Detection reagent for specifically detecting (1-3)-beta-D-glucan and preparation method thereof

Abstract

The invention belongs to the technical field of gene engineering, and specifically discloses a detection reagent for specifically detecting (1-3)-beta-D-glucan and a preparation method thereof. The nucleotide majorizing sequence of horseshoe crab G factor zymogen alpha subunit, beta subunit and prothrombin gene are obtained respectively and as shown as SEQ ID NO:1-3; the nucleotide sequence for jointly expressing the above three kinds of key factors is further obtained and as shown as SEQ ID NO:4; the eukaryon expression carrier for independently expressing or jointly expressing the three kinds of key factors is successfully constructed, finally an artificial horseshoe crab reagent similar to a natural horseshoe crab reagent is successfully obtained and can be used for detecting (1-3)-beta-D-glucan, the sensitivity is high, the specificity is high, the false positive can be reduced, the preparation technology is simple, proteins do not need to be extracted and purified, the productioncost is low, the demands for wild horseshoe crab sources greatly decreases, and the detection reagent has the wide marketing application prospects.

Description

technical field [0001] The invention belongs to the technical fields of biological detection and medical treatment, and in particular relates to a detection reagent for specifically detecting (1-3)-β-D-glucan and a preparation method thereof. Background technique [0002] The incidence of deep fungal infection is increasing year by year, and the mortality rate remains high due to the lack of effective early diagnosis methods. Currently, the methods for detecting fungal infection include blood culture, tissue biopsy, PCR technology and immunological methods. Among them, blood culture and tissue biopsy are not suitable for early diagnosis due to the long time-consuming culture method and low positive detection rate; PCR can only detect known pathogenic fungal infections, and is not suitable for early diagnosis of rare conditional pathogenic fungal infections; and immunology The method is to screen for multiple fungal antigens, which is easy to miss, time-consuming and unecono...

AI Technical Summary

Problems solved by technology

However, there are two different activation pathways for the coagulation process of Limulus horseshoe crabs, one is the endotoxin-mediated factor C pathway: Factor C (FC) is activated by binding to endotoxin, and then activates factor B, and the activated factor B will coagulate the zymogen (Proclotting enzyme) is converted into coagulation enzyme (clotting enzyme), coagulation enzyme converts coagulation protein into coagulation protein, and coagulation protein is cross-linked and dehydrated to form a gel; the other is 1,3-β-D-glucan The mediated factor G (FG) pathway can also cause similar agglutination reactions and produce false positive results. Therefore, natural sources of Limulus reagents cannot be directly used to detect fungal infections

[0005] At present, although there is a method to increase the detection specificity by adding anti-lipopolysaccharide substances to the LAL reagent, it cannot completely block the interference of endotoxin on the G test, and it is easy to produce false positive results

In addition, Limulus reagent can only be produced by collecting Limulus hemolymph. The preparation of Limulus reagent relies heavily on Limulus resources. And the rapid growth of production units such as medical devices (such as disposable syringes, implantable biomaterials), and the increasing demand for fungal infection detection reagents have gradually reduced the number of horseshoe crabs in the ocean

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