Molecular Cloning, Expression And Antimicrobial Resistance Of Transferrin From Asian Yellow Pond Turtle

We identified one cDNA encoding a Transferrin from the cDNA library (designated as MaTf). The MaTf cDNA was 2437 bp long and consisted of a 5'- UTR (untranslated region) of 47 bp, a 3'-UTR of 212 bp , and an open reading frame (ORF) of 2121 bp encoding a polypeptide of 706 amino acids. Forecasting molecular characteristics of the MaTf with some analysis software, it showed that N-lobe of MaTf was a signal peptide of 19 amino acids. The gene was composed of two similar domains, and each domain consisted of two subunits, which interacted to form a deep, hydrophilic iron-binding sites. The genomic DNA of MaTf has 17 exons and 16 introns, similar to that of known genes of other vertebrate animal species. It means structure conservation of MaTf. With by homology analysis, it showed that MaTf shared the highest homology 75%-97% with transferrin genes of birds and reptiles, and shared 65%-75% homology with mammalian, and shared some homology with Xenopus laevis and other amphibians. Quantitative PCR results showed that the expression of MaTf in the liver, spleen, kidney and heart tissues were increased after Serratia marcescens infection in different time, and the characteristics is similar with other animals which were infected by a pathogen.The specific primers are designed according to the full-length sequence of MaTf'cDNA, and the MaTf'Mature peptide sequence is obtained from the liver of the yellow pond turtle by using RT-PCR technology.Then it is inserted into PMD18-T and we obtain recombinant plasmid. The correct recombinant plasmid which is detected by enzyme digestion, PCR and sequencing is inserted into the prokaryotic vector pET-32a fused with the down stream 6-His·Tag. The expression vector is transformed into the E. coli BL21 (DE3) strain. after induction of IPTG,the MaTf fusion protein was successfully expressed.The purified fusion protein does antimicrobial agarose diffusion test for viscous marcescens, Staphylococcus aureus and Escherichia coli respectively. The results show that the antibacterial activity of recombinant transferrin is different to different strains, especially the antibacterial activity in the inhibition of Staphylococcus aureus is significant.The antibacterial activity is more significant in Gram-positive bacteria than the Gram-negative bacteria which reflects that the ransferrin of yellow pond turtle t is similar to other specific.The polyclonal antibodies is prepared using fusion protein as antigen.Sequencing analysis showed that, MaTf mature peptide sequence is inserted into the expression vector of pET-32a successfully. After Induced by IPTG, the fusion protein is expressed with a relative molecular mass of about 97 kD by SDS-PAGE electrophoresis and Western blot analysis which is consistent with the target protein in the size, and has a specific reaction with the monoclonal antibody with the His tag.It is purified by His Bind column and shows a single band by SDS-PAGE electrophoresis analysis.Polyclonal antibodies could react with the total protein which is extracted from the yellow pond turtle'tissue, and obtains a single band by Western blot experiments analysis. Experimental results show MaTf protein is extracted successfuliy, and polyclonal antibody shows the high price, high specificity, high affinity anti MaTf.This study is helpful for the further research of the Immunoassay of endogenous MaTf method, radioimmunoassay, enzyme-linked immunosorbent assay.These results provided important information for further exploring the roles of Asian yellow pond turtle MaTf in no-specific immunity.