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The Study Of Expression And Activity Characterization Of Fungal Immunomodulatory Protein FromFlammulina Velutipes (FIP-fve)

Posted on:2014-01-01Degree:DoctorType:Dissertation
Country:ChinaCandidate:X H KongFull Text:PDF
GTID:1260330401479631Subject:Biochemistry and Molecular Biology
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Immunoglobulin (Immunoglobin, Ig) is an important index to represent the humoral immune function with immunomodulatory function in vivo. Some products have been used in the treatment of immune-related diseases. Ig is composed of2heavy chains (H chains) and2light chains (L chains), which form Y-type structure. The variable area at the top of Ig is the main active region. Fungal immunomodulatory protein (FIP) is a small molecule protein isolated from the fungus and its structure and function are similar to Ig. At present,7kinds of FIPs have been identified and the FIP from Flammulina velutipes (FIP-fve) possesses immunomodulatory effects, including activating T lymphocytes to produce cytokines, preventing or treating asthma, rhinitis, eczema, allergies and inhibiting the proliferation of cancer cells.To investigate the immunomodulatory function of FIP-fve protein, first we expressed and purified the FIP-fve protein. The FIP-fve cDNA was obtained by RT-PCR method. The cloning vector pUC19-FIP-fve and expression vector pET30a (+)-FIP-fve were constructed. pET30a (+)-FIP-fve was transformed into Escherichia coli Transetta (DE3) by thermal conversion method after identification by enzyme digestion and sequencing, and recombinant protein was further expressed. Under the optimum conditions, the recombinant protein was induced by0.2mM IPTG for24h at28℃and the rate of expression was more than30%. The cell lysate method was optimized using ultrasonic method (200W,5S,30min) combined with lysozyme method, the cell lysis rate reached above98%. The protein purification process was established in large scale single protein by nickel ion affinity resin (affinity chromatography) and the His-FIP-fve yield reached29.1mg/L. In the mean time, its purity was more than97%. Additonally, the process of lyophilization of His-FIP-fve was established and the product was found that possessed stable morphology and good solubility, and it could be used as injection of freeze-dried powder. The secondary structure of His-FIP-fve was measured by circular dichroism spectra (CD) and the results showed that His-FIP-fve has correct folding and could be effective expressed in E. coli Transetta (DE3), in which it was formed with low content of a-helix (15%) and high levels of beta (38%) of secondary structure, and has a small amount of P-angle structure. It’s sturcture was similar to that of natural FIP-fve. The results substantiated that the correct structure of the recombinant protein is a prerequisite for biological activity on the secondary structure. The biological activity of the recombinant protein His-FIP-fve in vivo was tested by ELISA on three kinds of cytokines (IL-2, IL-4and IFN-γ) in the serum of mice. The results showed that the levels of IL-2and IFN-γ in mice serum were the highest in the lowest dose group (5mg/kg), and there existed significant difference (p<0.01) compared with the control group; however, the difference of IL-4level in mouse serum was not significant (p>0.05) between three dose groups and the control group. The results indicated that the recombinant His-FIP-fve can promote the production of IL-2and IFN-y in mice. At the same time, His-FIP-fve can induce the expression of IFN-y and IL-2. In addition, compared to FIP-fve expressed in other prokaryotic expression vector, the expression vector pGEX4t-1-FIP-fve was constructed and was applied in this study, and the expressed product was purified by Gst affinity chromatography and was cleaved by thrombin to remove Gst label and to obtain the pure FIP-fve protein. Yet the expression rate was low and the Gst-FIP-fve product was only5.8mg/L, and the process was so complex, the further study on the activity of expression product should be done.The studies on FIP-fve were mainly focused on the structure, function and immunomodulatory mechanism, while the research on its characterization and regulation mechanism of expression had not been revealed. Based on the theoretical research for improvement of varieties and its characterization of expression,8varieties of F. velutipes were cultivated to obtain the samples of fruiting bodies and were fermented in liquid medium to obtain the samples of mycelium. The internal control gene was selected. The expressive characterization was done by fluorescence quantitative PCR (Real-time PCR) among the different varieties of F. Velutipes. Using different part of the fruiting body, such as mushroom cap, middle stipe, basal stipe in the same varieties of F. velutipes, and mycelium (liquid fermentation), the difference of gene transcription was performed and it was found that the transcription level of FIP-fve was seen through the test. Results:The transcription level of FIP-fve gene was different in different breeds, the level of FIP-fve in white varieties was slightly higher than in the yellow variety; The expression level of FIP-fve in middle stipe was slightly higher than in the basal stipe and the cap of the mushroom; The expression level of FIP-fve in young buds of the mushroom was lower than that in the mature fruiting body. At the same time, His-FIP-fve polyclonal antibody was prepared by using purified His-FIP-fve as antigen to immunize New Zealand white rabbits, which has good specificity of rabbit anti His-FIP-fve polyclonal antibody with high titer of1:128000. The immunoblotting (Western-Blot) of FIP-fve in different breeds, different developmental stages of fruiting body, different parts of fruiting body and the mycelium were tested to investigate the expression characterization of FIP-fve by His-FIP-fve polyclonal antibody. Results:the expression of FIP-fve gene in white varieties was slightly higher than in the Yellow varieties; The expression level of FIP-fve in middle stipe was slightly higher than that of basal stipe; The FIP-fve gene was rarely expressed in mycelium; The expression level of FIP-fve was lower in young bud than in mature fruiting body.In the present study, the optimization of the expression and purification of recombinant FIP-fve was performed. The process of efficient expression and large-scale production of His-FIP-fve were obtained. And the activity experiment in mice showed that its biological activity was similar to natural protein. The study laid the foundation for the further study of fungal immunomodulatory protein and promoted the development of new products in allergy, antitumor and medical care and other aspects of the recombinant protein.
Keywords/Search Tags:FIP-fve, Recombinant protein, Activity characterzation, Enzyme-linkedimmuno sorbent assay, Expressed characterization
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