Gene Cloning And Prokaryotic Expression Of The Serine Protease Inhibitor RCSPI2 From The Skin Of Chinese Brown Frogs, Rana Chensinensis

Amphibian skin is a great potential treasure of biological resources. Over the past several decades, numerous neuromedins and antimicrobial peptides have been isolated from amphibian skin and its secretions. Comparatively, a few serine protease inhibitors have been identified from amphibian skin. These protease inhibitors are high performance implement on the research of protein metabolism relation to biology and medicine. Therefore, it has aroused wide interest on the medical profession.Based on the EST database of Rana chensinensis skin constructed by our laboratory, a cDNA fragment encoding serine protease inhibitor was obtained. The specific primers were designed according to the obtained cDNA sequence, and a full-length cDNA sequence encoding serine protease inhibitor including 3′UTR and 5′UTR was amplified from the total RNA of Rana chensinensis skin obtained from Xin Bin, Liaoning province by RT-PCR and 5′-RACE. The nucleotide sequences of cDNA encoding 200 amino acid composed of 876bp with 55bp 5′-UTR and 221bp 3′-UTR which is named RCSPI2. RCSPI2 belong to Kunitz family member containing five disulfide bonds. The cDNA sequence has been submitted to GenBank (Accession Number: GQ303267).The target sequence was inserted into the pMD19-T Simple cloning vector by T/A cloning. After identification by sequencing, the double digested DNA was cloned into prokaryotic expression vector pET32a and the recombinant plasmid containing RCSPI2 gene was transferred into E. coli BL21 (DE3) for expression under induction of isopropyl-N-D- thiogalactoside (IPTG) at final concentration of 1mM. The expressed recombinant protein with relative molecular weight of 36kDa was an expected size of protein existed in the form of inclusion bodies.The recombinant proteins were purified from the extract of transformed BL21(DE3) with affinity chromatography(Ni²⁺-NTA) according to the manufacturer's instructions. The purified His6-tagged protein isolated from the transformed BL21(DE3) cells was analyzed by 12% SDS–PAGE. After refolded by dialysis, the recombinant RCSPI2 showed a trypsin inhibitor activity. It is indicated that the prokaryotic expression vector for serine protease inhibitor RCSPI2 gene from the skin of Rana chensinensis was successfully constructed, and the recombinant serine protease inhibitor RCSPI2 was prepared. Two New Zealand rabbits were immunized with recombinant protein of RCSPI2. Indirect ELISA was used to detect the titer antibodies and recombinant protein of RCSPI2 shows strong immunogenicity. Using the obtained antiserum as the first antibody, the natural serine protease inhibitor was detected in Rana chensinensis skin protein crude extract administration by western blotting, the results also showed the recombinant protein of RCSPI2. This proved that the prepared antibody had a good antigen-specific.