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Recombinant Human Lysozyme Gene LYC4: Expression In Picha Pastoris And Its Antibacterial Activity

Posted on:2014-09-21Degree:MasterType:Thesis
Country:ChinaCandidate:X X LiFull Text:PDF
GTID:2180330464964263Subject:Biological engineering
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Human lysozyme is a kind of sterilization protein which is completely different from antibiotics on sterilization mechanism. Its antibacterial mechanism is that it hydrolyses the β-(1,4)-glycosidic bond in the alternating N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) of the peptidoglycan, than occured bacteriolysis by causing the rupture of bacteria cell walls under the effect of osmotic pressure. Our lab had cloned an immunoreactive protein gene taking the LYZ gene as the probes. This gene is named LYC4. The LYC4 gene encodes 127 amino acids and the 18 amino acids of its N-side is a strong signal peptide sequence. Homologous comparison shows that the homology between LYC4 and human lysozyme LYZ is 38% and the two-dimensional structure are extremely similar. The formation of length and the predicted molecular weight, along with the eight cysteine conservative structure fits the characteristics of C lysozyme. It belongs to the gene families of C type lysozyme.In the early research, we found LYC4 was expressed specifically in human testis by northern blotting analysis in16 kinds of tissues of the human body. In stiu hybridization and immunohistochemistry experiments showed that the expression abundance of LYC4 protein gradually weakened from caput to cauda in epididymis epithelium. A bound at 14kDa was detected in human seminal plasma and also was found in the protein extraction from mouse caput, corpus and cauda epididymis respectively by western blot analysis. And by fused with his-tag, recombinant LYC4 was expressed and purified.With previous study we express LYC4 protein in E.coli, both the expression quantity and activity of target protein are not high. On this basis, LYC4 protein was expressed using pichia system in this experiment. Pichia expression system with the characteristics of transformants genetic stability, accurate processing, efficient expression, and it was able to achieve stability to extend under the unselection pressure. It also can be directly express lysozyme with biological activity. Therefore, LYC4 gene was optimized according to the yeast codon preference. The code sequences optimized LYC4 gene was cloned under the control of the alcohol oxidase 1 promoter (AOX1) in the vector pPIC9K and integrated into the genome of the methylotrophic yeast Pichia pastoris (P. pastoris) strain GS115 by electroporation. The screening of multiple copies of transformants was operated on YPD medium adding different concentration of G418 geneticin. We could get the multiple copies after screening. After methanol inducing by the flasks, the supernatant was analyzed by SDS-PAGE electrophoresis. We could see the protein bands on 14.4 kDa. With human lysozyme (164071U/mg) as the standard and the red micrococcus as the substrate, we detected the activity of the human lysozyme LYC4. The recombinant protein of LYC4showed bactericidal activity to micrococcus and its activity was up to 38893U/ml.
Keywords/Search Tags:Human lysozyme, Pichia pastoris, Bactericidal activity
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