Construction Of CDNA Library Of Fasciola Gigantica And Expression And Purification Of Cathepsin L1 Gene

Fasciolasis is caused by the trematodes of genus Fasciola parasitizing in liver and bile duct of mammals such as cattles and sheep. It is widely distributed in China and brings animal husbandry a great economic loss. In addition, Human can be infected by Fasciola metacercaria and seriously endanger people'health, so Fasciolasis is an important zoonoses parasitic disease.We constructed the Fasciola gigantica cDNA library, the primary titer of the constructed cDNA library was 2.08×106 pfu/mL,while that of the amplified library was 6.23×109 pfu/mL. Cathepsin L1 secreted by Fasciola is a favorable diagnostic antigen for Fasciolasis infection . It was obtained from the Fasciola gigantica cDNA Library by PCR . The cDNA sequence has been submitted to GeneBank and its accession number is EF536689. and it was subcloned into pGEX-4T-2 expression vector, the recombinant plasmid pGEX/FgCL1/BL21was induced by 1mM IPTG at 30℃, the fusion protein was expressed in the form of inclusion bady. The molecular is 61KD. The proteins of target were purified by affinity chromatography with High-Affinity GST Resin. Western-blotting shows it can react with serum of cattle of naturally infected by Fasciola.we use the purified protein to establish ELISA. and the condition was optimized. the optimal concentration of FgCL1 for coating of plate was 4ug/ml, blocked with 5% skimmed milk, 37℃, 3h; the optimal condition of serum was 1:200, 37℃, 0.5h; the working condition of HRP-labeled goat anti-bovine IgG was 1:16 000, 37℃, 0.5h; the substrate for ELISA was incubated at 37℃, 20min. Following the determined of conditions of ELISA, the cut-off value was 0.143. Serum samples from other parasites infections were also detected. It showed that there were no cross-reaction with schistosome,Opisthorchis sinensis,Toxoplasma gondii. but have a little cross-reaction with Orientobilharzia. The result revealed that the indirect ELISA by the purified FgCL1 had good specificity for the detection of Fasciola gigantica antibady in serum. The above results indicated that FgCL1-ELISA developed in our laboratory could be used to detect Fasciolasis infection .