Study On The Preparation Of Recombinant Human PR3 By The Bombyx Mori Baculovirus-Based Expression System

Proteinase 3(PR3)is a neutral serine protease that is mainly located in azurophilic granules of neutrophils and specific granules as well as secretory vesicles or transferred to the plasma membrane.PR3 is a multifunctional protein involved in a variety of physiological activities,which can degrade extracellular matrix proteins,kill microbes,affect the activation of platelets and endothelial cells,regulate the biological activities of various cytokines and cell surface receptors,and participate in the immune regulation of inflammation sites.In recent years,a large number of studies have shown that PR3 is closely related to the development of various diseases such as granulomatosis with polyangiitis(GPA)and chronic obstructive pulmonary diseases(COPD).Anti-neutrophil cytoplasmic antibodies(ANCA)are specific autoantibodies that are associated with a variety of primary vasculitis and the titer of ANCA is consistent with disease activity.As one of the main target antigens of ANCA,PR3 and its autoantibodies(ANCA)have important significance in clinical diagnosis,guiding treatment and pathological mechanism research.Natural human PR3 protein is the best choice for clinical diagnosis of related diseases.However,due to limited natural PR3 resources available and complicated separation and purification process,its cost is prohibitive,which seriously hinders the application of in vitro diagnostic techniques for PR3 related diseases.Therefore the production of human PR3 protein by recombinant technology is a better chioce.At present,although human PR3 has been successfully expressed in various cell lines such as Escherichia coli,yeast and insect cells Sf-9,there are still many defects,such as complicated operation,low yield,poor antigenicity of recombinant protein,and high cost.This study attempts to express recombinant human PR3 in silkworm larvae by the silkworm-baculovirus expression system.The strategy was designed for recombinant human PR3 protein with a His-tag of 6 histidines at the C-terminus and an insect secretion signal peptide Mel at the N-terminus.The Mel-PR3-His fragment generated by PCR amplification was inserted into the donor plasmid pFastBac1 to prepare a recombinant donor plasmid pFastBac1-Mel-PR3-His.This recombinant donor plasmid was then transformed into E.coli BmDH10 Bac competent cells,and translocated with the BmNPV baculovirus shuttle vector Bacmid through the Tn7 transposable elements.The generated recombinant Bacmid DNA was isolated from selected white phenotype clones and transfected into bombyx mori BmN cells to yield recombinant baculovirus particles.The host insect,silkworm larvae at the five-instar stage,was infected with the recombinant virus by injection and the larval hemolymph was collected,in which the expressed recombinant human PR3 was released into the hemolymph with the help of insect secretion signal peptide.The collected hemolymph samples were subjected to the purification step by a nickel-nitrilotriacetic acid(Ni-NTA)-agarose column and further purified by two ion exchange columns.The highly purified recombinant human PR3 proteins were then validated by Western blotting and mass spectrometry analysis.Our strategy using BmNPV-based expression system to produce recombinant human PR3 in silkworm larvae not only has low cost,but also has high expression level,and is convenient for its large-scale production.It opens up a feasible new way for efficient production of recombinant human PR3 protein,which provides an important basis for the next exploration in the pathological research and diagnosis of PR3 related diseases.