Establishment of the Method of Immunohistochemistry Assay for the Detection of Scrapie in Chinese Short-Tailed Han Sheep by Monoclonal Antibody

The method of immunohistochemistry assay for the detection of scrapie in Chinese Short-tailed Han sheep was established using monoclonal antibody. Genomic DNA was isolated from Chinese Short-tailed Hart sheep blood. Using the polymerase chain reaction technique, PrP27-30 gene sequence was amplified from Chinese Short-tailed Han sheep genomic DNA. By recombinant DNA technology, the recombinant protein of Chinese Short-tailed Han sheep PrP27-30 was obtained. Then, using standard methodology of myeloma cell fusion, a panel of monoclonal antibodies was generated. With mAbs, scrapie in Chinese Short-tailed Han sheep was detected by immunohistochemistry assay. The recombinant protein of Chinese Short-tailed Han sheep PrP27-30 was obtained and a panel of six hybridoma cell lines secreting specific antibodies to Chinese Short-tailed Han sheep PrP27-30 related to scrapie was obtained with one fusion between myeloma Sp2/0 and spleen ceils from mice immunized with the purified recombinant protein. Four hybridoma cell lines can be used in immunohistochemistry assay for the detection of scrapie in Chinese Short-tailed Han sheep. So that the special monoclonal antibody developed in author＇s institute can be used to detect PrP^sc of scrapie in Chinese Short-tailed Han sheep by immunohistochemistry in China.

草原短尾羊Brachyury蛋白多克隆抗体制备

【目的】克隆草原短尾羊Brachyury基因(即T基因)编码区(CDS),并制备Brachyury蛋白特异性多克隆抗体,为草原短尾羊短尾表型机制研究提供重要的试验材料。【方法】提取草原短尾羊16 d胚胎的组织RNA,反转成cDNA,以cDNA为模板,采用PCR方法克隆草原短尾羊Brachyury基因CDS全长序列。将Brachyury基因CDS全长序列连接至pEASY■-Blunt E1载体,构建原核表达载体pEASY■-Blunt E1-Brachyury,并进行NheⅠ和EcoRⅠ双酶切鉴定。将原核表达载体导入RosettagamiB(DE3)大肠杆菌,并进行IPTG诱导表达,对表达产物进行回收纯化。利用纯化后的表达产物免疫6周龄日本大耳白兔,用间接ELISA(iELISA)法测定血清多克隆抗体效价。以制备的多克隆抗体为一抗,采用Western blot法检测草原短尾羊16,20,25和30 d胚胎中Brachyury蛋白的表达水平。【结果】克隆了1335 bp的草原短尾羊Brachyury基因CDS全长序列,成功构建了原核表达载体pEASY■-Blunt E1-Brachyury,表达获得了49.16 ku的重组Brachyury蛋白。成功制备了重组Brachyury蛋白多克隆抗体,抗体效价达1∶1500,该多克隆抗体可以特异性识别草原短尾羊胚胎中的Brachyury蛋白。草原短尾羊16 d胚胎中Brachyury蛋白的表达量最高。【结论】成功制备草原短尾羊Brachyury蛋白兔血清多克隆抗体,抗体效价为1∶1500,该多克隆抗体可以特异性识别草原短尾羊胚胎中的Brachyury蛋白。

草原短尾羊TBXT基因RACE克隆及胚胎各个时期表达量分析

为鉴定草原短尾羊TBXT mRNA在胚胎发育中的表达趋势,分析其TBXT cDNA全长序列。本研究使用实时荧光定量PCR(Quantitative Real time PCR)方法对草原短尾羊14、16、20 d和25 d胚胎中TBXT mRNA相对表达量进行分析,之后使用RACE(Rapid Amplification of cDNA Ends)技术克隆TBXT cDNA全长。结果显示:TBXT mRNA在16 d胚胎中相对表达量最高,草原短尾羊TBXT cDNA全长为2426 bp(GenBank登录号:MZ146381),其中开放阅读框(ORF)为1335 bp,编码444个氨基酸,3’非编码区(3’UTR)和ployA尾长度为763 bp,5’非编码区(5’UTR)长度为328 bp;系统进化树显示草原短尾羊Brachyury与山羊进化关系最近,与羊驼关系最远,氨基酸同源性比对结果显示不同物种之间Brachyury同源性很高,表明在脊索动物进化过程中Brachyury功能相对保守。