| Fugal Immunomodulatory Protein (FIP), isolated from higher Basidiomycetes, is a kind of small molecule protein with immune regulating activity. Until now, seven FIPs have been found and identified from Ganoderma lucidum (LZ-8 or FIP-glu), G. tsugae (FIP-gts), Flammulina velutipes (FIP-fve), Volvariella volvacea (FIP-vvo), G. japoncium (FIP-gja), G. microsporum (FIP-gmi), and G. sinensis (FIP-gsi). The function of FIPs has similarity with lectin. They could haemagglutinate red blood cells. While the structures and functions of FIPs have great similarity with immunoglobulin super family. They could promote proliferation of lymphocytes, and induce the expression of cytokines.In this study, the FIP-glu, FIP-gsi and FIP-fve gene were cloned from mycelia of G. lucidum, G. sinensis and F. velutipes respectively by homologous cloning, and then subcloned into the expression cassette vector pQE-30a. After induced expression in Escherichia coli M15, recombinant FIP-glu, FIP-gsi and FIP-fve were purified by Ni-NTA column respectively. On the one hand, the purified proteins were going to use for preparation of polyclonal antibodies, and on the other hand they were use in transcriptional expression of cytokine genes. The analysis of SDS-PAGE showed that the recombinant proteins were expressed in E. coli successfully. The analysis of BandScan V 5.0 showed that the yield of recombinant FIP-glu accounted for 51.1% of the total protein of E. coli, which soluble recombinant FIP-glu accounted for 55.4% of the total soluble protein of E. coli, and the FIP-glu reached a purity of 90% after purification; the yield of recombinant FIP-gsi accounted for 41.7% of the total protein of E. coli, which soluble recombinant FIP-gsi accounted for 46.1% of the total soluble protein of E. coli, and the FIP-gsi reached a purity of 90% after purification; the yield of recombinant FIP-fve accounted for 36.7% of the total protein of E. coli, which soluble recombinant FIP-fve accounted for 5.4% of the total soluble protein of E. coli, and the FIP-fve reached a purity of 90% after purification. MALDI-MS demonstrated that the proteins purification form the E. coli induced by IPTG were the mature FIP-glu, FIP-gsi and FIP-fve. Immunorecognition of recombinant proteins was demonstrated by Western Blot. The New Zealand white rabbits were immunized with the purity protein to prepare antibody, and got specificity anti-FIPs polyclonal antibody. The antibodies efficient values were 1:625,000 detected by indirect ELISA, and special bands were seen examined by Western Blot. Analysis of RT-PCR demonstrated that both of the recombinant FIP-glu and FIP-gsi protein could induce interleukin (IL)-2, IL-3, IL-4, interferon (IFN)-γ, tumour necrosis factor (TNF)-α, and IL-2 receptor (IL-2R) genes transcriptional expression; and the recombinant FIP-fve protein could enhance transcriptional expression of IL-2, IL-4, IFN-γ, TNF-α, lymphotoxin (LT), and IL-2R.In this thesis, two groups FIP genes (FIP-glu & FIP-gsi, and FIP-glu & FIP-fve & FIP-vvo) were shuffled through DNA shuffling. The results showed that two new genes from the two groups respectively were recombinant, and contained the fragments of the members of the two groups respectively.Using genomic walking technology, about 1 kb promoter of FIP-fve was cloned from genomic DNA of F. velutipes. There were not any same or similar sequences with the promoter's in NCBI database. Thus, the sequence of the promoter was a new. Bioinformatics analysis of sequence showed that there were seven TATA-boxes, seven CAAT-boxes, one G-box, one ABRE, three CGTCA-motifs, and six Skn-1 motifs. The transcriptional starting site was predicted in the 100 bp of upstream of ATG. In order to verify its function, the promoter was cloned into plant expression vector pCAMBIA 1304, and transferred into Nicotiana tabacum by agrobacterium-mediated transformation. After analysis of GUS staining and fluorescence, the GUS-GFP fusion protein was expressed in leaf epidermal hair and root by the promoter.
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