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Method for preparing gelsolin from Dendrorhynchus zhejiangensis

A technology of gelsolin and chitosan, which is applied in the field of preparing gelsolin, can solve rare problems and the like, achieves the effect of eliminating renaturation steps, maintaining natural activity, and being easy to popularize

Inactive Publication Date: 2012-01-18
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Nematode animals have strong regeneration ability, unique fat metabolism mode, and adaptability, but researches on various aspects of it are rare at home and abroad. Among them, the anti-tumor effect of gelsolin in nemophila and the preparation of the protein No purification method has been reported

Method used

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Embodiment

[0017] The method for preparing gelsolin from Nematodes zhejiang, comprises the following steps:

[0018]a. RNA extraction: Clean C. zhejiangensis to obtain C. zhejiangensis body wall, quickly freeze the C. zhejiangensis body wall in liquid nitrogen, grind into powder, and then extract total RNA with RNAiso plus RNA extraction kit, The specific method is operated according to the kit instructions: after the body wall tissue is ground into powder, weigh 5 grams, add 100ml of RNAiso lysate, homogenate, let stand at room temperature for 5 minutes, and centrifuge at 4°C for 5 minutes at 12,000 rpm / min. Take the first supernatant, add chloroform, the volume ratio of the first supernatant to chloroform is 5:1, after the solution is fully emulsified, let stand at room temperature for 5 minutes, centrifuge at 12000 rpm / min at 4°C for 15 minutes, and take Add isopropanol to the second supernatant, the volume ratio of the second supernatant to isopropanol is 1:1, let stand at room tempe...

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Abstract

The invention discloses a method for preparing gelsolin from Dendrorhynchus zhejiangensis, which is implemented by carrying out RNA (ribose nucleic acid) extraction, cDNA (complementary deoxyribonucleic acid) synthesis, enzyme digestion, connection and transformation, recombinant plasmid transformation, recombinant protein inducible expression, and recombinant protein separation and purification on the Dendrorhynchus zhejiangensis body wall, wherein after the cDNA is synthesized, a proper double-digestion primer is used for carrying out PCR (polymerase chain reaction) amplification, thereby obtaining a PCR product with the target segment; primers and a proper expression vector are subjected to double-digestion and connected, and are transformed onto a proper host; and the separation and purification are carried out under non-metamorphic conditions by an improved operation method using a Ni-NTAHis.BindResin kit, thereby saving the complex renaturation step, and being beneficial to keeping the natural activity of the recombinant protein and obtaining recombinant gelsolin with higher purity. The recombinant gelsolin prepared by the method disclosed by the invention can exist in a soluble form, and has an obvious inhibiting action on tumor cells; the concentration of the recombinant gelsolin is about 85%; and the method disclosed by the invention is scientific and reasonable, and is simple to implement and easy to popularize.

Description

technical field [0001] The invention relates to a preparation method of gelsolin, in particular to a method for preparing gelsolin from Nematode zhejiangensis. Background technique [0002] Gelsolin was first discovered in macrophages of rabbit lung, and it is an important actin-binding protein, which is involved in life activities such as cell movement and programmed cell death. At present, the research on gelsolin mainly focuses on the structure, mechanism of action and the impact on certain diseases; existing studies have shown that gelsolin has the effect of treating infection and rheumatic inflammation, and also has obvious tumor inhibitory effect. The expression in the tumor is significantly reduced, and it is closely related to the occurrence and development of malignant tumors. It is considered as a candidate tumor suppressor gene. [0003] Dendrorhynchus zhejiangensis belongs to Nematinea, Heteronemertea, Lineidae, and Dendrorhynchus. It was first found in Fenghua ...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/63C07K14/435
Inventor 李晔陈蕾苏秀榕李太武李振周君李成华
Owner NINGBO UNIV
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