step Cloning Kit试剂盒将ScFv基因片段与噬菌粒载体pCANTAB5E连接,构建库容约为3.5×106抗ι毒素噬菌体初级抗体库。以Ia、Ib为抗原进行Phage-ScFv表面展示文库的5轮亲和富集筛选,最终阳性率均可达70%。选取亲和力最强的3株阳性抗体(Ia-4、Ia-8和Ib1)进行可溶性表达,为ι毒素特异性抗体的大量制备奠定基础,并对产气荚膜梭菌ι毒素感染病例的检测和临床治疗研究具有一定的理论价值和实践意义。 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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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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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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