Objective: To express recombinant antithrombin-III (AT-III) in an E.coli expression system, prepare the antiserum of AT-III and determine its titer.
Methods: Gene segment of AT-III was acquired by cloning technology. Then prokaryotic expression plasmid pET32a(+)-AT-III was constructed and transformed into E.coli competent cells BL21. The cells were induced by IPTG to express AT-III. After purified, the product was used to immunize New Zealand rabbits. Then antiserum was detected using indirect ELISA and Western blotting.
Results: Specific bands appeared at about M(r); 77 000, indicating prokaryotic expression protein detected by SDS-PAGE and Western blotting. From the rabbits immunized by the purified fusion protein, we aquired AT-III antiserum, of which the highest titer was 1:12 800 as shown by indirect ELISA. Western blotting showed the antiserum had the ability of specific binding to AT-III protein expressed by 293T and CHO or purified AT-III protein.
Conclusion: Antiserum of human AT-III has been prepared successfully.