Preparation method of recombined human nerve growth factor based on insect baculovirus expression system

A growth factor and baculovirus technology, applied in the field of genes, can solve the problems of antigen reaction and life-threatening, and achieve the effect of high biological safety

Inactive Publication Date: 2010-12-08
JIANGHAN UNIVERSITY +1
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Problems solved by technology

Although the structure of mouse-derived NGF in this type of product is highly homologous to human NGF, and the biological effects have no obvious inter-species specificity, the mouse-derived protein products have both chemical and spatial structures. Different from human-derived protein products, and for humans, animal-derived proteins often have high immunogenicity, which is easy to cause antigenic reactions in the human body, and even life-threatening in severe cases

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  • Preparation method of recombined human nerve growth factor based on insect baculovirus expression system
  • Preparation method of recombined human nerve growth factor based on insect baculovirus expression system
  • Preparation method of recombined human nerve growth factor based on insect baculovirus expression system

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Embodiment Construction

[0039] The test materials used in the present invention are all commercially available products unless otherwise specified, and the ingredients and preparation methods of various reagents and culture media can refer to the operation in the routine experiment manual.

[0040] Construction and expression of recombinant human nerve growth factor insect baculovirus expression system:

[0041] 1. Acquisition of rhβ-NGF gene:

[0042] 1.1 Extraction of total RNA from human blood leukocytes

[0043] The method is carried out according to the conventional extraction method

[0044] 1) take sodium citrate anticoagulant blood, and centrifuge to precipitate white blood cells; 2) to about 7 × 10 6 Add 1 mL of Trizol to each cell EP tube, mix gently, let stand for 5 minutes, add 0.2 mL of chloroform, shake vigorously for 15s~30s, let stand on ice for 3 minutes, 4°C, 12000 rpm, 15 minutes; 3) Aspirate the upper layer of water Add an equal volume of isopropanol to another EP tube, invert ...

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Abstract

The invention discloses a preparation method of a recombined human nerve growth factor based on an insect baculovirus expression system. The method comprises the following steps: connecting a gene of a human nerve growth factor to an expression carrier Bacmid to construct a recombined expression carrier for expressing the human nerve growth factor; transfecting the recombined expression carrier to an insect cell to obtain recombined virosomes, thereby constructing the insect expression system for expressing the human nerve growth factor; and culturing the insect cells, and infecting with recombined viruses to obtain a great deal of recombined human nerve growth factors. The method can be used for industrial production of recombined human nerve growth factors. The expression system and the method can be used for preparing recombined human nerve growth factors of which the purity is greater than 98% and the biological specific activity is higher than 500000 AU / mg.

Description

technical field [0001] The invention belongs to gene technology, in particular to a method for preparing recombinant human nerve growth factor. Background technique [0002] Nerve Growth Factor (NGF) is the earliest discovered and most typical neurotrophic factor, and it is also one of the most important biologically active molecules in the nervous system. Both the expression of NGF and functional properties have an important regulatory effect, which can affect the survival and differentiation of certain neurons in the peripheral and central nervous systems, and play an important role in protecting the normal function of the nervous system. At present, NGF has been developed as a therapeutic agent for peripheral nerve injury. Drugs are used clinically, and are widely used in neurological diseases such as nerve injury, hemiplegia, stroke, craniocerebral injury, and cerebral palsy in children. [0003] However, the content of natural NGF is extremely low, which brings conside...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/866C12N7/01C07K14/48
Inventor 李佳楠汤华东
Owner JIANGHAN UNIVERSITY
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