Schwoebel E, Prasad S, Timmons T M, Cook R, Kimura H, Niu E M, Cheung P, Skinner S, Avery S E, Wilkins B
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
J Biol Chem. 1991 Apr 15;266(11):7214-9.
A full-length cDNA (rc55) encoding the major rabbit zona pellucida (ZP) glycoprotein (55 kDa) has been cloned and sequenced. A lambda gt11 expression library was constructed using poly(A)+ mRNA isolated from sexually immature rabbit ovaries which contain large numbers of developing follicles. The rc55 cDNA was identified using affinity purified polyclonal antibodies specific to ZP antigens which are shared among mammalian species. The deduced amino acid sequence of the full-length rc55 clone was matched to the NH2-terminal 25-amino acid sequence obtained for this protein. The predicted amino acid sequence consists of 540 amino acids including a putative signal peptide of 18-24 residues and six potential N-glycosylation sites. The cDNA hybridizes to a 2000-base species of mRNA from rabbit ovary which is not detected in other rabbit tissues. The message is present early in ovarian follicular development and is approximately 600-fold greater in sexually immature as compared with sexually mature rabbit ovaries. This cDNA was expressed as a cro-beta-galactosidase fusion protein using the pEX expression vector. Antibodies against native rabbit ZP, affinity-purified on the recombinant 55-kDa ZP protein, were found to recognize the native rabbit ZP glycoprotein, indicating partial conservation of native epitopes in the expressed recombinant protein.
已克隆并测序了编码主要兔透明带（ZP）糖蛋白（55 kDa）的全长cDNA（rc55）。使用从含有大量发育卵泡的性未成熟兔卵巢中分离的聚腺苷酸加尾（poly(A)+）mRNA构建了λgt11表达文库。利用对哺乳动物物种共有的ZP抗原具有特异性的亲和纯化多克隆抗体鉴定出rc55 cDNA。将全长rc55克隆的推导氨基酸序列与该蛋白获得的氨基末端25个氨基酸序列进行匹配。预测的氨基酸序列由540个氨基酸组成，包括一个推定的18 - 24个残基的信号肽和六个潜在的N - 糖基化位点。该cDNA与兔卵巢中一种2000个碱基的mRNA杂交，在其他兔组织中未检测到这种mRNA。该信息在卵巢卵泡发育早期出现，与性成熟兔卵巢相比，在性未成熟兔卵巢中的含量大约高600倍。使用pEX表达载体将该cDNA表达为一种cro-β-半乳糖苷酶融合蛋白。在重组55 kDa ZP蛋白上亲和纯化的针对天然兔ZP的抗体被发现能够识别天然兔ZP糖蛋白，表明在表达的重组蛋白中天然表位部分保守。
