Peptide combined with endotoxin and application thereof in endotoxin detection

Abstract

The invention discloses a peptide combined with endotoxin and application of the peptide in endotoxin detection, the peptide at least comprises the following sequence fragments: GCKK YRRF RWKF KGKF WFWG C, and the carboxyl terminal and / or nitrogen terminal of the peptide contains cysteine. The peptide provided by the invention can be used for quantitatively detecting the endotoxin, is low in detection limit, accurate in detection result, high in sensitivity and wide in detection linear range, covers the maximum value of the content of the endotoxin specified in Chinese pharmacopoeia, can be directly used for detecting the endotoxin in water for injection and injection, and does not need to be diluted; the method is high in precision, high in repeatability and short in detection time, and the single detection time of a single sample does not exceed 10 minutes; compared with a limulus reagent, the peptide is convenient to produce and low in cost, is not limited by limulus resources, the quality is stable, and the qualities of different batches have comparability; a prepared biosensor electrode can be repeatedly used for multiple times within a certain time, the result error is small, and the cost can be further reduced.

Description

technical field [0001] The invention relates to the technical field of endotoxin detection. More specifically, the present invention relates to a peptide that binds to endotoxin and its use in endotoxin detection. Background technique [0002] Endotoxin is lipopolysaccharide derived from Gram-negative bacteria and is an important component of the cell wall of Gram-negative bacteria. [0003] Because Gram-negative bacteria are widely distributed in the environment, although endotoxin is only released into the environment when the bacteria die and dissolve or the bacteria are destroyed, due to its relatively stable chemical properties, it can still maintain activity when heated at 100°C for 1 hour , It needs to be heated at 160° for 2-4 hours, or boiled with strong acid, strong base and strong oxidant for 30 minutes to inactivate. Therefore, it can exist in water, air, various raw materials, containers and other environments for a long time. [0004] Endotoxin is highly tox...

AI Technical Summary

Problems solved by technology

However, the Limulus reagent method also has many disadvantages, such as the source of raw materials is limited by the amount of wild resources, the quality is uneven, the stability of the reagent is poor, it is susceptible to interference leading to false negative / positive results, and the applicable detection range is narrow. It is increasingly prominent during use and difficult to overcome; (3) Different companies have developed kits based on endotoxin antibodies, but the detection limits, sensitivities, detection ranges and standards of different companies are inconsistent, and the test results are not comparable, making it difficult to popularize and apply

[0007] At present, the rapid detection of endotoxin is still based on the method of Limulus reagent. However, on the one hand, there is an irreconcilable contradiction between the production of Limulus reagent and the protection of endangered Limulus resources. Wild Limulus resources are exhausted and endangered, and the source of raw materials is limited; The long growth cycle makes it difficult to expand the population; the recycling of wild Limulus in captivity is unsustainable; on the other hand, the Limulus reagent has many problems and defects that are difficult to solve. There are many positive results, and the defect that the LAL method is susceptible to interference when detecting endotoxins has led to a narrow detection range of the LAL method; although there are reports that try to use genetic engineering technology to express the recombined LAL active protein factors separately, and then combine them into LAL , although it can solve the false positive problem caused by fungal dextran, it also cannot solve the complex multifactor reaction mechanism of Limulus reagent, which is easily disturbed and produces more harmful false negative problems; in addition, the cost of recombinant Limulus reagent is also relatively high. There are no related products for sale in the market. It is worth noting that even the Limulus reagent, which is composed of recombinant Limulus reagent active protein factors expressed separately by genetic engineering technology, is a protein itself, and the problem of poor chemical stability of the protein cannot be avoided.

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