[Study on the expression, purification and the anti-endotoxin activity of human endotoxin binding peptide and its mutant].
OBJECTIVE
To express endotoxin binding peptide and its mutant in E coli DH5alpha and detect the antiendotoxin activity of the purified peptides.


METHODS
(1 ) E coli DH5at containing pinpointXa3-EBP and pinpointXa3-mEBP was activated by IPTG to express biotin fusion protein. The fusion proteins were purified, and then digested by factor Xa for isolation of EBP and mEBP. The target peptide was purified with affinity chromatography and reversed-phase HPLC. Sequences of 10 amino acids at N-terminal were used for identification of mEBP. (2) PBMCs were isolated from blood of normal people, and they were stimulated with 5 mg/L FITC-LPS plus 2.0,5. 0 and 12. 5 mg/L EBP or mEBP. Then the mean fluorescent intensity was detected. PBMC was also stimulated with 1 mg/L LPS plus 2.0, 5.0 and 12.5 mg/L EBP or mEBP for 5 hours for the detection of the TNF-alpha and IL-6 level in the supernatant. (3) Thirty-five Kunming mice were randomized into normal control ( n = 5, with intraperitoneal injection of 0. 2 ml isotonic saline) , model group(n = 5, with intraperitoneal injection of LPS and 20% TBSA full-thickness burns), and treatment group (n = 15, with intraperitoneal injection of 5 mg/kg PMB or 10 mg/kg EBP or mEBP after burns). The serum contents of TNF-a and IL-6, and TNF-alpha mRNA level in hepatic tissue in each group were determined 6 hours after treatment.


RESULTS
( 1 ) EBP and mEBP were obtained after Xa digestion of biotin fusion protein, with purity reaching above 98% . The sequence of 10 amino acid at N-terminal was in accord with what expected. (2) The fluorescent intensity was decreased followed by an increase in mEBP or EBP concentration. Compared with normal PMBC, IL-6 and TNF-alpha level in the supernatant were obviously lowered in 1 mg/L LPS + 12.5 mg/L EBP group and I mg/L LPS +2. O0 , 5. 0, 12. 5 mg/L mEBP groups ( P < 0.01). (3) The serum level of IL-6 and TNF-ca in the therapeutic groups were obviously lower than that in model group ( P < 0.01 ) , and the levels of these cytokines were significantly lower in 10 mg/kg mEBP group than that in 10 mg/kg EBP group ( P <0. 01) , but they were similar to that in 5 mg/kg PMB treatment group ( P >0.05). (4) Relative optical density of TNF-alpha. mRNA in control, model, 10 mg/kg mEBP, 10 mg/kg EBP and 5 mg/kg PMB groups was 0.25, 0.93, 0.51 , 0.77 and 0.43, respectively.


CONCLUSIONS
Endotoxin binding peptides can be obtained by procaryon expression. Both EBP and mEBP have anti-LPS activity, but mEBP is more effective.
Sun
Ya-li
YL
Institute of Pathology Research, Southwest Hospital, Third Military Medical University, Chongqing 400038, PR China.
Liu
You-sheng
YS
Yang
Hai-jie
HJ
Wang
Chang-song
CS
Ma
Jian-zhou
JZ
D000818:Animals; D004731:Endotoxins / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008815:Mice, Inbred Strains; D050505:Mutant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides / Q000302:isolation & purification / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D004731:Endotoxins; D015850:Interleukin-6; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D050505:Mutant Proteins; D010455:Peptides; D014409:Tumor Necrosis Factor-alpha; C025357:endotoxin binding proteins