Cloning Of Nattokinase Gene And Expression In E. Coli And Yeast Pichia Pastoris

Nattokinase (NK) is a fibrinolytic enzyme that is extracted from natto—a traditional food in Japan. NK was discovered by Japanese scholar Hiroyuki Sumi in 1987. Compared with thrombolytic agents which is in common use such as streptokinase (SK), urokinase (UK), recombinant tissue-type plasminogen activator (rt-PA), anisoylated plasminogen streptokinase activator complex (APSAC) and prourokinase (pro-UK), Nattokinase is safer, easier to be absorbed by body, has more direct and more persistent effect on thrombosis and cost is low. It can be developed as a new and potent fibrinolytic medicine. In this study, gene coded NK is cloned and expressed in E. coli and Pichia pastoris respectively by recombinant DNA techniques. The results are as follow.In the first place, the primers were designed based on NK nucleotide sequences and properties of vector pBV220. The optimizing parameters for amplifying NK gene were determined by optimizing PCR conditions. They were: chromosomal DNA as template was 10ng, the concentration of Mg²⁺ was 1.5mmol/L and the annealing temperature was 60℃. Heating the PCR mixture under 95℃ for 3 minutes, running the process for 30 cycles as 94℃ 1 minute, 60℃ 1 minute, 72℃ 3 minutes. After that, keeping mixture in 72℃ for 15 minutes. Under these optimal conditions, specific and high performance PCR product were obtained.Vector pUC19-T was constructed and the PCR product was cloned on it. After being analyzed by restriction enzyme, PCR and sequencing, it was confirmed that heterogeneous gene spliced into vector pUC19-T was NK gene.After the NK gene from recombinant plasmid pUC19-NK was cloned on expression vector pBV220, it was transformed into host cell E. coli HB101. Recombinant plasmid pBV220-NK was extracted for identifying by analysis of restriction enzymes, PCR and sequencing. Studies on recombinant strain's growth and expression showed that heterogene have no distinct effect on host cell. The recombinant plasmid have excellent segregational stability but low structural stability. The results of kinetics of pBV220-NK expression in E. coli HB101 showed...