Cloning, Sequence Analysis And Prokaryotic Expression Of Lepus Capensis Zona Pellucida3

Hares (Lepus capensis) mainly distributed in the many areas of the north of the YangtzeRiver in China, and used to live in the forests of the hill, or nearby the farmland. The greenparts of the plants are their mainly food such as tender seedlings, stems and leaves and duringwinter and early spring they eat basal barks of the roots of the trees and bite the young trees.Since they reproduce fast, distribute widely, and have a big and wide appetite, hares havebring serious damage to China’s forestry and become one of the major disaster animal threatenforestry development. Although traditional hare control methods play a certain role incontrolling population of hares or reducing tree damage, they still constrained by the poorspecificity, rapid recovery of population, and environmental pollution problems.Immuno-contraception technology which means taking the key factor in the reproductiveprocess as target antigens to induce specific antibodies to block the key factors’ function hasbecome the focus of international research to control the pest. Several studies have shown thatZP3as the primary sperm receptor plays an important role in the process of fertilization, andis a potential candidate for the development of the contraceptive vaccine. In this study, hareZP3was selected as the research object. Through molecular biology techniques the codingsequence of ZP3was cloned and ZP3protein was expressed in E. coli. This study provides atheoretical basis for future immune-contraceptive study on hare control.Based on preliminary experiment, specific primers were designed according to the ZP3mRNA sequence of European rabbit (Oryctolagus cuniculus) in GenBank. Using total RNAisolated from hare(Lepus capensis) ovaries, the cDNA encoding hare ZP3was amplified byRT-PCR for the first time（designated as hZP3）and was submitted to GenBank (Accessionnumber：KC447289). The hZP3cDNA contains an open reading frame of1260nucleotidesencoding a polypeptide of419amino acid residues. Nucleotide identity between Lepuscapensis and Oryctolagus cuniculus was96%. The molecular weight was predicted to be45KDa with an isoelectric point at5.85. The predicted ZP3amino acids had the typicalcharacteristics of ZP family include a signal peptide at1–22aa(amino acid), a conserveddomain designated the ‘ZP domain’(45–305aa), A tetra basic consensus furin cleavage site(344–347aa)which present upstream of a hydrophobic transmembrane-like domain (382–405aa), and8conserved cysteine residues in ZP domain. Phylogenetic tree analysis suggested that Lepus capensis had the closest phylogenetic relationship with the Oryctolagus cuniculus,followed by rodents, in line with the laws of the evolution of species. Second structureprediction revealed the presence of51.31%random coils,25.30%Extended strand,18.62%alpha helices,4.77%Beta turn. Three-dimensional structure of ZP3was predicted byhomology modeling methods.According to hZP3cDNA sequence, specific primers with restriction sites were designedto amplify hZP3cDNA excluding the N-terminal signal sequence. Restriction enzymedigestion and sequencing verified that prokaryotic expression vector pET-28a-hZP3wassuccessfully constructed by homologous recombination technique. Prokaryotic expressionvector was transformed into E. coli BL21(DE). Expression of protein was then induced byaddition of IPTG. SDS-PAGE indicated the presence of protein bands at approximately53kDa which is slightly larger than expected. The possible reason was analysed and furthertesting and optimization method was proposed.