Study On The Breeding Of Innate Anti-Tachyplesin Ⅰ And The Induction Of Manual Anti-Tachyplesin Ⅰ In Feeding Bacillus

Tachyplesin I is a 17-amino-acid residues cationic antimicrobial peptite extracts from the blood of horseshoe crab, exhibiting broad-spectrum activities against bacteria, spirochetes、 fungus、mould、virus、protozoa, inhibition of tumor cell proliferation and induce cancer cells differentiation which applied high value.However, the strong inhibition to the host bacteria from tachyplesin I makes it difficult to high express through genetic engineering,which mostly restricts the research and applying of tachyplesin I.This experiment selects anti-tachyplesin I feeding baccilus strain by innate breeding and manually successive induction,expecting to obtain the host for genetic engineering of tachyplesin I. The following four research will be presented in this study, including study on the inhibiton to some common feeding baccilus from tachyplesin I;the study on anti-tachyplesin I mutationally microwave induced screening of bacillus subtilis BS168; antimicrobial induced screening of anti-tachyplesin I bacillus subtilis; the relative analysis on anti-tachyplesin I induced bacteria.We use the broth microdilution method to determinate the minimum inhibitory concentration(MIC) and minimum bactericidal concentration(MBC) of the fllowing feeding bacillus:Bacillus subtilis 168、Bacillus subtilis WB800、Bacillus cireldans CICC22015、 Bacillus lentus CICC10365、Bacillus firmus CICC23258、Bacillus natto CICC20443、 Bacillus pumilus CICC9003、Bacillus coagulans AS 1.2009、Bacillus megaterium AS 1.223、Bacillus licheniformis Chester AS 1.269 and Bacillus cereus.Frankland AS1.196 from tachyplesin I. The results showed their MIC were 20、20、20、2.5、40、 40、20、20、20、20、20ug/ml and MBC are 80、80、80、20、160、160、80、80、 80、80、80ug/ml repectively.The own advantages and low sensitivity to tachyplesin I of Bacillus subtilis make us to choose it to finish the following trials.In order to get Bacillus subtilis which resistant to tachyplesin I,we used the traditional microwawe induced methed and innovatively successive anti-tachyplesin I induced methed on Bacillus subtilis16S;the result indicated that the strain acquired from microwawe induced methed could grow up on the medium contained lethal concentration of tachyplesin I,but the subsequent study showed that the sensitivity of the accquired strain was lower than the original one,so it could not be used as host of tachyplesin I. Othervise,the strain we get use the another methed that could continuous grow up in the medium with the tachyplesin I concentration of 150ug/ml.By using drag susceptibility test in vitroand resistant stability test of high resistant strain,the result demonstrate that the strain has the risistance to tachyplesin I.On this basis, the antibacterial kinetics and cell membrane permeability of the original strain Bacillus subtilis168 and its induced strain were investigated in the study,meanwhile, Transmission and scanning electron microscopy were used to observe morphologic and ultrastructural changes among original strain and induced strain and their strains with 40ug/ml tachyplesin I at 37℃ for 2h.By comparing the difference between the original strain Bacillus subtilis168 and its induced strain,the results show that the antibacterial kinetics and cell membrane permeability and morphologic were difference among them.In order to study the reasons which caused the differences between the original strain and the induced one,we study the molecular mechanisms of the bacteria resistant to cationic antimicrobial peptide tachyplesin I according to the prior experience.we also compare the differences of acitivity and spectrum of the extracellular protease between the original strain and the induced one;analysising the extracellular proteomics between the original strain and the induced one by Two-dimensional electrophoresis technology;meanwhile we analysis the difference of the number of the negative charge on the cell wall and membrane by alcian blue (AB) staining method. The results suggest the acitivity and spectrum of the extracellular protease between the original strain and the induced one were different,and it is also the same as the number of the negative charge on the cell wall and membrane.It is just these differences that cause the resistance of the induced Bacillus subtilis168.