| In this study, we used existing epidermal growth factor receptor pathway substrate 15(Eps15), in gonad tissue from Hyriopsis schlegelii, transcriptome data from our laboratory, Hs-Eps15 gene’s specific primers were designed, and the PCR amplification were taken by template of gonadal tissue cDNA in H.schlegeli. The PCR products were cloned into pMD19-T vector, then turned into E.coli DH5, parts of middle segment was obtained after sequenced. Finally the cDNA sequence of Hs-Eps15 genes was obtained by PCR and 3’-RACE.The full-length cDNA sequence is3382 bp, including 923 bp 3’-UTR with a 24 bp poly(A) tail and a tailing signal(AATAA);the open reading frame(ORF) is 2454 bp, encoding 817 amino acids. The Hs-Eps15 protein domain analysis showed that N-terminal part is mainly composed of three EH(Eps15 homology) domains, no signal peptide, and the most remarkable characteristic of C-terminal is the existence of repeat sequence,multiple DPF(Glu, Pro, Phe);using relevant biological software analysis we obtained that the molecular weight is about 88.67 KDa,the theoretical isoelectric point is4.88, in secondary structure prediction, random coil and α-helical are the most, accounted for 41.74% and 41.49%, extension chain accounted for9.55,β-sheets are the least.The established phylogenetic tree analysis connected with MEGA4.0 software by NJ method showed that in many species, H.schlegeli Eps15 gene and the Crassostrea gigas Eps15 gene are the highest homology(about 64%).The Hs-Eps15 mRNA expression in different tissues in H.schlegeli were analyzed by Q-PCR technology.The results showed that the Hs-Eps15 gene in 11 tissues(heart, testis, ovary, intestine, liver, kidney, gill,adductor muscle,mantle, axe foot and blood) of H.schlegeli had obvious tissue specificity.It is highly expressed in testis, ovary is the second, but the expression levels in heart,gill and blood are relatively low,and heart is the lowest. It revealed that the EGFR pathway put Hs-Eps15 as the substrate can affect sperm production and development, it could be H.schlegelii ovarian local important regulatory factor, influencing the follicle growth and maturity.According to the full-length cDNA sequence of Hs-Eps15 gene, we obtained 3EH(Eps15 homology) from N-terminal sequence after blasted by NCBI, so we selected these complete and representative domains and designed a pair of primers to amplify the complete domain, pEASYTM-E1-Hs-Eps15 prokaryotic expression systems was constructed.Then the correct recombinant expression plasmid by sequencing were transformed into into E.coil BL21(DE3), and the protein were successfully expressed after induced by IPTG, so we obtained a molecular weight of approximately 32.6kDa fusion protein. With Ni-NTA resin, the expression products were purified by affinity chromatography and SDS-PAGE detection showed that the purified fusion protein was obtained, protein concentration was measured by Bradford content determination method and the results showed that protein concentration meet the requirements of preparation of polyclonal antibody. The purified Hs-Eps15 fusion protein was used as antigen to immunize Japanese long ear rabbit. The titer of the antiserum was determined by ELISA indirect method and the test result was that the titer was up to standard in 1: 512000.The specific identification of polyclonal antiserum was detected by Western blotting and recognized the purification of Hs-Eps15 fusion protein. Finally the frozen section of male,female gonad and liver in H.schlegelii were prepared, the expression of Hs-Eps15 protein in cells was located with rabbit antiserum prepared by immunofluorescence localization.We found that Hs-Eps15 protein is distributed mainly in the head of sperm, the nucleus membrane egg cell, but mainly distributed in the liver‘s nucleus membrane and cytoplasm. This is important to study the function of the Hs-Eps15 from the protein levels and the other related proteins’ foundations in this signal pathways. |
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