A monoclonal antibody raised to a Teladorsagia circumcincta 31-33 kDa doublet antigen was used to immunoscreen a T. circumcincta cDNA expression library. Sheep antibodies eluted from the proteins expressed by two clones immunopositive with the monoclonal antibody specifically recognised the doublet antigen on Western blots of third stage larval extract, confirming that these clones coded for the antigen. Database searches revealed high levels of similarity with beta-galactoside-binding lectin-like proteins (Ga1BPs or galectins) from Caenorhabditis elegans and Onchocerca volvulus. By analogy with these sequences, both T. circumcincta cDNA clones contain the full-length protein coding region. The native doublet proteins could be preferentially extracted from homogenates of third stage larvae with lactose and could be affinity purified on an asialofetuin column, confirming the identity of these bands as galectins. Reverse transcriptase-polymerase chain reaction amplification using a primer based on the C. elegans Spliced Leader SL1 sequence showed that the corresponding T. circumcincta mRNAs are also trans-spliced at their 5' ends. While there are considerable nucleotide differences between the two clones, the majority are located in the non-coding regions. Within the coding region there are 87 nucleotide differences but only three of these result in amino acid substitutions.

译文

使用针对teradorsagia cireccinta 31-33 kDa双峰抗原产生的单克隆抗体免疫筛选了T.Cireccincta cDNA表达文库。从两个克隆表达的蛋白中洗脱的绵羊抗体与单克隆抗体免疫阳性,特异性地识别了第三阶段幼虫提取物Western印迹上的doublet抗原,证实了这些克隆编码了该抗原。数据库搜索显示,秀丽隐杆线虫和盘旋虫的 β-半乳糖苷结合凝集素样蛋白 (Ga1BPs或半乳糖苷) 具有高度的相似性。与这些序列类似,两个T. circumcincta cDNA克隆均包含全长蛋白质编码区。可以优先从具有乳糖的第三阶段幼虫的匀浆中提取天然双峰蛋白,并可以在去唾液酸纤维蛋白柱上进行亲和纯化,从而确认这些条带与半乳糖蛋白的身份。使用基于秀丽隐杆线虫剪接的前导SL1序列的引物进行的逆转录酶-聚合酶链反应扩增表明,相应的T.Circucincta mrna也在其5' 末端被反式剪接。尽管两个克隆之间存在相当大的核苷酸差异,但大多数位于非编码区。在编码区内有87个核苷酸差异,但其中只有三个会导致氨基酸取代。

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