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Product for improving iron deficiency anemia and preparation method thereof

A technology for iron-deficiency anemia and products, applied in peptide preparation methods, chemical instruments and methods, medical preparations containing active ingredients, etc., can solve the problem that the stability and absorption rate of bovine lactoferrin is not as good as human lactoferrin, expression Inappropriate system expression and post-translational modification, restrictions on the application of recombinant proteins, etc., to achieve rapid recovery, simple preparation methods, and increased hemoglobin concentration

Inactive Publication Date: 2012-11-07
BEIJING GEFUCURE BIOTECHNOLOGY LIMITED COMPANY
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0009] However, the large-scale development of iron supplementation preparations using lactoferrin is still very limited. This is because bovine lactoferrin (bLF) is currently on the market. The content of bovine lactoferrin in milk is extremely low, and the production cost is extremely high. Moreover, as a heterologous protein, bovine lactoferrin is not as stable and absorbable as human lactoferrin in the human body. Therefore, the development and production of human lactoferrin in large quantities has always been the focus and difficulty of people's research.
At present, many attempts have been made to mass-produce recombinant human lactoferrin using 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.

Method used

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  • Product for improving iron deficiency anemia and preparation method thereof
  • Product for improving iron deficiency anemia and preparation method thereof
  • Product for improving iron deficiency anemia and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Preparation of transgenic cattle expressing recombinant human lactoferrin

[0056] According to the method disclosed in CN1873001 (invention name: production method of transgenic cloning large-scale livestock with transgenic human lactoferrin gene), the recombinant human lactoferrin transgenic cattle were prepared. The operation steps are as follows: (1) Use hLF BAC DNA containing the complete human lactoferrin gene as a mammary gland-specific expression vector; (2) Mix hLF BAC DNA with double-marker selection vector pEGFP-NEO or single-marker selection vector pNEO in proportion, Introduce into somatic cell nuclei of livestock, perform cell transfection, and obtain transgenic cells transformed with hLF BAC DNA; (3) cells are used as nuclear donors for somatic cell cloning, and obtain transgenic cattle transgenic with hLF BAC DNA.

[0057] The amino acid sequence of the recombinant human lactoferrin (rhLF) is shown in Seq ID No.1.

Embodiment 2

[0058] Example 2 Pilot purification and quality control of recombinant human lactoferrin

[0059] 1.1 Preparation of rhLF milk skim milk

[0060] The rhLF milk produced by the transgenic cow in Example 1 was heated to 40-45° C., and the milk was discharged into a milk fat separator to adjust the rotation speed to 65-75 rpm. Each batch of milk is skimmed twice consecutively to fully remove the fat.

[0061] 1.2 Sterilization by ceramic membrane filtration

[0062] Using ceramic membrane (pore size 1.4μm) for ultrafiltration of skim milk, because the size of bacteria is about 0-5μm, the effect of cold sterilization can be achieved.

[0063] The experimental operation is shown in Table 1.

[0064] Table 1 Operation steps of sterilization by ceramic membrane filtration

[0065]

[0066]

[0067] 1.3 Isolation and purification of rhLF from skim milk

[0068] 1.3.1 Buffer preparation

[0069] (1) Preparation of PBS stock solution

[0070] Stock solution 1: Na 2 HPO 4 ...

Embodiment 3

[0102] Example 3 Preparation of iron-saturated recombinant human lactoferrin (FerhLF)

[0103] The specific experimental steps are as follows:

[0104] 1) Dissolve 120 g of the purified and freeze-dried rhLF powder in distilled water to a volume of 12 L to prepare a 1% rhLF solution.

[0105] 2) Prepare 1.4% FeCl respectively 3 Solution 28L and 10% NaHCO 3 Solution 28L.

[0106] 3) Mix 12L 1% rhLF solution with 28L 1.4% FeCl 3 ,28L10%NaHCO 3 Mix, stir constantly to mix thoroughly, and let stand at room temperature for 1 hour.

[0107] 4) Centrifuge the reacted solution to collect the supernatant, which turns brownish red.

[0108] 5) Using Millpore's ultrafiltration concentration system, 60L supernatant solution was subjected to ultrafiltration concentration and buffer replacement (20mM PB), 6 cycles per batch, the volume was compressed to 8L and the solution after replacement was lyophilized to obtain FerhLF powder.

[0109] 6) Determination of iron content: the iron c...

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Abstract

The invention provides a method for purifying a large amount of recombinant human lactoferrin from cloned transgenic milk, and also provides a development of a product for improving iron deficiency anemia by using the purified recombinant human lactoferrin as a raw material, in particular provides a novel iron preparation-iron-saturated recombinant human lactoferrin (FerhLF). By utilizing the novel iron preparation, the effect of improving the iron deficiency anemia is greatly enhanced, the hemoglobin concentration of rates suffered with the iron deficiency anemia is obviously improved, the obvious adverse reaction does not occur, the use is safe, the effect is obvious and the preparation method is simple.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a product for improving iron deficiency anemia and a preparation method thereof. Background technique [0002] Iron is an important component of hemoglobin, 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 will weaken the immune system, accompanied by symptoms such as fatigue, irritability, irritability, and memory loss. More importantly, iron deficiency in the body can cause iron deficiency anemia (Iron deficiency anemia, IDA). Iron deficiency anemia (IDA) is one of the most common nutritional deficiency diseases worldwide. About 1 / 3 of the world's 5 billion people are anemic. And IDA or iron malnutrition accounted for about 500 million people. The incidence of iron deficiency (ID) in developing countries is four times that in developed countries. my country is one of the cou...

Claims

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Application Information

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IPC IPC(8): C07K14/79C07K1/36C07K1/34C07K1/18A61P7/06
CPCC07K14/79C07K1/36C07K1/34A61K38/40C07K1/18A61P7/06
Inventor 于添
Owner BEIJING GEFUCURE BIOTECHNOLOGY LIMITED COMPANY
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