Study On Cloning And Expression Of Araneus Ventricosus Cathepsin B-like CDNA Avg1

Avgl cDNA (GenBank AY302573) screened from the major ampullate gland of Araneus Ventricousus cDNA library is a new cathepsin B-like gene. Cathepsin B is a lysosomal cysteine endoproteinase, which is implicated, in many physiological and pathological events, especially in tumor invasion and metastasis. So the relate studies on CB are increasingly focused on.The Avg was amplified from the tatal RNA of the major ampullate gland of Araneus Ventricousus by RT-PCR with the primer AvgS and AvgX designed according to Avgl cDNA. The PCR product and The prokaryotic expression vector pET-28a(+) and pET-20b(+) were digested by the restriction enzyme of EcoR I and Nco I .Two recombinant expression plasimids were constructed by T4 ligase and transformed into host strain E.coli DH5a. The Avg was completely identical to free signal peptide part of Avgl by sequencing and had the correct open reading frame. The two recombinant expression plasimids Avg-28a and Avg-20b were successfully constructed.Avg-28a in E.coli BL21 (DE3) was induced by 1PTG and the special protein (about 36 kDa) was detected by SDS-PAGE. It amounted to 39.6%of the total protein of E.coli DE3 by thin-layer scan. The pattern of expression was mainly inclusion body. SDS-PAGE of inducing Avg-20b-DE3 by IPTG did not show the special protein but the Avg mRNA transcription was detected by RT-PCR. The experiment of assaying the expression protein enzyme activity was carried on. The result indicated that the inclusion body protein treated in denaturing and renaturing agent was not shown the activity of cathepsin. But the supernatant of Avg-20b-DE3 by supersonic treatment showed the high activity.The inclusion body protein was purified by SDS-PAGE and electrophoresis elution and immunized rabbits to prepare anti-serum. The result of EL1SA showed obvious positive reaction. The special band of 36kDa appeared in Western-blot. It indicated that the inclusion body protein effectively induced the body to produce the antibody and had excellent antigenicity. The findings of this experiment will lay foundations for further studying the application of CB.