Heterogeneous Expression And Protein Purification Of The Rabbit Defensin Gene NP-l

Defensins, a family of small peptides, which have been found in a large number of organisms, are a kind of antimicrobiol and cytotoxic peptides. They can be used as antimicrobiol drugs for a variety of microorganisms, such as, bacteria, fungi, enveloped virus (including HIV virus), and some tumour cells. It has great potentiality to produce defensins using bioreactor,Rabbit defensin NP-1, which derived from the neutrophile cells of rabbits, is one of the defensins that has the strongest antimicrobiol activity among more than 30 kinds of defensins extracted from nature. The transformation of NP-1 gene into E.coli and C.ellipsodiea were studied in this paper. NP-1 was expressed in different gene engineering host cells, and purified by affinity chromatography. In order to improve expression efficiency, activities of five different promoters were compared when we transferred the NP-1 into C.ellipsodiea, and then the strongest promoter was selected. All these studies make a well basis for further studies, such as, the medical and the antimicrobiol mechanisms studies of defensins. The detail contents and results are stated as followings:1. The NP-1 gene encoding rabbit defensin was inserted into prokaryotic expression vector. Recombination plasmids were transformed into E.coli BL21, which could express fusion protein when induced by IPTG. The defensin was purified by affinity chromatography. Tris-Tricine protein electrophoresis showed the defensin protein was existed as dimer. This showed that E.coli could express the NP-1. However, antimicrobiol assay showed that this defensin has no biological activites. It is unpractical to produce the defensin protein through prokaryotic system.2. So, we transfer the NP-1 gene to C.ellipsodiea. First, a his-tag was added to the ahead of NP-1 by PCR, in order to simplify target protein purification. Then, the His-NP-1was constructed into C.ellipsodiea expression vectors, which were transformed into C.ellipsodiea by electroporation. G418-risistance analysis, PCR analysis, southern blot analysis, protein function analysis, and protein electrophoresis indicated the stable integration and correct transcription of NP-1 in C.ellipsodiea.3. In order to find the most effective promoter in C.ellipsodiea. Five different promoters were constructed into five different C.ellipsodiea expression plasmids These plasmids were transferred into C.ellipsodiea. By comparing the different strengths of antimicrobiol function of NP-1 under the regulation of the five different promoters, the strongest promoters in C.ellipsodiea was selected.4. Target protein was purified with the help of his-tag through affinity chromatography. Make a good basis for the further study.