Chacterization of biological activity of ferritin protein by mass production using the recombinant vector

A mass production method of Periserrula leucophryna-derived ferritin protein using a recombinant vector for mass production of ferritin protein, the recombinant vector comprising a gene encoding Periserrula leucophryna-derived ferritin protein represented by an amino acid sequence of SEQ ID NO:

• 1, the method comprising:
• 1) cloning, into a T vector, a DNA fragment of the replication origin obtained by performing PCR by using a pRBASFer vector, which is a vector comprising a ferritin gene of Periserrula leucophryna, as a template;
• 2) obtaining a DNA fragment of the replication origin from the T vector into which the DNA fragment is cloned;
• 3) preparing new recombinant secretion vector (pRBASFer-ori) in which the replication origin is introduced in a tandem array by digesting the existing pRBASFer vector with a restriction enzyme and filling with a Klenow enzyme, and again digesting with another restriction enzyme and ligating the DNA fragment obtained in 2) above;
• and
• 4. culturing, in a medium, a Bacillus subtilis transformant into which the recombinant vector is introduced,
• wherein, in 1) above, PCR is performed with the existing pRBASFer vector as a template and with a forward primer of SEQ ID NO:
• 5 and a reverse primer of SEQ ID NO:
• 6, which have an introduced restriction enzyme site and which are specific to a replication origin in the pRBASFer vector.
• wherein, in 4) above, the Bacillus subtilis transformant to which the recombinant vector is introduced is cultured at a culture temperature of 28 to 35° C. at a stirring speed of 150 to 250 rpm, at an air injection rate of 0.5 to 2 vvm for 36 to 96 hours incubation by adding, into the medium, soy peptone 1% to 4% (w/v) as a nitrogen source and barley 1% to 3% (w/v) as a carbon source.