| As alternative antibiotic, antibacterial peptides has been shown attractive potential application in medicine. Silkworm antibacterial peptide Cecropin B belongs to traditional cationic antibacterial peptides, with a propensity to form a -helices. The sterilizing mechanism of the Cecropin B is that the peptide can permeate the lipid bilayer membranes of most Gram-positive and gram-negative bacteria to cause cell death.Thanatin is a bactericidal and fungicidal at physiological concentration, exhibiting the broadest antibacterial spectrum and strongest activity so far observed among insect antibacterial peptides. The structure of thanatin has a two stranded beta -sheet stabilized by one disulfide bridge. The C-terminal residues, Met21, Arg20, were involved in the hydrogen-bonding network. The possible mechanism of thanatin is to depress the respiration of the bacteria.In this study, health silkworm pupae at the third day old were induced with LPS. After incubated two hours at 27℃, total mRNA was extracted and the silkworm structure Cecropin B gene was cloned with RT-PCR method and checked by nucleotide sequencing. Thanatin gene was PCR amplified and cloned with three contiguous primers. GST fusion expression of the antibacterial peptides Cecropin B and Thanatin were carried out. SDS-PAGE showed that the expression level was approximately 22%. The result of agarose diffusion assay indicated that the fusion protein exhibited partly antibacterial activity.An in vivo antibacterial activity monitoring system was established in the sense that E.coli is intrinsically sensitive to the attack of the antibacterial peptides. The degree of growth inhibition of transformant cells was found to depend on expression of GST-fusion protein. With the 0.5mmol/L IPTG, OD610=0.1-0.15 transformant cell concentration and JM109 transformant cell, the inhibition of the transformant can be used as an index of antibacterial activity of peptides.The results of activity analysis of truncated form of Thanatm showed that the N-terminal region was rather benign compared to those in the C-terminal region. Mutant variants Thanatin' and Thanatin" were synthesized. Thanatin' is a mutant that a fragment, Pro-Gly-Pro-Arg-Ser-Tyr, inserted behind the fourth amino acid (Lys4) of native Thanatin. Thanatin" is the V6G/I8R/I9S mutant of native Thanatin.The antibacterial activity of mutant Thanatin' and Thanatin" were analyzed with the in vivo monitoring assay system. In Thanatin", the polar amino acid substitution will change the hydrophobic core of the molecule, vanishing the hydrophobic cluster defined by the side-chains of Pro? and Ile9 of native Thanatin, in which the Arg20 side-chain embedded. The mutation will surface the Arg20 positive side-chain and consequently give rise to free rotation of the main-chain. From the experimental evidence that the activity remained in this variant, suggesting the hydrophobic cluster in the C-terminal was not indispensable for the anti-Gram-negative activity of Thanatin. In Thanatin", because of the insert fragment, the two stranded P -sheet were shorted in simulated molecular, resulting in a decreasing in antibacterial activity revealed by in vivo monitoring system.Different antibacterial mechanism of Cecropin B and Thanatin led to the difference in the growth pattern of transformants in the in vivo monitoring system. Growth curve of transformants carrying pGEX 4T-Cec B showed slow increasing. However the pattern of pGEX 4T-Thanatin showed increasing in the first 2-3 hours after inducing, after then, a slow decreasing in bacterial density.10 kinds of hybrid antibacterial peptides were designed on the basis of Cecropin B and Thanatin with the help of computer analysis. The N-terminal of the designed peptides contained a amphiphilic a -helix and the C-terminal contains the P -sheet sequence of Thanatin.4 high activity hybrid antibacterial peptides, CB-Th c, CB-Th e, CB-th b and CB-Th g were selected with the in vivo monitoring system. The hybrid peptide CB-Th e was selected for further study. Fu...
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