Method for screening snake venom nerve growth factor (vNGF) active polypeptides

A technology of nerve growth factor and active polypeptide, applied in nerve growth factor, growth factor/inducing factor, biochemical equipment and method, etc., can solve the problems of difficult to obtain vNGF raw materials, unknown activity maintenance and effect, complicated purification process, etc.

Inactive Publication Date: 2012-12-12
SHENYANG MEDICAL COLLEGE
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Problems solved by technology

At present, a lot of basic research on vNGF has been carried out at home and abroad, but there are few researches and practices on clinical application, mainly due to the following reasons: due to the dual effects of NGF on promoting growth and inducing apoptosis, the curat

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  • Method for screening snake venom nerve growth factor (vNGF) active polypeptides
  • Method for screening snake venom nerve growth factor (vNGF) active polypeptides

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Embodiment 1

[0019] The method for screening active polypeptides of snake venom nerve growth factor according to the present invention, by analyzing the original sequence of 28 registered vNGF reference sequences, and comparing the 11 registered vNGF mRNA sequences, it is found that vNGF has a high degree of homology, Between 93% and 99%, the variation points are mainly concentrated at 441, 541 and 555. The amino acid and nucleotide homology analysis of 11 vNGF mRNA sequences were carried out, and based on this , using vNGF as a template to analyze the breakpoints of functional regions, based on this, the screening steps are as follows:

[0020] 1. Construction of fusion protein: It is proposed to use vNGF as a template to construct two fragment sequences of different lengths by PCR primer method, which are: v said two fragment sequences, respectively: vNGF F1 fragment, corresponding to 20-388 genes site, 369bp in total; vNGF F2 fragment, corresponding to 389-751 gene site, 363bp in total,...

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Abstract

The invention relates to the biotechnological field, and concretely relates to a method for screening snake vNGF active polypeptides. The method comprises the following steps: constructing fusion proteins with sputa NGF (vNGF) as a template; constructing a plasmid expression system through pBluscriptII SK+ and pQE30; carrying out separation, identification and purification through a 6X histidine recovery purification technology to form a stimulant stock solution of the vNGF active polypeptides; and verifying the biological activity of prokaryotic expression vNGF beta fragments. According to the invention, the sputa NGF is adopted as the template to carry out the breakpoint analysis of a functional zone, a design scheme that different polypeptide stimulants with two histone labeled fusion proteins as research models are constructed through gene recombination with the sputa NGF as the template is provided, and the activities of the vNGF polypeptides are researched to screen the vNGF active polypeptides and provide molecular biological supports for the basic researches of the sputa NFG application.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a screening method for snake venom nerve growth factor active polypeptides. Background technique [0002] Nerve Growth Factor (NGF) has a wide range of clinical application prospects, mainly in: promoting the development and differentiation of neurons, reducing neuron damage in pathological conditions, promoting neuron repair and regeneration; inducing vascular endothelial cells, promoting Angiogenesis; increase the activity of various hydrolytic enzymes in cell lysosomes, enhance the phagocytosis of macrophages, and induce cell apoptosis, etc. The homology between Venom NGF (vNGF) and human NGF is between 66% and 80%, and it has a reliable positive regulatory effect on human nerve cells. The mechanism of action is similar, and there are similar peptide stimulation and activation fields. . At present, a lot of basic research on vNGF has been carried out at home and abroad, but ther...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/63C07K14/48
Inventor 徐军吴鹏祁荣张忠
Owner SHENYANG MEDICAL COLLEGE
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