碩士 / 東吳大學 / 微生物學系 / 98 / A specific primer was designed to perform 3’- and 5’-RACE PCR (rapid amplification of cDNA ends by PCR), which was used to clone the full length (780 bp) of cDNA suspected to be an anti-lipopolysaccharide factor (ALF). The polyadenylation signal ranged from nucleotides 752 to 762. For translation of the fragment, the 5’UTR consisted of nucleotides 1 to 482 and the 3’ UTR contained nucleotides 483 to 780. The open reading frame (ORF) included nucleotides 102 to 482, yielding 124 amino acids. The putative signal peptide consists of amino acids 1 to 25 (Met1 �� Ala25). The mature peptide is composed of 99 amino acids and is a cation, with a molecular charge of 2.84 at pH 7. Its putative molecular weight is 11.5 kDa and has a predicted pI of 9.07. The putative lipopolysaccharide (LPS) binding domain is composed of 22 amino acids (Cys55 �� Cys76), which is a highly conserved region. The N-terminal also has a highly conserved, hydrophobic region made up of 5 amino acids: WPCGW (W75 �� W79). Structural analysis projected that this specific ALF consists of 3 α-helices and 4 β-sheets, and the LPS binding domain is a β-hairpin.
To produce the ALF, an E. coli HMS174 (DE3) prokaryotic protein expression system was used and the induction results indicated that after 0.5 mM IPTG induction, OD600 decreased with time and then it rised after 4 hours induction. In addition, Western blotting result was unable to detect the ALF. However, using the yeast Pichia pastoris SMD1168 eukaryotic protein expression system, the results indicated that, after 0.5 % methanol induction, it was can produce the recombinant ALF from extracellular detected by Westen blotting.
The recombinant ALF (a His-tag fusion protein) from extracellular solution was purifed by Ni-NTA agarose, it was can product 145 mg/L ALF after purification. The purified recombinat ALF showed antibacterial activity against the prawn Gram-negative pathogen Aeromonas hydrophila (MIC=8 μM, MBC=16 μM), but not the Gram-positive pathogen Lactococcus garvieae; in addition, the recombinant ALF also has a result of bactericidal effect and inhibited the growth of E.coli HMS174 (DE3) (MIC = 16 μM，MBC = 128 μM).
| Identifer | oai:union.ndltd.org:TW/098SCU05381003 |
| Date | January 2010 |
| Creators | Chien-Pang Chung, 鍾建邦 |
| Contributors | Hung-Hung Sung, Chia-Chen Liu, 宋宏紅, 劉佳振 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 103 |
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