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Method of extracting lacto ferritin from transgene lactoferritin paddy rice

A lactoferrin and transgenic technology, which is applied to the preparation method of peptides, chemical instruments and methods, plant peptides, etc., can solve the problems of high NaCl content in products, affect the application of lactoferrin, and not meet actual needs, etc., to achieve enhanced functions Increased effect on food performance and feed quality, easy and fast extraction, cost savings

Inactive Publication Date: 2006-08-30
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But such a concentration of NaCl will affect the practical application of lactoferrin
For example, using lactoferrin with a high concentration of NaCl directly as a food additive or as a raw material to prepare various beverages will cause high NaCl content in the product, which does not meet actual needs
And adopt desalination step, promptly increase cost and may cause the loss of part output (lactoferrin)

Method used

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  • Method of extracting lacto ferritin from transgene lactoferritin paddy rice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1, the acquisition of the transgenic rice seed expressing lactoferrin:

[0029] The human lactoferrin gene (hLF) was synthesized by Shanghai Sangong, and the promoter was the rice Gt promoter. Gt and hLF were cloned in plasmid pCAM1300 to obtain pCAM1300-GT-hLF. pCAM1300-GT-hLF was transformed into rice varieties Xiushui 11 and Taipei 309 by Agrobacterium. hLF is controlled by the promoter Gt1 and can be expressed in rice seeds. Among the obtained 40 transgenic lines (2801-2840), two of them, lactoferrin 2824 and lactoferrin 2831, can express a relatively high level of lactoferrin and are used as materials of the present invention .

[0030] Of course, all transgenic lactoferrin rice seeds can be used as materials in the present invention.

Embodiment 2

[0031] Embodiment 2, the extraction of human lactoferrin in rice seeds, carry out the following steps successively: (the water referred to in this embodiment refers to the water not containing NaCl)

[0032] Method A:

[0033] Step 1. Add water or buffer solution to the rice seeds obtained in Example 1, mix and pulverize to form a wet powder; or directly pulverize the rice seeds obtained in Example 1, add water or buffer solution after pulverization and mix to form a wet powder. Powder; the buffer solution used is a buffer solution with a concentration of NaCl<0.1mol / L and a pH value of 6-7.5, and the temperature of the above pulverization process is kept below 70°C. The feed ratio of water or buffer solution to the rice seeds is 1 milliliter / gram.

[0034] Step 2. Mix and shake the above wet powder at a frequency of 250 rpm for 2-3 hours, centrifuge at 5000g for 10 minutes, and separate the precipitate and supernatant solution.

[0035] Step 3. Collect the precipitate obtai...

Embodiment 3

[0042] Embodiment 3, lactoferrin content and purity analysis:

[0043] The content and purity of the extracted lactoferrin can be analyzed by SDS-PAG. According to the general method of SDS-PAGE (Osper, F Brent, R Kingston, et al. Hardcover Molecular Biology Experiment Guide [M]. Beijing: Science Press, 1998, 334-338). The lactoferrin sample extracted in Example 2 was analyzed. Western analysis showed that the main protein band on the SDS-PAGE gel was lactoferrin. Densitometric scanning analysis found that it accounted for 40-60% of the total protein content, specifically as figure 1 Shown:

[0044] When selecting lactoferrin 2824 to carry out embodiment 2, 1 represents the lactoferrin extracted from lactoferrin rice 2824;

[0045] When selecting lactoferrin 2831 to carry out embodiment 2, 2 represents the lactoferrin extracted from lactoferrin rice 2831;

[0046] 3, 4, and 5 represent negative controls;

[0047] When selecting lactoferrin 2803 to carry out embodiment 2,...

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Abstract

The present invention discloses a method for extracting lactoferrin from transgenic lactoferrin rice. Said method includes the following steps: 1), adding water or buffer solution in transgenic lactoferrin rice seed, mixing them, pulverizing to form wet powder; or directly pulverizing transgenic lactoferrin rice seed, then adding water or buffer solution to form wet powder; the buffer solution contains NaCl, whose concentration is less than 0.1 mol / L and whose pH value is 6-9, the temperature of the above-mentioned pulverization process is below 70deg.C, the material-adding ratio of water or buffer solution and transgenic lactoferrin rice seed is 1-10ml / g; and 2), mixing and shaking the above-mentioned wet powder, then making centrifugal separation for 10min, and separating precipitate and supernatant.

Description

technical field [0001] The invention relates to a method for extracting lactoferrin from transgenic lactoferrin rice. Background technique [0002] Lactoferrin (LF) is one of the most important active substances in breast milk. It is a protein with various biological functions (B. Lo¨nnerdal, S. Iyer, Lactoferrin (1995) Molecular structure and biological function, Annu. Rev. Nutr. 15: 93-110). It not only participates in the transport of iron, but also has functions such as antimicrobial, antioxidation, anticancer, and immune system regulation. It is considered to be a new type of antibacterial and anticancer drug and a food and feed additive with great development potential. [0003] LF can promote the sterilization and phagocytosis of neutrophils or macrophages, and regulate the activity of NK cells and the proliferation of lymphocytes and neutrophils. Under normal circumstances, serum contains 0.30-0.5 μg / mL of LF; but once infection occurs, LF will be released from act...

Claims

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Application Information

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IPC IPC(8): C07K1/14C07K14/415
Inventor 沈志成
Owner ZHEJIANG UNIV
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