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URN etd-0906110-144855 Statistics This thesis had been viewed 2381 times. Download 773 times. Author Yen-Chieh KO Author's Email Address No Public. Department Bioengineering Year 2009 Semester 2 Degree Master Type of Document Master's Thesis Language zh-TW.Big5 Chinese Page Count 96 Title Production of recombinant LL-37 and preparing SAW-MIP Biosensor for detection of LL-37 Keyword LiTaO3 Protein Immunosensor surface acoustic wave molecularly imprinted polymer LL-37 cathelicidin Antimicrobial peptides Antimicrobial peptides cathelicidin LL-37 molecularly imprinted polymer surface acoustic wave Protein Immunosensor LiTaO3 Abstract It’s well known that bacteria have ability to develop antibiotic-resistance in short
time after treatment. To find therapeutic agents with different mode of action than
conventional antibiotics is a necessity. To meet the above requirement, antimicrobial
peptides (AMPs) represent a good alternative for infective antibiotics. AMP LL-37 with positive charges belongs to an immune system that has a capacity to distinguish
membrane composed by different lipids in human. This unique property enables LL-37 to destroy the negative charged bacteria membranes. In consequence the above mechanism is effective to bacteria those have developed resistance to antibiotics. Neither bacteria nor LL-37 exists in healthy individuals, except suffering urinary tract infection (UTI).
The DNA sequence encoding full-length LL-37 was chemically synthesized and cloned into the pET-32a(+) vector for protein expression in BL21(DE3) Escherichia coli. The expressed LL-37-containing fragment was purified by metal affinity chromatography. LL-37 peptide was released by chemical cleavage in 50% formic acid at 50 ℃ for 32 h. and separated by reverse-phase HPLC.
We applied the Surface Acoustic Wave–Molecularly Imprinted Polymers principle - a molecular templating polymer aggregation on the Surface Acoustic Wave, to form imprints of the cathelicidin LL-37. LL-37 was polymerized together with functional monomer Methacrylic acid ( MAA )/Poly (ethylene glycol) dimethacrylate（PEG400DMA, Mn=550） (5:95 by volume), in 10 minutes at ambient temperature. The polymerization was caused by bonding protein and polymer together by MAA. The template with identifiable hole of protein was formed. Using this characteristics to screen the structure of LL-37, and detect the frequency variations of surface acoustic wave by network analyzer. We polymerized the concentration 0.02 mg/ml, 0.08 mg/ml, and 0.14 mg/ml of LL-37 on the SAW–MAP sensor area. Removed the target molecule after washing the sensor by NaOH and then measured the frequency as start frequency. The star frequency of concentration 0.02 mg/ml is 106,437,500 Hz, then the frequency decrease to 106,421,875 Hz after rebinding the LL-37. The star frequency of concentration 0.08 mg/ml is 105,125,000 Hz, then the frequency decrease to 105,093,750 Hz after rebinding the LL-37. The star frequency of concentration 0.14 mg/ml is 106,250,000 Hz, then the frequency decrease to 106,202,750 Hz after rebinding the LL-37. So we can ascertain that the SAW–MIP binding higher concentration protein then the frequency would decrease more. Besides, the interference HSA didn’t influence the change of SAW–MIP frequency.
Advisor Committee Chung-Yih wang - advisor
Hsin-Chun Lu - co-chair
I-Ching Kuan - co-chair
Wen-Ching Shih - co-chair
Files Date of Defense 2010-07-30 Date of Submission 2010-09-08