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研究生:尤恬娜
研究生(外文):Yustina Yusuf
論文名稱:Mycbacterium abscessus 分泌性蛋白質 MPT53 之探討
論文名稱(外文):Characterization of MPT53, a secreted protein from ‎ Mycobacterium abscessus
指導教授:李晏忠
指導教授(外文):Yen-Chung Lee
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:生物農業科技學系碩士班
學門:農業科學學門
學類:農業技術學類
論文種類:學術論文
畢業學年度:100
語文別:英文
論文頁數:59
中文關鍵詞:Mycobacterium abscessusMPT53secreted protein‎
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Mycobacterium abcessus is a Non-tubercolous Mycobacteria (NTM) from rapid growing ‎group (RGM) that causes more than 80% RGM-related lung diseases in human all over the world. ‎Its wide spectrum of disease, distribution, and resistancy to antibiotic made it one of the most ‎emerging pathogens. The genome of M. abscessus encodes many suspected virulence genes and ‎secreted proteins to combust immune response. One of its most important secreted proteins is ‎MPT5 that also found in M. tuberculosis and oher Mtb complex and NTM. The first characterized ‎MPT53 is from M. tuberculosis that functions as a Disulfide Bond-forming (DsB) protein and ‎induces strong immune response in tuberculous guinea pig. Dsb maintain the correct folding of ‎many secreted protein, so it is involved in virulence indirectly. The aim of this study was to ‎characterize MPT53 of M. abscessus to reveal its biological function and role in pathogenesis. In ‎this study, the gene encoding M. abscessus MPT53 was cloned and overexpressed in E. coli for ‎further characterization. The full-length mpt53 has 534bp nucleotides, encoding 178 amino acids. ‎It contains 44 amino acids signal peptides in its N-terminal followed by ~15 kDa mature protein. ‎The full-length protein was insoluble and it was purified under denaturing condition followed by ‎re-folding. N-terminal sequencing and Western blot were used to confirm the expression of the ‎protein. MALDI-TOF analysis showed that the molecular weight of full-length MPT53 is ~20 ‎kDa and the mature MPT53 is ~15 kDa. Full length protein was processed into mature one in E. ‎coli recombinant bacteria. Site direct mutagenesis of the signal peptide showed that alanine ‎residue at position 44 was critical for the cleavage. Amino acids alignment showed that MPT53 of ‎M. abscessus has the active site motif of thioredoxin like superfamily, CxxC. Mature protein and ‎its variant C38A was able to be purified in native condition with affinity chromatography. The ‎invivo analysis by spotted titer of cadmium showed that MPT53 has oxidative relative ability. The ‎mutation on C38A residue showed decreasing oxidative ability especially in high concentration of ‎cadmium.‎
ABSTRACT i
Keywords: Mycobacterium abscessus, MPT53, secreted protein‎ ii
ACKNOWLEDGEMENT iii
TABLE OF CONTENTS iv
LIST OF TABLES viii
INTRODUCTION ‎9‎
‎1.1‎ Mycobacterium genus ‎9‎
‎1.1.2‎ Non-tuberculous Mycobacteria ‎10‎
‎1.1.3‎ Mycobacterium abscessus ‎11‎
‎1.2‎ Secreted Protein ‎11‎
‎1.2.1‎ MPT53‎ ‎12‎
‎1.3‎ Disulfide bond forming protein (Dsb)‎ ‎12‎
MATERIALS AND METHODS ‎15‎
‎2.1‎ Bacterial Strains and Plasmid ‎15‎
‎2.2‎ Growth and storage of bacterial culture ‎15‎
‎2.3‎ DNA techniques ‎15‎
‎2.3.1‎ Polymerase chain reaction (PCR)‎ ‎15‎
‎2.3.2‎ Isolation of Plasmid DNA from E. coli ‎16‎
‎2.3.3‎ Extraction of DNA from agarose gel ‎16‎
‎2.3.4‎ PCR product clean up ‎16‎
‎2.3.5‎ Agarose gel electrophoresis ‎16‎
‎2.4‎ Cloning procedures ‎17‎
‎2.4.1‎ Ligation ‎17‎
‎2.4.2‎ Competent cell preparation ‎17‎
‎2.4.3‎ Transformation ‎17‎
‎2.5‎ Protein Techniques ‎18‎
‎2.5.1 ‎ Protein expression ‎18‎
‎2.5.2‎ Purification under native condition ‎18‎
‎2.5.2‎ Purification under denatured condition followed by refolding ‎19‎
‎2.5.3‎ SDS-PAGE ‎19‎
‎2.5.4‎ Western Blot ‎20‎
‎2. 6‎ N-terminal Sequencing ‎20‎
‎2.7‎ Sample preparation for Mass analysis ‎21‎
‎2.8‎ Site direct mutagenesis ‎21‎
‎2.8.1‎ Mutant strand synthesis ‎21‎
‎2.8.2‎ DpnI digestion of the amplification products ‎22‎
‎2.8.3‎ Transformation of XL10-Gold® ultracompetent cells ‎22‎
‎2.9‎ Disulfide Bond-forming Protein analyses ‎23‎
‎2.9.1‎ In vivo analysis of oxidative protein activity ‎23‎
RESULTS ‎24‎
‎3.1‎ Full length MPT53‎ ‎24‎
‎3.1.1 ‎ Cloning of full length mpt53‎ ‎24‎
‎3.1.2 ‎ Expression, Purification, and Refolding of full-length MPT53‎ ‎24‎
‎3.1.3 Amino acids alignment of MPT53 from M. abscessus and MPT53 from M. tuberculosis.‎ ‎25‎
‎3.1.4‎ N-terminal amino acid sequencing of full-length MPT53‎ ‎25‎
‎3.1.4‎ Mass determination of the purified protein.‎ ‎26‎
‎3.1.5‎ Critical residue for cleavage on signal peptide of full-length MPT53‎ ‎26‎
‎3.2‎ Mature MPT53‎ ‎27‎
‎3.2.1‎ Cloning of mature MPT53‎ ‎27‎
‎3.2.2‎ Expression and purification of mature MPT53‎ ‎27‎
‎3.2.3‎ ‎ Site direct mutagenesis of active site on mature MPT53‎ ‎27‎
‎3.2.4‎ In-vivo analysis of oxidative activity ‎28‎
DISCUSSION ‎29‎
REFERENCES ‎32‎

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