Construction Of Horseshoe Crab Coagulation Factor Reaction System For Detecting Single Pyrogen

Tachypleus amebocyte lysate coagulation cascade reaction can be activated by trace amounts of bacterial endotoxin or fungal(1,3)-β-D-glucan.Respectively,the gel is produced through the factor C pathway and the factor G pathway,thus allowing the body to defend against pathogens.Tachypleus amebocyte lysate reagent(TAL)made from blood can be activated simultaneously by endotoxin and(1,3)-β-D-glucan,so it will lead to non-specific in vitro detection of TAL due to cross-reaction,resulting in false positive detection results.Therefore,the preparation of TAL that identifies a single pyrogen is an urgent technical problem to be solved in this technical field.At the same time,horseshoe crab is a secondary protected animal in China.In recent years,the resources of horseshoe crab have been excessively consumed and are on the verge of exhaustion,and natural horseshoe crab blood is difficult to supply a large number of TAL for production.Therefore,a recombinant TAL preparation in place of the natural TAL is required to meet the detection requirements for endotoxin or(1,3)-β-D-glucan.However,the heterologous expression of eukaryotic proteins has always been a bottleneck in protein engineering,and its expression level in eukaryotic expression cells is low,and the heterologous protein expression level in prokaryotic cells is large,but it is easy to form inactive inclusion body precipitation.The purpose of this study is to improve the soluble expression of monomeric protein of horseshoe crab coagulation factors in prokaryotic cell supernatant and prepare a highly active recombinant TAL for the detection of single pyrogen.In this paper,the prokaryotic expression vector of the optimized sequences of G factorαsubnit(FGα)、G factorβsubnit(FGβ)and proclotting enzyme(PCE)genes of TAL were constructed.we obtained biologically active protein FGα’、FGβ’and PCE’by fusing horseshoe crab coagulation factors with anchor tag and coexpression with chaperonin in Escherichia coli.Three kinds of combined reaction system of diferrent horseshoe crab coagulation factors were constructed by using the prepared recombinant horseshoe crab coagulation factors,which were FGα’and FGβ’two-factor reaction system、FGα’and PCE’two-factor reaction system、FGα’、FGβ’and PCE’three-factor reaction system,and the peptidase activity of the reaction systems were determined.Finally,a multi-enzyme reaction system that could specifically detect(1,3)-β-D-glucan was constructed,and the reaction system was optimized.The results showed that a large number of recombinant horseshoe crab coagulation factors FGα’、FGβ’and a small amount of PCE’with biological activity were successfully expressed in E.coli,and a large amount of soluble PCE’was obtained after renaturation of PCE’inclusion body.The yield of soluble protein of FGα’、FGβ’and PCE’was 82.4%、50.5%、29.4%.And about 81.2%of PCE’inclusion body after renaturation.Two and three-factor recombinant horseshoe crab coagulation reaction systems for the specific determination of(1,3)-β-D-glucan were constructed,respectively,of which the constructed three-factor recombinant horseshoe crab coagulation reaction system had the best effect,and its standard curve has a good linearity in the concentration range of(1,3)-β-D-glucan of[0.39-100]pg/m L,with a curve R~2 of 0.9948.The results showed that the recombinant horseshoe crab coagulation factor reaction system was equivalent to commercial TAL in the detection of(1,3)-β-D-glucan,and the sensitivity of the former(0.39 pg/m L)is higher than that of the latter(12.5 pg/m L).The results of substrate cross experiment showed that the constructed three-factor recombinant horseshoe crab coagulation factor reaction system could specifically detect(1,3)-β-D-glucan.In this study,the recombinant horseshoe crab coagulation factor reaction system for the specific detection of(1,3)-β-D-glucan was successfully constructed using the prepared recombinant horseshoe crab coagulation factors FGα’、FGβ’and PCE’,which provided a new scheme to replace the natural TAL,reduced the demand for wild horseshoe crab resources while improving the detection specificity,and had positive significance for protecting wild horseshoe crab resources.