Studies On Expression And Biological Activity Of Antibacterial Peptides Riched In Different Cation Amino Acids

Bacterial resistance to existing drugs is a constantly growing problem that, combinedwith a decline in the development of new antibiotics, presents a significant threat to animaland human health. The identification of new antimicrobial agents is therefore ofconsiderable importance.In recent years, antibacterial and antifungal peptides have gained a lot of interest, dueto their potential use as a new generation of therapeutic agents. But It is not actual for thepeptides be used clinically because many native antibacterial peptides were separated fromorganisms or synthesized artificially, which have low activity to pathogens and toxic toeukaryotic cells. In this syudy, the author expressed six peptides(Sheep MyeloidAntibacterial Peptide SMAP-29, Metalnikowin -ⅡA, Thanatin, Moronecidin, Protegrin-1and 4 kD Scorpion Defensin) in series and investigated the biological activity. The contentsand results are as followes:1 In order to obtain a higher activity and lower toxicity antibacterial peptide, thesequences of six antimicrobial peptides - Protegrin-1, 4 kD Scorpion Defensin,MetalnikowinⅡA, SMAP-29, Thanatin and Moronecidin- were exploited to generate asynthetic antimicrobial peptide cp gene, which was then cloned into the yeast expressionvector pPICZa-A. The constructed recombinant expression vector pPICZa-cp wastransformed into P.pastoris X-33, in which the CP peptide was expressed and secreted intothe medium and the contents of CP in the medium was 271μg/mL. In vitro activityexperiments showed that the recombinant antimicrobial peptide CP could keep antibacterialactivity after being boiled at least 30 minutes and had higher acid stability.The MIC of CPto S.aureus is 1.624μg/mL. The hemolytic rate of the CP was 3.47％at the concentrationof 135.5μg/mL.2 In order to compare the activity and toxicity of combinant peptide CP, theMetalnikowinⅡA(MET) and Scorpion Defensin(SD) were expressed in P. pastoris respectively. According to the sequences from GenBank AAB27599 and AAB27538, usingthe preferential condon of P. pastoris, the Met and Sd gene were obtained by PCR andTD-PCR respectively. The two modified antibacterial peptides' genes were cloned into theyeast expression vector pPICZa-A to construct the recombinant expression vectorpPICZa-Met and pPICZa-Sd and then transformed into yeast host strain X-33, respectively.The results showed that the two peptides were expressed in yeast and the contents of METand SD in the medium were 223μg/mL and 131μg/mL, respectively. The combinantantibacterial peptide CP's antibacterial activity was 3～7 times higher than that of MET andSD. The heat stablity of CP was similar to that of MET, They could tolerate to be boiled atleast 30 minutes. The acid and alkaline stability of CP and MET were similar too. Thehaemolysis of CP was between that of MET and SD.3 In order to understand the synergic interaction between antibacterial peptide andantibiotics, nine clinically used antibiotics (ampicillin, streptomycin, kanamycin, neomgcin,tetracycline, polymyxin B, norfloxacin, cephamycin and ciprofloxacin) were adopted. Invitro antibacterial activities of three antibacterial peptides, alone or in combination with theantibiotics were measured by MIC. Synergy was observed when the peptides werecombined with ampicillin, streptomycin, kanamycin, neom gcin, tetracycline, polymyxin B,norfloxacin, cephamycin and ciprofloxacin by FIC. The antibacterial peptides andantibiotics had different activity to different bacteria. The three antibacterial peptides hadsynergic or enhancement relationship with kanamycin, streptomycin, tetracycline andpolymyxin B, and indifferent or antagonistic relationship with other antibiotics. The resultsindicate that the interaction in antibacterial peptides, antibiotics and bacteria isextraordinary complex, except synergism, there are still indifference and antagonism.4 To study the effects of combinent antibacterial peptide CP on the growthperformance and immunity function of the mice, 180 ICR mice with similar weight bodywere separated into six groups(low, middle and high dosage of peptide CP, blank control,blank vector control and ciprofloxacin group) at random. All mice were fed under the sameconditions for 35 days and were granted different drugs by oral administration every day.Some indices such as body weight, feed intake, spleen index et al were detected and thedata were analysed using the analysis software (SPSS13.0). The average daily weight gainof every group was not significant difference (P＞0.05), and the feed/weight-gain ratio ofthe low and middle dosage groups mice were significantly lower than that of the controlgroup mice(P＜0.01). The T lymphocytes transformation rate of all dosage of CP groups's mice increased remarkably than that of blank control group (P＜0.01), and the cytokine ofIFN-γand IL-2 in the routine serum of low and middle dosage groups increased than that ofthe control group. All dosage of CP groups protected partially mice from lethal E.coli K99challenges, and the protection rate of the low dosage group was higher than that of themiddle and high dosage groups. The results indicate that combinant antibacterial peptideCP has a promoting effects on the growth and immune functions of mice, and also providesan experimental basis for the clinical application.5 To study the effect of antibacterial peptide CP on the entero-microflora andabsorbability of small intestinal tract in mice, 180 ICR mice with similar weight body wereseparated into six groups at random. All mice were fed under the same conditions for 35days. The villus length, crypt depth and villus/crypt (V/C) value of duodenum and jejunumwere determined every week, respectively, and the entero-bacteria of Rectal contents wereseparated on the 35th day. The quantities of entero-bacteria of the experimental groupswere lower than that of control group significantly, and the quantity of entero-bacteria andthe dosage of CP showed inverse correlation. The villus length,crypt depth and V/C valueof duodenum and jejunum of the antibacterial peptide CP groups' mice were similar tothose of the control group mice(P＞0.05) on the zero day. On the 35th day, they werehigher than those of the control group's mice remarkly (P＜0.01). The results show that theantibacterial peptide CP can effect the entero-bacteria double facially. It can improve thesmall intestinal absorbability and enhance feed conversion ratio at proper concentration.The results of this study provide valuable support for further development ofantibacterial peptide.