Analysis Of Lepus Capensis Zona Pellucida Protein 2 And 3 Expression

As a wild rabbit, Lepus Capensis cause amount of economic loss in agriculture and forestry. Applicable reproductive antigen will be benefit to develop immunocontraceptives for wild rabbit’s population control. Mammalian zona pellucida(ZP) has good antigenicity, and the antibody of ZP can effectively hinder sperm-egg interaction and trigger immune infertility. In order to understand more about an antigenicity of Lepus Capensis ZP2 and map its epitope, we designed a special reverse transcription primer, which 5’ ends was phosphorylated, basing ZP2 cDNA sequence of Oryctolagus cuniculus. The first strand of cDNA was synthesized and was acted as template to amplify the open reading frame of Lepus Capensis ZP2. The sequence of ZP2 cDNA contained 2,184 nucleotides, and its homology was identified. Its signal peptide and transmembrane helices was respectively located at ZP235-56 and ZP2698-717. The hZP2 cDNAs was amplified and contained 954 nucleotides. 1,716 bp ZP2 f cDNA, excluding the signal peptide and transmembrane helix domain, was inserted into the pET-28a(+) vector to construct a recombinant vector pET-28a-ZP2 f. After inserting the hZP2 c DNA into pET-28a(+) plasmids, the recombinant pET-28a-ZP2 f or pET-28a-hZP2 vector was then transformed into E.coli BL21 competent cells. Temperature, IPTG concentration and time was optimized to increase the level of expression of ZP2 f or hZP2 gene, and the optimum expression of ZP2 f growed at 28℃ and was induced 8 h using 1.2 mM IPTG; the optimum expression of ZP2 f growed at 37℃ and was induced 15 h using 0.7 mM IPTG. The recombinant E.coli BL21 was induced and expressed dissoluble hZP2 or hZP2 f usion proteins, which molecular weight were 68.46 kDa or 41.17 kDa. The level of expression of hZP2 fusion proteins was much more than expression of ZP2 f fusion proteins. 2,194 bp ZP2 c DNA and the pEGFP-N1 vector were used to construct a recombinant vector pEGFP-N1-ZP2. After transfecting into a rabbit kidney cell line(RK 13), the recombinant RK 13 cells was then cultured in geneticin(G418) 14 days. Screening a stably recombinant fluorescent RK13 cells, its genomic DNA was extracted and was confirmed the presence of ZP2 using polymerase chain reaction(PCR). It could successfully transcribe ZP2 gene, which was confirmed by reverse transcription-polymerase chain reaction(RT-PCR). An inducible GFP-fused ZP2 was expressed in the extraction of fluorescent RK 13 cells. Because large numbers of membrane proteins expression could hinder cell growth and cause the ZP2 gene deletion in some recombinants. Hence, its fluorescence became weaker.A recombinant SFV encoding hare ZP3 was engineered by homologous recombination between SFV and the pSC11 insertion vector inserted the ZP3 cDNA in RK13 cells. The recombinant SFV was enriched basing the β-galactosidase screening system. Genom or total RNA of the recombinant SFV acted as template, and a deduced band was observed after both PCR reactions. SDS-PAGE and western blot analysis also have a band similar to the size of ZP3, and ZP3 was found in cell extracts. At the same time, the recombinant SFV viral titer is about 4.2×107 pfu/mL on day 4. All these findings indicate the presence and expression of the hare ZP3 gene.