| Tachypleus tridentatus is an ancient marine life.The innate immune system of Tachypleus tridentatus is completely dependent on a unique and very effective blood coagulation system.The coagulation cascade of the coagulation system is mainly composed of four coagulation ptoteins such as Factor C(FC)?Factor G(FG)?Factor B(FB)and Proclotting Enzyme(PCE)and one kind of coagulogen.There are two ways to activate the system,one is Factor C activated by endotoxin,and the other is Factor G activated by(1,3)-?-D-Gurdlan.Natural Limulus amebocyte lysate blood products are widely used in the detection of endotoxin and fungal glucan in drugs and food,medical devices,clinical medical research and other fields.However,due to the excessive consumption of Limulus resources,the number of Limulus decreased sharply.In 2019,the Tachypleus tridentatus has been listed in the Red list of International Union for Conservation of Nature(IUCN)--Endangered(EN).Therefore,the preparation of Limulus reagent requires other methods to replace natural Limulus blood to obtain the raw materials of Limulus reagent.The purpose of this experiment is to construct a reaction system for the detection of endotoxin or(1,3)-?-D-Gurdlan by expressing foregin protein in E.coli engineering bacteria,so as to make the detection more sensitive and specific,so as to replace the extraction of natural Limulus blood and solve the problem of shortage of raw materials in the production of Limulus reagent.In this paper,the coagulatiton factor proteins such as FG ?,FG ? and PCE were heterogeneously expressed in E.coli expression system,and a single pathway reaction system for specific detection of(1,3)-?-D-Gurdlan was constructed.The soluble expression of FG ? and PCE was improved by changing the induction conditions,co-expression with molecular chaperone CpkA and designing fusion tag peptide.The renaturation experiment of the induced inclusion body was carried out in vitro,and the refolding protein was used to construct the reaction system to detect(1,3)-?-D-Gurdlan sysytem with different combinations of coagulation factors(two factors and three factors).We also tried to use molecular chaperones to assist the renaturation of FG ? and PCE in vitro.The results showed that FG ?,FG ? and PCE recombinant proteins were successfully expressed in Esherichia coli(DE3),but mainly in the form of inclusion bodies.Molecular chaperone can assist the folding Limulus coagulation factor in vivo/ in viro.Labeling peptide/ molecular chaperone co-expression system could increase the soluble exprssion of FG ?: the soluble expression of FG ?' was increased by 2 times,and the soluble expression of FG ?' and molecular chaperone MA4386 was increased by 7.5 times.The renaturation of three kinds of Tachypleus amebocyte lysate coagulation factor proteins were all active,it had peptidase activity in both the 1M and 2M urea concentration range,and had the highest activity in the 2M urea environment,and the CpkB-assisted renaturation FG ? + PCE two-factor detection system had the highest peptidase activily induced by(1,3)-?-D-Gurdlan(9.6 U/mg),and the specific activity was 0.9 U/mg higher than that of Limulus amebocyte lysate.In addition,non-glucan sugars could not induce peptidase activity,which showed that the two-factor detection system had high specificity for(1,3)-?-D-Gurdlan.In this experiment,three kinds of Tachypleus amebocyte lysate coagulation factor proteins were successfully expressed,and the soluble expression of FG ? was improved by using label peptide /molecular chaperone co-expression system.Finally a high activity and high specificity system was constructed for the detection of(1,3)-?-D-Gurdlan. |
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