Fusion Expression,Condition Optimization And Antimicrobial Activity Analysis Of CAP18

Such argue on the remnant of antibiotic and antibiotic-resistant strains were increasinglyvigorous with the wide and misused application of traditional antibiotics. The great concern todevelop effective but human, animals and environmental compatible antibiotic alternatives is one ofthe focus researches. Antimicrobial peptide, one of the most important constituent components ofhost denfence system, is a kind of small peptides produced by inducing in organism. It posssesesantimicrobial activities against a wide variety of microorganisms including G^-,G⁺,fungi,protozoon,enveloped virus and cancer cell etc, and exhibits unique mechanism agaist bacteria.These properties make it as great potential and can be applied in many fields as one of alternativeantibiotics. CAP18 is a novel 18 kDa cationic protein originally purified from rabbit granulocytes.The C-terminal 37 amino acids of CAP18 (CAP18_106-142) make up the lipopolysaccharide-bindingdomain. CAP18_106-142 has broad antimicrobial activity against both gram-positive andgram-negative bacteria and has unique LPS-binding activity. Also, CAP18_106-142 still remains itsantimicrobial activity in some extreme condition. So CAP18_106-142 may have therapeutic potentialfor sepsis and andotoxin shock and may as a new therapeutic drug treat infectious disease causedby bacteria especially aiming at those antibiotic-resistant strains. Otherwise, CAP18_106-142 may havea therapy role as an adjunctive therapy in combination with conventional antibiotics in the treamentof infectious disease caused by gram-negative bacteria. But according to our knowledge, there is noreport on expression of CAP18_106-142 in any type of expression system. The functional CAP18_106-142was successfully expressed in E.coli by gene engineering method in this experiment, which hasinsignificance in the development of basis of theory and practice to expressing CAP18_106-142 andother small peptides, and also in the exploration of universal method and technology of geneengineering of peptides and antibacterial peptides research.The following results were obtained in this study:1. According to the biased codon used in E. coli, the gene sequence encoding CAP18 wasdesigned. The gene fragment of CAP18 was first obtained through gene SOEing;2. In order to identify the DNA, CAP18 gene fragment was inserted into the pMD18-T vector,and the DNA sequence confirmed by sequencing was identical to the purpose. Then the DNAsequence was inserted into the pET-32a vector, constructed the fusion expression vectors of p-cap;3. The construct p-cap was used to transform into E. coli BL21 (DE3) as expression host. After IPTG induction, the results of SDS-PAGE and Westem-bloting analysis showed that the CAP18fusion protein was successfully expressed;4. The optimized induction condition was established that IPTG were added to the finalconcentration of 0.6mmol/L when the bacterial cells density reached OD=0.8, then continuouslyculture the cells for 4h. The expression level of soluble CAP18 fusion protein exceeded 40% of thetotal soluble proteins under the induction of the optimized condition;5. A new efficient media (Complex auto-inducing media-4, CAI-4) was obtained byoptimizing the key component of complex auto-inducing media. The expression level of solubleCAP18 fusion protein was 40 times higher in CAI-4 than in LB, and 16 times higher than in LBwhose induction condition had been optimized6. CAP18 fusion protein was higher than 85% purity by chromatography purification asjudged SDS-PAGE, but the production is not ideal and only 200mg fusion protein was obtained perliter culture of induced bacteria;7. The CAP18 fusion protein was partially cleaved by recombinant enterokinase. Therecombinant CAP18 has inhibition activity to the growth of E.coli DH5αestimated by liquidgrowth inhibition assay, which suggested the functional CAP18 has been achieved and first realizedthe heterologous expression of LfcinB by gene engineering method.