CN1257931A - Ferritin for supplementing iron to human body and its preparing process and application - Google Patents

Abstract

A ferritin for supplementing iron to iron-deficiency patient is prepared through high-density culture of engineering bacteria and preparing ferritin. The culture medium used in high-density culture contains ammonium carbonate (5g/L), dipotassium hydrogen phosphate (6g/L), potassium bihydrogen phosphate (3g/L), magnesium sulfate (1.5 g/L), tryptone (10-20 g/L), yeast extract (2-10 g/L), glucose (80-100 g/L) and trace elements (Cu, Co, Ca, Zn, Mo and Mn) (10 to the power -4- -5 gm/L for each). Its advantages are simple process, high yield and high iron content up to 2-5%.

Description

A kind of ferritin that can be used for compensating iron for human and its production and application

The present invention relates to a kind of ferritin that can be used for compensating iron for human and its production and application.

The easy iron deficiency of human body, pharmaceutically replenish human body at present, cause anemia because of iron deficiency with minimizing with inorganic iron, but poor effect, because of inorganic iron is difficult for absorbing, and mouthfeel is bad, easily causes nausea and reduces appetite, and all mending iron with ferritin in the world is being research direction, ferritin is a kind of protein that extensively exists in the plant, contain 4 iron atoms in its each molecule, its molecular weight is 12000, and iron claims non-haemin iron in this albumen.

The object of the invention provides a kind of ferritin that can be used for compensating iron for human and its production and application, the ferritin expression amount height that this method obtains, and iron-holder is many.

For achieving the above object, the present invention by the following technical solutions: this ferritin that can be used for compensating iron for human comprises protein and iron, iron content 2-5% wherein, all the other are protein.

A kind of preparation method who can be used for the ferritin of compensating iron for human, its feature comprises following step:

1. engineering bacteria high-density culture;

2. the preparation of ferritin.

Embodiment 1.

1. engineering bacteria high-density culture

The engineering bacteria that contains ferritin gene is e. coli bl21 (DE3), and the carrier that is loaded with ferritin gene is pSE280.Colibacillus engineering is by three grades of cultivations, firsts and seconds is on 200 rev/mins of shaking tables, cultivate on the LB substratum with trilateral down at 37 ℃, each was cultivated 6-8 hour, and the inoculum size according to 1% goes up a jar cultivation for three grades, fermentor tank is 20 liters, use high-density culture medium when jar is cultivated, 37 ℃ of cultivations are cultivated when the OD600 value is for 13-15 after 5 hours approximately, induce with IPTG (isopropylthiogalactoside), every liter adds 0.5 milliliter of 0.5mol/L IPTG solution.Induce after 3-4 hour and put jar.Final bacterium liquid OD600 value is about 18.Ferric ammonium citrate begins to add 0.1 grams per liter, after with the IPTG chemically inducible expression, does not extremely influence the maximum of thalli growth according to the heavy amount of increasing input gradually of thalline.Finally adding to every liter of bacterium liquid is 0.35 gram.Obtaining the wet thallus amount is the 60-70 grams per liter.

Used high-density culture based formulas is:

Ammonium sulfate 5.0 grams per liters, dipotassium hydrogen phosphate 6.0 grams per liters,

Potassium primary phosphate 3.0 grams per liters, sal epsom 1.5 grams per liters,

Tryptones 10-20 grams per liter, yeast extract 2-10 grams per liter,

Glucose 80-100 grams per liter,

The trace element (copper, cobalt, calcium, zinc, molybdenum, manganese) each 10 ^-5-10 ^-4Mol.

2. product preparation

Cultivation back 12000 rev/mins of centrifugal collection thalline of continuous centrifuge in jar, wet thallus can be placed on-20 ℃ of preservations down as do not handle at once.

Wet thallus suspends with 5 times of distilled water, smudge cells under 1000Psi pressure (or with the fragmentation of ultrasonic disruption instrument), descended centrifugal 15 minutes at 10000 rev/mins, supernatant liquor is with the analytical pure ethanol sedimentation albumen of 5 times of volumes, measure thing at 40 ℃ of drying under reduced pressure, product is pulverized again, promptly gets the ferritin goods.The iron-holder of this kind ferritin goods is about 2-5%.

3. the acute toxicity test of product, effect experiment and iron-holder mensuration are as follows, and can see annex 1,2,3 for details.

1, the acute toxicity test of product: making the maximum tolerated dose of albumen pulvis mouse stomach administration is 8000mg/kg, in continuous 7 days observe animal generally in order, no abnormality seens such as behavior, no dead animal, and body weight gain.

2, effect experiment: per os gavages 160mg/kg and 80mg/kg, and ferritin has the effect that obvious inhibition anaemia forms to atmosphere iron anaemia rat.

3, iron-holder is measured: Fe content is 5.23%.

Advantage of the present invention:

1. the product iron-holder height that obtains of this method, about 2-5%, duplicate experimental results show that and can be used for replenishing Iron is used for the treatment of the hypochromia that causes because of iron deficiency;

2. can increase wet thallus output and product output with high-density culture medium;

3. product can be used for normal human's the replenishing of iron, and also can be used for the iron deficiency patient and mends iron.

Claims (4)

1, a kind of ferritin that can be used for compensating iron for human is characterized in that it comprises protein and iron, iron content 2-5% wherein, and all the other are protein.

2, a kind of preparation method who can be used for the ferritin of compensating iron for human, its feature comprises following step:

1. the high-density culture of engineering bacteria;

2. the preparation method of ferritin.

3. by the described ferritin preparation method of claim 2, it is characterized in that:

1. used high-density culture based formulas is:

Ammonium sulfate 5.0 grams per liters, dipotassium hydrogen phosphate 6.0 grams per liters,

Potassium primary phosphate 3.0 grams per liters, sal epsom 1.5 grams per liters,

Tryptones 10-20 grams per liter, yeast extract 2-10 grams per liter,

Glucose 80-100 grams per liter,

The trace element (copper, cobalt, calcium, zinc, molybdenum, manganese) each 10 ^-5-10 ^-4Mol.

4, a kind of described preparation that can be used for the ferritin of compensating iron for human of claim 1 that contains is characterized in that it contains ferritin.