Yamasaki N, Tsubamoto H, Hasegawa A, Inoue M, Koyama K
Department of Obstetrics and Gynecology, Hyogo College of Medicine, Japan.
J Reprod Fertil Suppl. 1996;50:19-23.
The pig zona pellucida (pZP) is composed of glycoproteins pZP1, pZP3 alpha and pZP3 beta. Previously, we showed that pZP1 was proteolytically cleaved into two components (pZP4 and pZP2), and suggested that pZP1 was a homologue of mouse ZP2. We now report the genomic organization of pZP1 gene, and expression of the recombinant protein in mammalian cells. Two genomic clones, pZP4g and pZP4.1, were isolated by screening the pig genomic library with an already cloned cDNA encoding pZP4 as a probe. These clones covered 25 kb of the genome, including the entire pZP1 locus. The pig ZP1 genome consists of 18 exons and 17 introns. For production of the recombinant proteins of pZP1, a minigene containing a genomic fragment covering exon 6 to exon 17 was constructed and cloned into the expression vector, pBCMGS-neo. Mammalian cell lines, CHO (for stable expression) and 293T (for transient expression), transfected with the plasmid construct synthesized pZP1 recombinant proteins, probably as a membrane-bound protein.
猪透明带（pZP）由糖蛋白pZP1、pZP3α和pZP3β组成。此前，我们发现pZP1被蛋白水解切割成两个组分（pZP4和pZP2），并提出pZP1是小鼠ZP2的同源物。我们现在报告pZP1基因的基因组结构，以及重组蛋白在哺乳动物细胞中的表达。通过用已克隆的编码pZP4的cDNA作为探针筛选猪基因组文库，分离出两个基因组克隆pZP4g和pZP4.1。这些克隆覆盖了25kb的基因组，包括整个pZP1基因座。猪ZP1基因组由18个外显子和17个内含子组成。为了生产pZP1的重组蛋白，构建了一个包含覆盖外显子6至外显子17的基因组片段的小基因，并将其克隆到表达载体pBCMGS-neo中。用该质粒构建体转染的哺乳动物细胞系CHO（用于稳定表达）和293T（用于瞬时表达）合成了pZP1重组蛋白，可能是一种膜结合蛋白。
