Cloning of a New Sperm Membrane Protein sp18 Gene and Its Expression in E. coli
Bo Jialin1 Gou Kemian1,2 An Xiaorong1,2 Li Ruiguo1 Hou Jian1,2 Chen Yongfu1,2**
(1. State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, China; 2.College of Biological Science, China Agricultural University， Beijing 100094, China)
Abstract Abstract: cDNA encoding for a sperm membrane antigen, designated sperm membrane protein sp18, was cloned and sequenced from murine testis λgt11-cDNA expression library using sp18 mAb. Computr-generated translation analysis of 1 468 bp cDNA yielded an open reading frame(ORF) of 429 bp which deduced 142 amino acids with the first ATG(Met) start codon at nucleotide 694 bp, the stop codon TAA at nucleotide 1 122 bp and a small polyadenylation tail at nucleotide from 1 245 to 1 250 bp. The translated protein, which consists of 142 amino acids, has a calculated molecular mass of 15.7 kD, 8 potential N-linked glycosylation sites and 12 O-linked glycosylation sites. The hydropathy plot generated from deduced amino acids sequence indicated it to be a sperm span-membrane peptide. Comparing the nucleotides of sp18 cDNA with other nucleotides deposited in GenBank , the 10 to 651 bp of sp18 cDNA and 532 to 1 173 bp of human Hr44, the 10 to 816 bp and 864 to 1 444 bp of sp18 cDNA and 1 408 to 2 208 bp and 2 250 to 2 309 bp of bovine Hr44 had 98% similarity respectively. This indicated that sp18 cDNA was a new gene(GenBank accession number:AF129872) specifically localized in reproductive system.The sp18 cDNA was subcloned into pET-30a(+) vector and expressed in BL-21(DE3) of E.coli with molecular mass of 16.5 kD. Western blot indicated that sp18 mAb could recognize sp18 protein and protein possesse immunogenicity. All above laid a foundation of further investigating the function of sp18 gene.
