Screening Endotoxin-binding Peptides By Phage Display Technology To Establishing A New Method For Detecting Endotoxin

Endotoxin is a Gram-negative bacterial cell wall of an integral part,also known as lipopolysaccharide.Endotoxin into the blood,there will be fever,lower blood pressure,disseminated intravascular coagulation,endotoxin sepsis and a series of clinical response,it will lead to shock,or even death.Therefore,some food,medicine,medical equipment in the factory should be carried out endotoxin testing to avoid the harm caused by endotoxin.Chinese Pharmacopoeia included bacterial endotoxin test method includes two methods:gel method and photometry,the use of limulus reagent to qualitative or quantitative detect endotoxin.LAL is extracted from the blood of horseshoe crab,scarcity of raw materials,expensive,unstable.In this paper,phage clones were obtained from phage display technol ogy by three rounds of dodecapeptide library,then the phage ssDNA was extracted and the sequence was analyzed by DNASTAR and BLAST software.Finally,the dodecapeptide sequences were synthesized by solid phase synthesis method.Using molecular interaction technology to identification the binding ability of dodecapeptides to endotoxin and establish enhanced turbidimetric method to detect endotoxin.The phage display peptide library was co-incubated with the plate coated with the target molecule.After two rounds of nonspecific elution and amplification and one round specific elution,finally the phage clones were screened successfully.The phage ssDNA was extracted and sequenced.The phage ssDNA was extracted and sequenced.The sequence of the peptide was QVTPQVPRSTQM and QVNGLGERSQQM.The purity of the synthesized peptide was 95% by solid-phase synthesis.Compatibility analysis of synthetic dodecapeptides using molecular interaction techniques.The dissociation equilibrium constant(KD)were 3.52 × 10-9 and 8.012 × 10-9 and Full^R2 were 0.9979 and 0.9969,indicating that the synthesized dodecapeptide has good affinity with endotoxin.The endotoxin-enhanced turbidimetric method was used to establish endotoxin-enhanced turbidimetric method.The linear range of endotoxin was 0.03 ~ 0.48 EU / mL,R2 = 0.9925.The method can be simple and accurate detection endotoxin and lay the foundation for rapid detection of endotoxin.