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Cereus sinensis verrill soluble cell peptide CCT-I and separating and purifying method and application thereof

A technology of CCT-I and separation method, which is applied in the field of CCT-I, a sea anemone cytolytic polypeptide and its separation and purification, can solve the problem of the difficulty of separation and purification of sea anemone toxin, the separation and purification of cytolytic toxin, and the research on its composition and properties. There have been no reports, activity reduction or even loss and other problems

Active Publication Date: 2013-11-27
张朝晖
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

my country's coastal sea anemones are rich in resources, especially with the successful artificial breeding and breeding of Chinese fairy shadow sea anemones by the Jiangsu Provincial Institute of Marine Fisheries, which makes it possible to use sea anemone resources for industrialized comprehensive development. Chinese fairy shadow sea anemones ( Cereus sinensis Verrill) belongs to the genus Cereus sinensis Verrill, which is an edible and poisonous sea anemone widely distributed in the coast of my country. The separation, purification, composition and properties of its cytolytic toxin have not been reported yet.
[0004] The separation and purification of sea anemone toxin is relatively difficult, the main reasons are as follows: first, sea anemone crude toxin contains many kinds of protein components, and some of the properties between proteins (such as: molecular weight, isoelectric point, hydrophobicity etc.) are very close; second, sea anemone toxins are mostly protein substances, which are easily affected by changes in external environmental factors and cause their activity to be reduced or even lost; third, different species of sea anemones, due to their different growth environments, cause There are also large differences in their physiological structure and the toxin components and properties contained in the body, which also makes it difficult to directly imitate the separation and purification of toxins from different species of sea anemones, and can only be obtained through tedious experiments.

Method used

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  • Cereus sinensis verrill soluble cell peptide CCT-I and separating and purifying method and application thereof
  • Cereus sinensis verrill soluble cell peptide CCT-I and separating and purifying method and application thereof
  • Cereus sinensis verrill soluble cell peptide CCT-I and separating and purifying method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Embodiment 1 Separation of the Cytolytic Polypeptide of Sea Anemone chinensis

[0035] In the first step, the sea anemone is crudely prepared. Cut off the tentacles of the live Anemone chinensis, and quickly freeze them in a -80°C refrigerator. Before preparation, take out the tentacles that have been frozen and thawed twice, and after thawing at room temperature, weigh 20g accurately and cut them into pieces with scissors. After fully homogenizing with a glass homogenizer, add a certain amount of ultrapure water according to the material-to-liquid ratio of 1:5, seal it with a parafilm, and place it in a refrigerator at 4°C for 2 hours. During this period, stir once every 15 minutes, pay attention to stirring gently so as not to generate a lot of foam. Then centrifuge at 4°C and 10000r / min for 1 hour, take out the supernatant, and then centrifuge for 1 hour under the same condition, and the obtained supernatant is the sea anemone crude venom. The protein concentration...

Embodiment 2

[0039] The detection of embodiment 2 hemolytic activity detection

[0040] The first step is blood sample preparation. Healthy adult SD rats were taken, blood was collected from the heart, the blood was stored in an anticoagulant tube, and stored at 4°C for future use.

[0041] In the second step, 2% erythrocyte suspension of SD rats was prepared. A certain amount of SD rat blood was taken out from the anticoagulant tube, and 10 mmol·L -1 Phosphate buffered saline (PBS pH=7.0 containing 0.15mol·L -1 Sodium chloride) washing, 1500r·min at 4°C -1 Centrifuge for 5 minutes, discard the supernatant, and repeat 4 to 5 times until the supernatant is clear and transparent without red. The red blood cells are diluted with PBS to a 2% red blood cell suspension, and stored at 4°C for later use.

[0042] The third step is the detection of hemolytic activity. Add 200 μL of CCT-I solutions of different concentrations to 200 μL of 2% erythrocyte suspension, then dilute to 1 mL with PBS,...

Embodiment 3

[0046] Example 3 Identification of Cytolytic Polypeptide CCT-I of Sea Anemone chinensis

[0047] The first step is SDS-PAGE electrophoresis to check the purity of CCT-I and estimate the molecular weight. The sample was mixed with 5× loading buffer (v:v=4:1) and boiled at 95°C for 5 minutes, then separated with 5% stacking gel and 12% separating gel, and finally fixed and stained with Coomassie brilliant blue R-250, Molecular weights are determined by comparison to standard proteins. The standard proteins contained in the standard protein marker are: phosphatase b 97.2kDa, bovine serum albumin 66.4kDa, ovalbumin 44.3kDa, carbonic anhydrase 29.0kDa, trypsin inhibitor 20.1kDa and lysozyme 14.3kDa. The results show that CCT-I mainly contains two protein bands, the purity is high, and the molecular weight is about 80kDa ( Figure 4 ).

[0048] In the second step, the purity of CCT-I is checked by HPLC. CCT-I was dissolved in 10% acetonitrile (acetonitrile: 0.1% TFA = 1:9), filt...

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Abstract

The invention discloses a cereus sinensis verrill soluble cell peptide CCT-I and a separating and purifying method and an application thereof. The separating and purifying method comprises the following steps: after cereus sinensis verrill tentacle crude toxin is salted out and fractionally precipitated with ammonium sulfate, active sites are separated and purified through Sephadex G-100 and superdex 75 gel filtration chromatography columns in sequence, and the soluble cell peptide is obtained through separation from a cereus sinensis verrill tentacle at the first time and is finally authenticated as the novel cereus sinensis verrill soluble cell peptide that has not been reported through SDS-PAGE and LC-MS, wherein two peptides (alpha and beta) in the novel cereus sinensis verrill soluble cell peptide are similar in molecular weight of about 80 kDa, and the HPLC test shows that the two peptides are high in purify and similar in polarity. The half soluble cell rate is tested to be 5.75 micrograms*mL<-1>, and the MTT process test displays that the CCT-I has strong antitumor activity in vitro, and can be used for preparing tumor-treating drug.

Description

technical field [0001] The invention belongs to the technical field of natural medicines in traditional Chinese medicine, and relates to the cytolytic polypeptide CCT-I of Chinese fairy shadow sea anemone and its separation and purification method and application. Background technique [0002] Statistics show that since the 1980s in my country, the incidence of malignant tumors has shown an obvious upward trend, and the incidence of common tumors has increased at a rate of 5-10%. There are 1.3 million patients in China, and the current number of cancer patients is 5.4 million. The incidence rates of lung cancer and breast cancer are the highest in large and medium-sized cities; the incidence rates of gastric cancer and esophageal cancer are the highest in rural areas. It can be seen that the market potential of antineoplastic drugs in my country is huge. The relevant research work on the treatment of malignant tumors is making continuous progress. There are 49 commonly used...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K1/36C07K1/30C07K1/16A61K38/17A61P35/00
Inventor 张朝晖秦松史文军吴建平崔红利万夕和杨海萍王李宝
Owner 张朝晖
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