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A kind of preparation method of snake venom thrombin-like protein

A thrombin-like and snake venom-like technology is applied in the field of preparation of snake venom-like thrombin-like proteins, which can solve the problems of difficult protein biochemical properties and the like

Inactive Publication Date: 2011-12-28
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Apart from the tedious protein purification, it is quite difficult to determine the biochemical properties of the resulting protein

Method used

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  • A kind of preparation method of snake venom thrombin-like protein
  • A kind of preparation method of snake venom thrombin-like protein
  • A kind of preparation method of snake venom thrombin-like protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Construction of a phage display library of Agkistrodon akistrodon venom glands

[0046] The construction of the phage display library of Agkistrodon venom glands exemplarily adopts the following technical scheme:

[0047] 1. Extraction of RNA from the venom glands of Agkistrodon halys

[0048] Carefully separate the venom glands of the fresh Agkistrodon akistrosus, peel off the muscle tissue, weigh (about 0.18-0.20 mg / piece), and store at -80°C. Place the venom gland tissue of Agkistrodon akistrodon in a mortar pre-cooled with liquid nitrogen, and carefully grind it to powder. Then use the Trizol method (CHOMCZYNSKI P.A reagent for the single2step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples [J]. Bio Techniques, 1993, 15: 53225371) to extract tissue RNA. Take 2 μl RNA, dilute it 25 times, measure the OD value at 260nm and 280nm wavelength under the ultraviolet spectrophotometer, and calculate the concentration of the total RN...

Embodiment 2

[0080] Example 2: Analysis of Thrombin Gene Sequence and Protein Structure of Agkistrodon Agkistrodon Venom

[0081] 1. Acquisition of thrombin-like gene from Agkistrodon akistros venom.

[0082] (1) According to the aforementioned method, the phage liquid after three rounds of screening was 1:10 with LB liquid medium 8 After dilution, take 100 μl of phage liquid, 250 μl of freshly shaken BLT5403 bacteria (Novagen, Germany), 3ml of top layer agar kept at 55°C, mix well and immediately pour into a plate covered with LB, after cooling, place in a 37°C incubator Incubate for 2 hours. A single phage plaque can be seen.

[0083] (2) Scrape a single phage plaque from the LB, put it into an EP tube, and add 100 μl of 10 mM EDTA (disodium ethylenediaminetetraacetic acid), pH 8.0 solution.

[0084] (3) Vortex the centrifuge tube and heat at 65°C for 10 minutes.

[0085] (4) Cool to room temperature and centrifuge at 14000 g for 3 minutes.

[0086] (5) Aspirate the supernatant for PC...

Embodiment 3

[0094] Embodiment 3: construction of prokaryotic expression plasmid of Agkistrodon akistrodon venom thrombin-like gene (see Figure 7 )

[0095] A. Construction of expression plasmid pET-32(a)-TLE

[0096] Design and synthesis of primers (synthesized by Invitrogen):

[0097] TLE(up): 5'-GGAATTCATGGTGCTGATCAGAGTGCTAGCAAACCTTCT-3' (SEQ ID NO: 5)

[0098] TLE (down): 5'-CCCAAGCTTTCACGGGGGGCAAGCCGTAGTTG-3' (SEQ ID NO: 6)

[0099] PCR conditions: 94°C for 3min

[0100] 94°C 30sec, 55°C 30sec, 72°C 1min 35cycles

[0101] 72℃6min

[0102] PCR system

[0103]

[0104] The PCR products were separated by 1.0% agarose gel electrophoresis, and the amplified fragments were recovered from the gel.

[0105] After the PCR product gel was recovered, it was digested with restriction endonucleases EcoRI (TaKaRa Company) and HindIII (Takara Company) respectively, and the plasmid vector pET-32(a)-c(+) ( In the figure -(a)+ in pET-32, Novagen Company) was ligated to pre...

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Abstract

The invention relates to the field of biomedicine, in particular to a preparation method of snake venom thrombin-like protein. Firstly, the venom tissue of Agkistrodon akistrosus is isolated, and a phage display library is constructed. Using fibrin / fibrinogen as a substrate, three rounds of panning selected, a new thrombin-like gene from Agkistrodon venom was obtained. The present invention also constructs the prokaryotic expression vector pET-32(a)-TLE of the viper venom thrombin gene of Agkistrodon akistrodon, and obtains the recombinant snake venom thrombin with biological activity after induced expression, affinity purification and molecular sieve purification protein. The enzyme can be used to prepare cardiovascular thrombolytic drugs.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a preparation method of snake venom thrombin-like protein. Background technique [0002] Cardiovascular and cerebrovascular diseases such as cerebral apoplexy, coronary heart disease and peripheral vascular thrombosis seriously affect people's quality of life and cause heavy economic burden. As one of the wild resources in nature, snake venom contains many biologically active peptides and proteins, including components that have an important impact on the body's circulatory system. Using snake venom to research and develop new drugs for treating thrombosis has become one of the hotspots of domestic and foreign research. Due to the limitations of natural snake venom resources and product purity, simply relying on improved technology can no longer meet the needs, and a large number of snake venom recombinant proteins can be obtained through modern genetic engineering technology, which i...

Claims

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Application Information

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IPC IPC(8): C12N9/74C12N15/70C12N15/57C12N15/10C12R1/19C12R1/92
Inventor 陆一鸣王俊杰胡振林王凯慧魏玉保孙树汉
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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