Vol 48(2014) N 5 p. 646-654; DOI 10.1134/S0026893314050148
D.-D. Sui, J.-L. Wu, H. Zhang, H. Li, Z.-M. Zhou, D.-H. Zhang, C.-X. Han*
Molecular Cloning, Structural Analysis, and Expression of Zona Pellucida Glycoprotein ZP3 Gene from Chinese Zokor, Myospalax fontanieriiCollege of Forestry, Northwest A&F University, Yangling, Shaanxi 7122100, China
Received - 2014-01-17; Accepted - 2014-04-01
The zona pellucida 3 (ZP3) plays a crucial role in reproductive immunology. We obtained a fulllength cDNA encoding Chinese zokor ZP3, using rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The cDNA contains an open reading frame of 1269 nucleotides encoding a polypeptide of 422 amino acid residues. The amino acid sequence has a high degree of homology with those of hamster (78%), mouse (76%), and rat (74%). XhoI and SacI sites after restriction give an1158 bp fragment of zokor ZP3 cDNA, excluding the signal sequence and transmembrane-like domain, which was cloned under the phage T7 promoter-lac operator control in the pET-28a(+) vector. Recombinant pET-zokorZP3(r-ZP3) was expressed as a poly-histidine fusion protein in E. coli strain BL21 (DE3). Optimum expression of r-ZP3 was observed at 28оC, 1 mM IPTG and 2 h of inducing. The purified protein was tested by Western blot.
Chinese zokor, expression, recombinant ZP3, structural analysis, zona pellucida