Preparation and identification of recombinant human neutrophil elastase / 中华男科学杂志

ABSTRACT

<p><b>OBJECTIVE</b>To prepare a purified recombinant human neutrophil elastase (HNE) using genetic engineering technology, and pave the way for the preparation of the antibody to HNE and establishment of semen HNE detection methods.</p><p><b>METHODS</b>HNE mRNA was obtained from human peripheral blood granulocytes with specific HNE primers, and the cDNA of HNE was cloned into the plasmid pGEX-2T to derive a recombinant plasmid pGEX-2T/HNE. After PCR identification, double-enzyme digestion and gene sequencing, the recombinant plasmid was transferred into competent Escherichia coli DH5alpha and further induced to express the recombinant fusion protein GST/HNE by isopropyl beta-D-thiosulfate galactosidase (IPTG). The recombinant fusion protein was cleaved by thrombin and further purified with glutathione agarose beads to obtain purified recombinant HNE.</p><p><b>RESULTS</b>The recombinant plasmid pGEX-2T/HNE was successfully prepared and transferred into E. coli DH5o; the expression of the recombinant fusion protein GST/ HNE was successfully induced by IPTG at 18 degrees C overnight; and the purified recombinant protein HNE was successfully obtained by thrombin cleavage and purification of glutathione agarose beads.</p><p><b>CONCLUSION</b>The acquirement of purified recombinant HNE has prepared the ground for the preparation of the antibody to HNE and establishment of semen HNE detection methods.</p>