WO2014005466A1

WO2014005466A1 - Product for mitigating iron deficiency anemia and method for preparing same

Info

Publication number: WO2014005466A1
Application number: PCT/CN2013/075663
Authority: WO
Inventors: 于添; 王建武; 李宁
Original assignee: 北京济福霖生物技术有限公司
Priority date: 2012-07-02
Filing date: 2013-05-15
Publication date: 2014-01-09
Also published as: CN102766206A

Abstract

Disclosed is a product for mitigating iron deficiency anemia, which is iron-saturated recombinant human lactoferrin. Further disclosed is a method for preparing the product for mitigating iron deficiency anemia.

Description

Product for improving iron deficiency anemia and preparation method thereof

The present invention belongs to the field of biotechnology, and in particular to a product for improving iron deficiency anemia and a preparation method thereof. Background technique

Iron is an important component of hemoglobin, which plays a role in transporting oxygen in the human body and is one of the essential nutrients for the human body. Iron deficiency in the body can reduce immunity, accompanied by symptoms such as fatigue, irritability, irritability, and memory loss. More importantly, iron deficiency in the body can cause iron deficiency anemia (IDA). Iron deficiency anemia (IDA) is one of the most prevalent disease-deficient diseases in the world. About one-third of the world's 5 billion people are anemic. The number of people with IDA or iron malnutrition is about 500 million. The incidence of iron deficiency (ID) in developing countries is four times that of developed countries. China is one of the countries with a high incidence of iron deficiency anemia in the world. The average incidence of IDA in various groups is 15%-20%, and the incidence rate of women and children is over 20%. The incidence of IDA in children in severe areas is as high as 70%, and the incidence of IDA in women of childbearing age is as high as 35%. Anemia is a serious threat to women's health.

Although iron is more common in food, the form of iron in food is not conducive to the absorption and utilization of the body. Currently, the medical profession generally uses iron supplements to treat patients with iron deficiency anemia. At present, there are two main types of iron supplements in the market: one is the traditional chemical synthesis of iron preparations including inorganic iron and organic iron (non-heme iron); the other is biological heme iron. Inorganic iron mainly includes ferrous salts, such as ferrous sulfate, ferrous gluconate, ferrous fumarate, ferrous lactate, ferrous succinate, etc., although such iron supplementation has better iron-reinforcing effect, Its low bioavailability, poor taste and long-term use will stimulate the gastrointestinal mucosa, which may cause side effects such as nausea, bloating, digestive disorders, diarrhea, constipation, especially for patients with stomach problems, pregnant women or children. . Due to the adverse side effects of inorganic iron preparations, a new generation of iron supplement research has begun. In the 1990s, the National Center for Disease Control and Prevention successfully developed a new iron supplement, ethylenediamine tetraacetate, which has inorganic The obvious advantage of iron preparation: not only good taste, high absorption rate and stable nature without gastrointestinal irritation. Subsequently, organic iron preparations of similar nature and similar structure were continuously developed, such as iron glycinate, iron threonate and the like. However, due to the high production cost of amino acid trace element complexes and the high cost of preparations, it is not widely used in China. Heme iron (porphyrin iron) is the best absorption rate in the field, and the most bioavailable iron supplement is mainly separated from the qualified blood of livestock, and the blood cell part is further digested by protease to remove blood cells. Porphyrin-rich ferritin obtained after protein. However, due to its low iron content, limited blood sources also limit its widespread use.

At present, the iron supplements used in medicines and food additives in the domestic market are mainly ferrous preparations. Although the absorption rate of organic iron preparations is slightly improved, it is still unsatisfactory, and it is difficult to popularize the price. Therefore, the development of iron supplements with better stability, higher bioavailability and lower prices has been the focus of research.

Long-term studies have shown that lactoferrin (LF) can promote the body's absorption and utilization of iron, and maintain the body's iron metabolism balance. Lactoferrin is a multifunctional metal-binding protein with a molecular weight of 80 kD and is widely found in mammalian milk and various other tissues as well as in its exocrine fluids. It can participate in a variety of physiological processes, including host defense, inflammation regulation, growth stimulation, and promotion of iron absorption in the gastrointestinal tract. Lactoferrin is structurally and biochemically similar to the height of transferrin, but its affinity for iron ions is 260 times that of transferrin and maintains iron over a wide range (pH 4-ll). Combination of. Since transferrin does not have the function of transporting iron under extremely acidic conditions in the stomach, only lactoferrin can carry iron ions in the intestinal tract of animals.

Lactoferrin can maintain iron balance in the body by regulating the iron absorption of intestinal mucosal cells according to the body's demand for iron. Fe ³⁺ is insoluble in water at physiological pH and is difficult to use by organisms. Lactoferrin is capable of chelation of Fe ^{3+ to} increase its solubility, and 1 mole of lactoferrin can dissolve 70 moles of Fe ³⁺ much higher than its iron ion binding capacity. Lactoferrin binding to Fe ³⁺ alters its chemical form and promotes uptake of Fe ^{3+ by} cellular cells. After the iron-binding lactoferrin enters the small intestine, iron ions are transported to the blood through receptors on the intestinal epithelial cells; the intestinal cells can also be straight A large amount of lactoferrin is absorbed to obtain iron ions. At the same time, lactoferrin can also improve the bioavailability of iron and maintain the body's iron metabolism, so it can be used to prevent and treat anemia caused by iron deficiency. Rat feeding experiments show that lactoferrin-binding iron can significantly improve iron deficiency anemia and the effect is far superior to FeS0 ₄ and FeCl ₃ , and other inorganic iron ions, which not only improves the bioavailability of iron but also avoids the inorganic iron pair. Damage to the gastrointestinal mucosa. In 2010, the International Journal of Immunopathology and Pharmacology reported that Italian researchers in the clinical study of iron deficiency anemia in pregnant women found that lactoferrin increased serum ferritin, hemoglobin, and iron levels significantly better than FeS0 ₄ , and had no significant side effects. It was further confirmed that lactoferrin can be used for the treatment of iron deficiency and iron deficiency anemia. In addition, clinical studies have shown that lactoferrin also has the effect of preventing sports anemia and muscle fatigue.

Lactoferrin can maintain iron balance in the body by regulating the iron absorption of intestinal mucosal cells according to the body's demand for iron. Lactoferrin promotes iron absorption mainly by increasing the solubility of Fe ³⁺ and specifically binding to the lactoferrin receptor ( LFR ) of animal intestinal mucosal cells, increasing iron absorption efficiency and avoiding iron ions on the intestinal tract of animals. Direct stimulation. Lactoferrin recognizes and binds to LFR after reaching the intestinal tract and is immersed in cells by endocytosis and releases Fe ³⁺ . The cell has a negative feedback regulation mechanism for the absorption of iron. When there is iron deficiency in the cell, the LFR on the cell surface increases, increasing the chance of binding of LF to its receptor. If the intracellular iron content is high, the LFR expression on the cell surface is reduced, and the iron intake is reduced. To reduce the adverse reactions caused by iron ions to the body. Studies have shown that lactoferrin can regulate the body's absorption of iron by regulating the inflammatory response and reducing the expression of pro-inflammatory factor IL-6 in the blood.

In summary, lactoferrin can not only enhance the absorption rate of iron but also reduce the amount of available iron and reduce the negative impact of iron on the body. Therefore, lactoferrin can be used as a good iron supplement.

However, the large-scale development of iron supplements using lactoferrin is still limited. Because of the current presence of bovine lactoferrin (bLF), bovine lactoferrin is extremely low in milk and the cost of production is extremely high. High and bovine lactoferrin as a heterologous protein, its stability and absorption rate in human body are not as good as human lactoferrin, so it has been developed and produced in large quantities. Human lactoferrin has always been the focus and difficulty of people's research. At present, many attempts have been made to produce recombinant human lactoferrin by prokaryotic and eukaryotic expression systems. However, the expression levels and post-translational modifications of many expression systems are not suitable, which limits the application of these recombinant proteins. Recently, researchers have successfully obtained human lactoferrin transgenic cloned cattle using somatic cell cloning technology. Recombinant human lactoferrin (riiLF) has a high expression level of up to 2.5 g/L in milk. Recombinant human lactoferrin has similar biological activities as natural human lactoferrin, including sensitivity to trypsin, iron binding release ability, and antibacterial activity. The use of this recombinant human lactoferrin for iron supplementation can greatly reduce the cost of replacing bovine lactoferrin, and its ability to bind to human intestinal epithelial cell-specific receptors will greatly enhance its iron transport efficiency and increase iron absorption efficiency. . Summary of the invention

It is an object of the present invention to provide a product which improves iron deficiency anemia.

Another object of the present invention is to provide a method of preparing the improved iron deficiency anemia product. The present invention utilizes recombinant human lactoferrin expressed in transgenic cloned milk as a raw material to improve the development of iron deficiency anemia products, and provides a novel iron supplement preparation product, iron-saturated recombinant human lactoferrin (FerhLF).

The present invention also provides a method for mass-purifying recombinant human lactoferrin from transgenic milk and a method for preparing a iron-iron-saturated recombinant human lactoferrin.

The method for mass purification of recombinant human lactoferrin from transgenic milk comprises: preparation of transgenic bovine expressing recombinant human lactoferrin, defatting of milk, filtration sterilization, and ion exchange chromatography to separate and purify recombinant human lactoferrin from skim milk. Specifically, the following steps are included:

1) Preparation of transgenic cattle, including the following steps:

i) using hLF BAC DNA containing the complete human lactoferrin gene as a mammary gland expression vector;

Ii) mixing hLF BAC DNA with the double-marker selection vector pEGFP-NEO or the single-marker selection vector pNEO, introducing into the nucleus of the somatic cell, performing cell transfection, and obtaining transgenic cells transformed into hLF BAC DNA; Iii) performing somatic cell cloning as a nuclear donor to obtain transgenic cattle transfected with hLF BAC DNA;

2) The riiLF milk produced by the above transgenic cattle is heated to 40-45 °C, and then subjected to cream separation, and continuously degreased twice at 65-75 rpm; then the defatted milk is microfiltered by a filter having a pore size of 1.4 μm. Bacteria

3) using a BPG140/500 column, cation exchange chromatography using SP Sepharose Big Beads as a column packing, eluting with a PBS solution having a pH of 6.5, and collecting the eluate having the peak elution peak of the same rhLF activity;

4) The eluate is subjected to ultrafiltration concentration and freeze-drying to obtain a recombinant human lactoferrin powder. In the above method, in step 3), premixing is preferably carried out with 20 mM PBS containing 0.4 M NaCl and pH 6.5 to remove the heteroprotein, and then eluted with 20 mM PBS containing 1 M NaCl, pH 6.5, and collected and combined with the same rhLF. The elution of the peak of the active elution peak.

In the above method, the ultrafiltration in the step 4) is specifically: the eluate having the same peak elution peak of the same riiLF activity obtained in the step 3) is pumped into the ultrafiltration membrane through a peristaltic pump for ultrafiltration concentration, and added to the pH. A solution of 6.0 mM 20 mM phosphate buffer was circulated 7 times until the concentrate outlet conductivity was below lmS/cm, and the solution was further concentrated.

The method for preparing the iron deficiency anemia product is as follows: recombinant human lactoferrin (rhLF, amino acid sequence such as Seq ID No. 1) expressed by transgenic cloned bovine milk and Fe ³⁺ in NaHC0 ₃ solution (or KHC0 ₃₎ Chelation is carried out in solution). Specifically, it is a recombinant human lactoferrin dissolved in water formulated as a solution, the solution was then mixed with a solution of FeCl ₃ or KHC0 ₃ and NaHC0 ₃ solution, iron saturation is made of recombinant human lactoferrin solution stirred, then after After desalting and lyophilization, iron-saturated recombinant human lactoferrin (FerhLF) powder was obtained, wherein the iron content was 6.62 mg/g.

The present invention is the first attempt to develop iron supplement products using recombinant human lactoferrin expressed by transgenic cloned cow's milk. The use of this new iron supplement greatly enhances the effect of improving iron deficiency anemia, significantly improves the hemoglobin concentration in rats with iron deficiency anemia, and its recovery effect is much faster and less obvious than the positive control (FeS0 ₄ ) group. Adverse reactions, using safety Full, the effect is obvious, and the preparation method is simple. DRAWINGS

Fig. 1 is a chromatogram showing the effect of the SPLF purification of SP Sepharose Big Beads in the rhLF of the present invention. Figure 2 is a graph showing the purity of rhLF by SDS-PAGE electrophoresis of the present invention.

Fig. 3 shows the results of rhLF SDS-PAGE electrophoresis detection of 8 consecutive purifications of the present invention. Figure 4 is a comparative analysis of riiLF and hLF using high efficiency gel filtration in the present invention.

Figure 5 is a comparison of the secondary structure of rhLF and hLF by circular dichroism.

6 is a blood routine test result of a rat anemia recovery experiment using FeriiLF, A is a hemoglobin concentration, B is a red blood cell count, C is a hematocrit, D is a red blood cell mean hemoglobin content, and different lowercase letters are identified between groups. There are significant differences.

Figure 7 shows the results of other indicators in the rat anemia recovery experiment using FeriiLF. A is the average red blood cell capacity, B is liver iron, and C is spleen iron. There are significant differences between different lowercase letters. detailed description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

The reagents used in the following examples and their sources:

Na ₂ HP0 ₄ · 12H ₂ 0, Na3⁄4P0 ₄ · 2H ₂ 0, NaOH, 95% ethanol, NaCl, Fe _s 0 ₄ , FeCl ₃ , NaHC0 ₃ were all domestically analyzed and purchased from Beijing Chemical Reagent Company. Other reagents are of domestic analytical purity. Natural human lactoferrin (hLF) was purchased from Sigma. Bovine lactoferrin was purchased from Tatua, New Zealand (90% protein content with 95% lactoferrin content). Bovine lactoferrin detoxification was purchased from the Talaydon Science Laboratory (Taradon Laboratory, Belgium).

The instruments used are as follows:

Ceramic membrane ultrafiltration system, Taupson

Easy load Masterf ex , Millpore Chromatography column (BPG 140/500), GE Healthcare

Chromatography media (SP Sepharose Big Beads), GE Healthcare

Chromatography System (AKTA Pilot), GE Healthcare

Creamer (Χ5-Π :ι a B a -100 ), Russia

Ultrafiltration Concentration System, Millpore

Automatic blood cell analyzer, MEK-6318k

The circular dichroism spectrometer is Jasco 810 (Jasco, Japan).

Example 1 Preparation of Transgenic Bovine Expressing Recombinant Human Lactoferrin

A recombinant human lactoferrin transgenic cow is prepared according to the method disclosed in ZL 200610076240.5 (Invention name: Method for producing animal cells transfected with human lactoferrin gene). The steps are as follows: (1) using hLF BAC DNA containing the complete human lactoferrin gene as a mammary gland specific expression vector; (2) mixing hLF BAC DNA with the double marker selection vector pEGFP-NEO or the single marker selection vector pNEO, It is introduced into the nucleus of the somatic cells of the livestock, and transfected to obtain transgenic cells transformed into hLF BAC DNA; (3) The cells are cloned as nuclear donors to obtain transgenic cattle transfected with hLF BAC DNA.

The amino acid sequence of the recombinant human lactoferrin (rhLF) is shown as Seq ID No. 1. Example 2 Pilot Purification and Quality Control of Recombinant Human Lactoferrin

1.1 Preparation of ALF milk skim milk

The rhLF milk produced by the transgenic cow in Example 1 was heated to 40-45 ° C, and the milk was discharged into a cream separator to adjust the number of revolutions to 65-75 rpm. Each batch of milk is continuously degreased twice to fully remove fat.

1.2 Ceramic membrane filtration sterilization

The defatted milk is ultrafiltered using a ceramic membrane (pore size 1.4 μηι), and the effect of cold sterilization can be achieved because the size of the bacteria is around 0-5 μm.

The experimental operation is shown in Table 1.

Table 1 Steps of using a ceramic membrane to filter and sterilize

Pressure (MPa) flow rate (ton h) temperature

Remarks membrane inlet membrane outlet membrane back pressure circulation flow CO 0.38 0.14 0.28 5 70 Add 80 °C double distilled water to keep warm and sterilize to cool down (with cooling circulating water) with milk ultrafiltration, flux is 1.84L / min;

0.36 0.12 0.10 5 30

315L/m2/h

Alkaline wash once (0.5%, 0.8M NaOH) 15L

0.28 0.10 0.22 5 50

Left and right, 50 ° C

Rinse with water 2-3 times

Alkaline wash twice (1%, 1.6M NaOH) 15L left

0.36 0.14 0.3 5 80

Right 80 ° C

0.30 0.08 0.16 5 55 Pickling 0.5% nitric acid 15L or so Wash twice PH neutral water flux 12L/min,

0.38 0.13 0.11 5 30

2057 L/m2/h

1.3 Separation and purification from skim milk rhLF

1.3.1 Buffer preparation

(l) PBS stock solution preparation

Stock solution 1: Na ₂ HP0 ₄ ·12Η ₂ 0 (0.2 M, Mw: 358.14, 16 L): Weigh 1146g

Na ₂ HP0 ₄ .12H ₂ 0 was poured into a 20 L vessel, and 16 L of deionized water was added and stirred for use.

Stock solution 2: NaH ₂ P0 ₄ · ₂ Η ₂ 0 (0.2 M, Mw: 156.01, 18 L): Weighed 561.636g

NaH ₂ P0 ₄ *2H ₂ 0 was poured into a 20 L vessel, and 18 L of deionized water was added and stirred for use.

( ² ) Preparation of buffers A and B

Buffer A (Buffer A, 100 L): Use the amount of simplification to measure 8.77 L of stock solution 2 and

1.231^0.2^[Liquid 1 in the 1001^ container, which is 10 x Buffer A, spare. Add 90 L of deionized water before use, stir and dissolve completely, adjust the pH to 6.5 with IMNaOH (or 1MHC1), and store in two 50LPP barrels for use.

Buffer B (Buffer B, 50 L): Measure the amount of 0.265 L of stock solution 2 and

4.735 L stock solution 1 In a 100L container, weigh 2922g of NaCl, which is 10 x Buffer

Β, 0.45μηι filter microfiltration and use; add 45 L of deionized water before use, stir and dissolve completely, adjust the pH value to 6.5 with IMNaOH (or 1MHC1), store in 50LPP bucket for use.

1.3.2 Packing column

(1) Shake the ion exchange medium SPSepharose BigBeads (new medium, stored in ²⁰ % ethanol) and set aside; (2) Take the BPG140/500 column, wash it and place it vertically. Drain the bubble of the outlet end and connect it to the column. Add ultra-pure water about 1cm in height, then stir evenly by the glass rod. The ion exchange medium is slowly introduced into the column, and then the adapter at the inlet end of the discharged bubble is connected to the column;

(3) After the column is installed, flush it with ultrapure water through an atmospheric pressure chromatography system (rinse the ethanol in the storage medium) 5 column volumes (flow rate: l L/min), and then use 2 column volumes. BufferA (equilibration buffer) equilibrates the column to the pH of the outlet of the column;

(4) Finally lower the inlet end adapter to the surface of the media, paying attention to air bubbles. The final column height was recorded and the bed volume was controlled at around 3L.

1.3.3 ion exchange chromatography

Column Model: BPG140/500 Chromatography Media: SP Sepharose Big Beads Column Height: 20 cm; Column Volume: 3 L; Chromatography System: AKTA Pilot; Total Time: 304 min.

Load 8 times in parallel. (Table 2 )

Table 2 Elution steps of ion exchange chromatography

Step column volume volume line speed volume flow time balance BufferA: 20mM PB pH 6.5 2CV 6L 150cm / h 384ml / min 16min loading skim milk 5CV 15L 150cm / h 384ml / min 40min rinse BufferA 3CV 9L 150cm / h 384ml / min 24min

40% Buffer B: 20mM

Pre-elution 10CV 30L 150cm/h 384ml/min 80min

PB+0.4M NaCl pH 6.5

Buffer B: 20mM PB+1M

Elution 4CV 12L 150cm/h 384ml/min 32min

NaCl pH 6.5

CIP lM NaOH 3CV 9L 50cm/h 128ml/min 72min Pre-equilibration Buffer B 3CV 9L 150cm/h 384ml/min 24min Balance BufferA 2CV 6L 150cm/h 384ml/min 16min The results of ion exchange chromatography are shown in Figure 1.

During the chromatography, after the skim milk starts to load a column volume, the medium on the column appears yellow; after the loading is completed, the yellow whey flows through, and about a quarter of the volume of the upper part of the column is light gray. . The sample of the breakthrough peak was sampled. The sample obtained at the beginning of penetration was cloudy and grayish yellow. The mid-pass peak was sampled as white, which was the casein flowing through. The breakthrough peak obtained when using Buffer A flushing medium was clear. yellow. 40% Buffer B pre-elution The elution peak is yellow, and the lactoperoxidase which is weakly bound to the medium is eluted. After pre-elution 10 column volumes, the impurity whey protein and endotoxin are removed, and the column is shallow. The gray part turns pale red. After starting to elute with 100% Buffer B, the light red band moves down the column and elutes, and the resulting LF target elutes reddish.

1.3.4 SDS PAGE analysis of rhLF purity

The samples collected by hLF, bLF, 100% Buffer B eluted rhLF and 40% Buffer B eluted were electrophoresed on 10% SDS-PAGE using raw milk as a control. The electrophoresis time was 50V, lh; 90 V, 2 h. It can be seen from the electrophoresis pattern 2 that the rhLF purified product eluted by 100% Buffer B in the pilot process has the same molecular weight as the natural hLF standard (Sigma), and the sold hLF standard (Sigma) and bLF standard (purchased) New Zealand Tatua lactoferrin sold by Nanjing Tianyi Trading Co., Ltd., endotoxin bLF standard (purchased from Labrador Laboratory, Thalen Laboratory, Lactoferrin-NFQ, sold by New Bailey Hengkangyuan International Trading Co., Ltd.) Higher purity than MW, the LF purity is greater than 95%. The 40% Buffer B pre-eluting peak has less hLF loss, and the band with similar molecular weight to LF may be lactoperoxidase (Fig. 2).

The electrophoresis results of LF target peaks obtained from 8 parallel chromatography in the pilot test showed that the pilot process was reproducible, and the purity of the target protein obtained by 8 chromatograms was higher (Fig. 3).

1.4 Ultrafiltration concentration and freeze drying of rhLF purification solution

1.4.1 Ultrafiltration condensate desalination:

(1) Take the rhLF active elution peak component (volume about 50 L), pump it into the ultrafiltration membrane through a peristaltic pump for ultrafiltration, and concentrate the above solution to a volume of 8 L.

(2) 4 L of 20 mM PB, pH 6.0 was added 7 times to sufficiently remove the sodium chloride in the solution until the concentrate outlet conductivity was lower than lmS/cm.

( ³ ) The above solution was concentrated to a volume of 4 L.

(4) Sampling 2 mL 2, measuring the protein concentration (A280 method, or Coomassie brilliant blue method A595), weighing the volume of the legal concentrated desalting solution. (The density of the protein solution was 0.99 g/ml).

The ultrafiltration buffer solution used in the ultrafiltration in step (1) is prepared as follows: 4.385 L stock solution 2 and 0.615 L 0.2 M stock solution 1 in a 100L container, add 45L of deionized water, stir and dissolve completely, then adjust the value of 11 to 6.5 with 1M NaOH (or 1M HC1), store in 50L PP barrel for use.

1.4.2 Vacuum freeze drying:

Operating conditions: Freeze-drying is divided into three stages, the first stage is pre-frozen-4 (TC 4 hours; the second stage is lyophilized and sublimated for 5 times, each time for 1 (TC takes 1 hour, then directly rises to 30 °). Maintaining the temperature for 400 minutes, the pressure 300mtorr is equivalent to 0.399 mbar, corresponding to the sample temperature of -29 ° C. The third stage freeze-drying condition 3 (TC, pressure is 0, 240 minutes.

1.5 purity and physical and chemical properties of rhLF

1.5.1 Comparative analysis of high efficiency gel filtration rhLF and hLF

The gel was filtered using an Agilent 1260 chromatography system and a SuperSW3000 (4.6 mm (ID) x 30 cm (L), TOSOH) column with a buffer of 0.1 M PB-0.1 M Na ₂ S0 ₄ at a flow rate of 0.5 ml/min. Sample size 20μΙ^. Efficient gel filtration analysis showed that the rHLF obtained by the pilot purification was consistent with the peak time of the natural HLF standard and had a very similar molecular weight (Fig. 4).

1.5.2 Circular dichroism analysis rhLF and hLF structure

Use circular dichroism. The scanning wavelength is 190-260 nm and the resolution is lnm. The same system as the solution in the sample to be tested was used as the blank, and the CD spectrum was the average of the three measurements. The results showed that the circular dichroic chromatogram of recombinant human lactoferrin purified from the pilot test was basically coincident with that of natural human lactoferrin, indicating that the recombinant human lactoferrin expressed in bovine mammary gland was basically consistent with the secondary structure of natural lactoferrin. Adsorption and desorption on the ion exchange medium does not cause irreversible changes in the secondary structure of the protein, which facilitates the acquisition of intact biologically active recombinant proteins.

(Figure 5 ).

Example 3 Preparation of iron-saturated recombinant human lactoferrin (FerhLF)

The specific experimental steps are as follows:

1) The purified lyophilized 120 g of rhLF powder was dissolved in distilled water to a volume of 12 L to prepare a 1% rhLF solution.

2) Prepare 28L of 1.4% FeCl ₃ solution and 28L of 10% NaHC0 ₃ solution, respectively.

3) Mix 12L 1% rhLF solution with 28L 1.4% FeCl ₃ , 28L 10% NaHC0 ₃ Mix, mix thoroughly and stir constantly, and let stand for 1 hour at room temperature.

4) The supernatant after the reaction was centrifuged to obtain a supernatant, and the supernatant was brownish red.

5) Using a Millpore ultrafiltration concentration system, 60 L of the supernatant solution was subjected to ultrafiltration concentration and buffer replacement (20 mM PB), 6 times per batch, the volume was compressed to 8 L, and the displaced solution was lyophilized to obtain FerhLF powder.

6) Determination of iron content: The iron content of FerhLF was determined by atomic emission spectroscopy, and the iron content was 6.62 mg/g.

Example 4 FerhLF improves iron deficiency anemia in rats

Experimental principle:

According to the "Improving Nutritional Anemia Function Test Method", an experimental iron deficiency anemia model can be formed by feeding animals with low-iron feed, and then "iron-saturated recombinant human lactoferrin" is administered to observe blood cytology and blood production of animals. The influence of the chemical and other indicators can determine the effect of the test sample on improving nutritional anemia in animals.

Experimental animals:

Healthy virgin milk SD rats, male, 8-12 rats in each group during formal anemia recovery.

1.1 Establish a rat model of iron deficiency anemia

Rats were acclimated for 3-5 days in the experimental environment, fed with low-iron feed (see Table 3 for formula) and double distilled water, using stainless steel cages and food cans to avoid iron contamination during the experiment. Partial rat appendix blood was selected weekly to determine hemoglobin (HGB) content from week 3, and the body weight and HGB of all rats were determined until most animals had HGB below 100 g/L. After 4 weeks, the average hemoglobin of rats fed iron-deficient diet was lower than 90g/L, red blood cell count (RBC), hematocrit (HCT), red blood cell mean volume (MCV), and red blood cell mean hemoglobin content (MCH also significant Below normal (Table 4), the rat anemia model was established successfully.

Table 3 Low iron feed formula

Ingredient Weight percent, %

DTA treatment of casein 15 rice flour (stalk rice) 70 corn oil 5.0 AIN- ⁷⁶ M mixed salt 3.5

AIN- ⁷⁶ M Mixed Vitamin 1.2

Gelatin 5.0

DL-methionine 0.3 Table 4 Blood routine after preparation of iron deficiency anemia model in rats

Red abalone number, hematocrit, red cell average volume, red blood cell average blood red, blood reward rule, group

(g/D ( X 10 /L) (fL) protein amount ' (pg) model group 8:! soil li 4.9 soil 0.4 20.7 soil 3.0 41 ±3 16.6 soil 1.3 xEf ffl 128 + 9 6,4 + 0.3 32, 0 + 3,0 57 ± 2 224 soil 15

1.2 FerhLF for hemoglobin recovery in a rat model of iron deficiency anemia

Rats with Hb<100g/L were selected as experimental animals. They were randomly divided into low-iron control group and experimental group according to Hb level and body weight of anemia rats. All groups continued to feed low-iron feed, and low-iron control group gave corresponding For the solvent, the experimental group was given different doses of the test sample at 8:00 am every morning. The test sample was given for 30 days, and if necessary, extended to 45 days, and the body weight and various hematological indexes were measured. A conventional feed-feeding group was also established, and an anemia model was not established. During the experiment, it was administered with double distilled water (Table 5).

Table 5 Experimental animal grouping and agent t selection

Number group feeding water

1 normal control group (n=10) normal feed double distilled water double distilled water

2 anemia model group (n=10) iron deficiency feed double steamed water double distilled water

3 positive control group (n=10) iron deficiency feed double distilled water 2mg/kg/d FeS0 ₄

4 FerhLF high dose group (n=10) iron deficiency feed double distilled water containing 2mg/kg/d (Fe ³⁺ ) and lOOmgrhLF

5 FerhLF medium dose group (n=10) iron deficiency feed double distilled water containing lmgkg/d (Fe ³⁺ ) and 50mgrhLF

6 FerhLF low dose group (n=10) Iron deficiency feed double distilled water containing 0.5mgkg/d (Fe ³⁺ ) and 25mg rhLF

7 rhLF+Fe ²⁺ (n=10) iron-deficient feed double distilled water containing Fe ²⁺ 2mgkg/d and lOOmgrhLF

8 BLF+Fe ²⁺ (n=10) Iron-deficient feed double distilled water contains Fe ²⁺ 2mgkg/d and lOOmgbLF

1.3 Hematology index test

The blood of each group of animals was analyzed by MEK-6318k automatic blood cell biochemical analyzer. The hemoglobin concentration, the number of red blood cells, the hematocrit, the average hemoglobin content of red blood cells, the average red blood cell volume, the liver iron, the spleen iron and other indicators to evaluate the effect of iron-saturated lactoferrin on iron deficiency anemia are shown in Fig. 6 and Fig. 7. (Different lowercase letters identify significant differences between groups).

After the 28-day feeding experiment, the hemoglobin concentration of the rats in each experimental group recovered significantly and increased to more than 100 g/L compared with the model control group. The recovery of hemoglobin concentration in the FerhLF (high) and FerhLF (middle) groups was faster and more effective than the other groups. The hemoglobin concentration in the FerhLF (high) group had no significant difference from the hemoglobin concentration in the normal control group, indicating that the FerhLF (high) group had returned to normal. At the same iron content, the hemoglobin recovery ability of the positive control (FeS0 ₄ ) group was slower and the effect was inferior to FerhLF (high). The concentration of hemoglobin in FerhLF (low), rhLF+Fe ²⁺ and bLF+Fe ²⁺ groups was not significantly different from that in FeS04 group, indicating that the addition of Fe ^{2+ to} lactoferrin did not increase iron absorption better. This may be due to lactoferrin. Proteins do not bind to Fe ²⁺ and cannot be transported into the blood by the mechanism of lactoferrin. In addition, FerhLF (high) can significantly increase the number of red blood cells, hematocrit, red blood cell average hemoglobin, red blood cell average capacity, liver iron and spleen iron levels and significantly better than other groups to further confirm that FerhLF (high) can be obvious Improve iron deficiency anemia.

According to the "Method for Improving the Function of Nutritional Anemia", the Hb concentration of the test group was increased by more than 10 g/L compared with the model control group, and it was judged that the test substance had an elevated hemoglobin effect. If the concentration of Hb in the test group is close to the normal level, it indicates that the test substance has a strong effect of increasing the Hb concentration. Therefore, both medium and high doses of FerhLF have a strong effect of increasing the concentration of Hb. FerhLF (low), rhLF+Fe ²⁺ and bLF+Fe ²⁺ and FeS0 ₄ groups also have elevated hemoglobin effects.

The above experiments show that FerhLF has the most prominent effect in improving iron deficiency anemia, and the effect of iron preparation supplemented with rhLF can also improve iron deficiency anemia. The use of ALF as a supplement for iron supplements and blood medicines not only enhances the effect of iron supplementation, but also enhances the body's immunity. Therefore, the development of rhLF will have broad commercial prospects and should Although the present invention has been described in detail above with reference to the preferred embodiments of the present invention, it will be apparent to those skilled in the art. Therefore, such modifications or improvements made without departing from the spirit of the invention are intended to be within the scope of the invention. Industrial Applicability The present invention provides a novel iron supplement preparation product--iron saturated recombinant human lactoferrin

(FeriiLF). The use of this new iron supplement greatly enhances the effect of improving iron deficiency anemia, significantly improves the hemoglobin concentration of iron-deficient anemia rats, and has no obvious adverse reactions, safe use, obvious effect, and simple preparation method. .

Sequence table

<110> Beijing Jifulin Biotechnology Co., Ltd.

<120> A product for improving iron deficiency anemia and preparation method thereof

<130> KHP13312210. 3

<160> 1

<170> Patentln version 3. 5

<210> 1

<211> 711

<212> PRT

<213> Artificial sequence

<400> 1

Met Lys Leu Val Phe Leu Val Leu Leu Phe Leu Gly Ala Leu Gly Leu 1 5 10 15

Cys Leu Ala Gly Arg Arg Arg Arg Ser Val Gin Trp Cys Thr Val Ser

20 25 30

Gin Pro Glu Ala Thr Lys Cys Phe Gin Trp Gin Arg Asn Met Arg Arg 35 40 45

Val Arg Gly Pro Pro Val Ser Cys lie Lys Arg Asp Ser Pro lie Gin 50 55 60

Cys lie Gin Ala lie Ala Glu Asn Arg Ala Asp Ala Val Thr Leu Asp 65 70 75 80

Gly Gly Phe lie Tyr Glu Ala Gly Leu Ala Pro Tyr Lys Leu Arg Pro

85 90 95

Val Ala Ala Glu Val Tyr Gly Thr Glu Arg Gin Pro Arg Thr Hi s Tyr

100 105 110

Tyr Ala Val Ala Val Val Lys Lys Gly Gly Ser Phe Gin Leu Asn Glu 115 120 125

Leu Gin Gly Leu Lys Ser Cys Hi s Thr Gly Leu Arg Arg Thr Ala Gly

130 135 140

Trp Asn Val Pro lie Gly Thr Leu Arg Pro Phe Leu Asn Trp Thr Gly 145 150 155 160

Pro Pro Glu Pro lie Glu Ala Ala Val Ala Arg Phe Phe Ser Ala Ser

165 170 175 Cys Val Pro Gly Ala Asp Lys Gly Gin Phe Pro Asn Leu Cys Arg Leu

180 185 190 Cys Ala Gly Thr Gly Glu Asn Lys Cys Ala Phe Ser Ser Gin Glu Pro

195 200 205

>

Se Seerr li

Claims

Claim

A product for ameliorating iron deficiency anemia characterized by being iron-saturated recombinant human lactoferrin.

2. A method of improving the product of iron deficiency anemia, according to claim 1, wherein the recombinant human lactoferrin with Fe ³⁺ in chelated NaHC0 ₃ or KHC0 ₃ solution; wherein said recombinant human milk The amino acid sequence of ferritin is shown as Seq ID No. 1.

3. The method recited in claim 2, characterized in that it is a recombinant human lactoferrin powder was dissolved in aqueous solution is formulated, and then the solution was mixed with a solution of FeCl ₃ or KHC0 ₃ and NaHC0 ₃ solution, stirred An iron-saturated recombinant human lactoferrin solution is prepared, followed by desalting and lyophilization to obtain iron-saturated recombinant human lactoferrin powder.

4. The method according to claim 3, wherein the preparation of the recombinant human lactoferrin powder comprises the following steps:

1) Preparation of transgenic cattle, including the following steps:

Ii) mixing hLF BAC DNA with the double-marker selection vector pEGFP-NEO or the single-marker selection vector pNEO, introducing into the nucleus of the livestock somatic cell, and transfecting the cells to obtain transgenic cells transformed into hLF BAC DNA;

Iii) performing somatic cell cloning as a nuclear donor to obtain transgenic cattle transfected with hLF BAC DNA;

4) The eluate is subjected to ultrafiltration concentration and freeze-drying to obtain a recombinant human lactoferrin powder.

5. The method according to claim 4, wherein in step 3), pre-elution is carried out with 20 M PBS containing 0.4 M NaCl, pH 6.5 to remove the heteroprotein, and then 20 mM containing 1 M NaCl, pH 6.5. The elution was carried out with PBS, and the eluate having the same peak elution peak of the same rhLF activity was collected.

The method according to claim 4 or 5, wherein the ultrafiltration in the step 4) is specifically: the eluate having the peak elution peak of the same riiLF activity obtained in the step 3) is peristaltic The pump was pumped into an ultrafiltration membrane for ultrafiltration concentration, and 20 mM phosphate buffer of pH 6.0 was added and circulated 7 times until the concentrate outlet conductivity was lower than lmS/cm, and the solution was further concentrated.