Redmond D L, Knox D P, Newlands G, Smith W D
Moredun Research Institute, Edinburgh, UK.
Mol Biochem Parasitol. 1997 Mar;85(1):77-87. doi: 10.1016/s0166-6851(96)02812-5.
Antisera from lambs immunised with the Haemonchus contortus integral membrane protein complex, Haemonchus galactose-containing glycoprotein (H-gal-GP), the lambs being refractory to subsequent challenge, were used to identify several clones from an adult H. contortus lambda gt11 cDNA library. Using gene-specific oligonucleotide primers in conjunction with primers directed to a conserved nematode Spliced Leader (SL) sequence and to the polyA+ tail of mRNA, the remaining 5' and 3' sequences of one of these clones, metallopeptidase-1 (MEP1), were amplified. The 2.4 kb full-length coding sequences was subsequently amplified in a single reaction. Sequence analysis identified MEP1 as encoding a putative zinc metallopeptidase, which shared limited homology with the mammalian type II integral membrane protein neutral endopeptidase (NEP). Southern blotting indicated that MEP1 belonged to a multigene family. MEP1 was expressed in bacteria as a glutathione-S-transferase (GST) fusion protein, and a specific antiserum raised in sheep. This antiserum recognised several polypeptide components of H-gal-GP. Immunolocalisation studies showed that MEP1 encoded a protein located on the luminal surface of the nematode gut. Both MEP1 mRNA and protein are developmentally regulated with expression being limited to the blood-feeding stages of H. contortus.
用捻转血矛线虫完整膜蛋白复合物、含半乳糖的捻转血矛线虫糖蛋白（H-gal-GP）免疫的羔羊血清（这些羔羊对后续攻击具有抗性），用于从成年捻转血矛线虫λgt11 cDNA文库中鉴定几个克隆。使用基因特异性寡核苷酸引物，结合针对保守线虫剪接前导序列（SL）和mRNA的聚腺苷酸尾巴的引物，扩增了其中一个克隆金属肽酶-1（MEP1）的其余5'和3'序列。随后在单一反应中扩增出2.4 kb的全长编码序列。序列分析确定MEP1编码一种假定的锌金属肽酶，它与哺乳动物II型膜蛋白中性内肽酶（NEP）具有有限的同源性。Southern印迹表明MEP1属于一个多基因家族。MEP1在细菌中作为谷胱甘肽-S-转移酶（GST）融合蛋白表达，并在绵羊中产生特异性抗血清。该抗血清识别H-gal-GP的几个多肽成分。免疫定位研究表明MEP1编码一种位于线虫肠道腔表面的蛋白质。MEP1 mRNA和蛋白质在发育过程中受到调控，表达仅限于捻转血矛线虫的吸血阶段。
