Study On The Preparation Of Human Myeloperoxidase By The Bombyx Mori Baculovirus-based Expression System

Myeloperoxidase(MPO) is a soluble enzyme contained in glycosylated hemoglobin that is presented in normal human Polymorphonuclear Neutrophils(PMN) Azurophil granules. It plays an important role in immune system by forming strong oxide and hypochlorite as a catalytic to kill aerobic microorganisms. It has become an established tool for autoimmune systemic vasculitis and inflammatory diseases diagnosis by anti-neutrophil cytoplasmic antibodies(p-ANCA). Antigens commonly used in clinical diagnosis are human MPO and proteinase 3(PR3) by detecting these antigens corresponding to antibodies derived from neutrophils. By antigen-specific diagnostic equipment, it can be easily diagnosed for those disease related to the antibodies against neutrophil cytoplasm. As the main target of p-ANCA immunofluorescence spectrum, antibodies against human MPO point out the extent of certain diseases, such as idiopathic crescentic glomerulonephritis, Churg-Strauss syndrome, and classic nodular type multiple arteritis and other related diseases.The antigen for clinical diagnostic related to immune diseases in vitro is better to be from human source. But the native source proteins are limited and appear diversity, resulting in lower purity, inconsistency in activities amaong preparations, and high price. Due to the obstacles of techniques in recombinant protein expression and the folding of the target, the price of recombinant human MPO obtained is high.In this study, a full-length cDNA of human myeloperoxidase(MPO) was cloned to the donor plasmid containing histidine. The obtained recombinant plasmid containing human MPO gene was then transformed into E. coli BmDH10 Bac competent cells. The transposition occurred through the Tn7 elements. High molecular weight Bacmid DNA was prepared from a selected white phenotype E. coli and used to transfect silkworm BmN cells for the production of recombinant baculovirus particles. The host larvae were infected with prepared recombinant virus containing human MPO gene. Different amounts of heme precursor were fed before infection. After 5 days post injection, the hemolymph of infected larvae was collected. Collected larval hemolymph was disrupted by sonicating in the presence of lysis buffer, centrifuged at high speed. The supernatant after centrifugation was then purified by Ni-NTA affinity chromatography combined with ion exchange chromatography. The analytic experiments showed that our obtained recombinant human MPO was well-expressed and assembled in larvae of Bombyx mori with proper post-translational modification. The specific activity of the purified protein was 396.7 ± 41.7 milliunits/mg. Importantly, it could be recognized by the sera of patients.The specific activity of recombinant human MPO by Bombyx mori baculovirus expression system was equivalent to the expression of MPO in human neutrophils. The expression level is high in this system. Aaveragely, as much as 1.7 micrograms of target protein could be obtained from per larva. This study provides a simple, fast, reliable and economic way for the large-scale production of recombinant human MPO by use of Bombyx mori as a bioreactor.