RECOMBINANT EXPRESSION AND ANTIMICROBIAL ACTIVITY ANALYSIS OF TRANSFERRIN IN ASIAN YELLOW POND TURTLE

Transferrin has a variety of biological functions such as iron transportation, anti-microbial, immunoregulation, etc. In this paper, the expression pattern and the antibacterial activity of purified recombinant protein of Mauremys mutica transferrin (MaTf) were analyzed. The ORF of MaTf were obtained by RT-PCR, then ligated into pET-32 a (+) vector to construct recombinant vector for prokaryotic expression, then transformed into E. coli BL21 competent cells. The putative protein was successfully expressed with the induction of IPTG (0. 8 mmol/L) at 30℃ for 4 hours. The solubility of the expressed products was tested by PAGE electrophoresis of supernatant and precipitation separately resulted from smashed bacterial. The results indicated that the protein was in inclusion body and insoluable. The precipitation of smashed bacterial including the recombinant protein was washed with 1×binding buffer including 2M urea, resolved in 1×binding buffer including 6 M urea at 37℃ for 1h, purified with His bind column and then renatured by dialysis. The purity was tested by PAGE electrophoresis and western blot, which confirmed the high purity of the recombinant protein of MaTf obtained. The biological activity of anti-microbial was tested in vitro by assessing the effect on bacterial growth repression. According to the diameter of repressed bacteria circle on plate for equal dose of purified protein, the antibacterial effects to three kind of bacteria tested are different, which are Staphylococcus aureus Escherichia coli Serratia marcescens. The expression pattern in different tissues in M. mutica were quantified by western blot, which showed that the pattern at protein level is similar to the mRNA level, which is: liver spleen kidney heart. This research provides an important information for exploring the biological function of transferrin in non-specific immune response.