Gene sequence and amino acid sequence of snake venom plasmin of agkistrodon blomhoffii ussurensis

A white-browed pit viper and plasmin technology, which can be used in genetic engineering, plant genetic improvement, enzymes, etc., can solve the problems that the drug cannot exert its maximum effect, cannot be administered intravenously, and the purity is not high.

Inactive Publication Date: 2012-10-10
CHINA AGRI UNIV +1
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Problems solved by technology

[0003] At present, the plasmin used clinically in China is extracted from animals, the purity is not high, and it has certain antigenicity. After entering the human body, it may induce a series of antigen-antibody reactions. Drug eruption and severe erythema multiforme drug eruption, and intravenous administration cannot be achieved, so that the drug cannot exert its maximum effect

Method used

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  • Gene sequence and amino acid sequence of snake venom plasmin of agkistrodon blomhoffii ussurensis
  • Gene sequence and amino acid sequence of snake venom plasmin of agkistrodon blomhoffii ussurensis
  • Gene sequence and amino acid sequence of snake venom plasmin of agkistrodon blomhoffii ussurensis

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Experimental program
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Effect test

Embodiment 1

[0024] 1. Total RNA extraction

[0025] Cut off the snake head, take out the venom gland immediately, freeze it in liquid nitrogen, and store it at -70°C for later use. Take out 100mg venom gland tissue, add 1ml solution D (containing 4M guanidine isothiocyanate, 25mM sodium citrate, pH7.0; 5% sodium lauryl sarcosine (Sarcosyl), 0.1M β-mercaptoethanol), After fully homogenizing in a tissue homogenizer, transfer the homogenate into a 10ml centrifuge tube, then add 0.1ml of 2M sodium acetate (pH4), 1ml of water-saturated phenol and 0.2ml of chloroform: isoamyl alcohol (49: 1) The mixture was thoroughly shaken and mixed, and then left to stand on ice for 15 minutes. Samples were centrifuged at 10,000 g at 4°C for 20 min in a Hitach refrigerated centrifuge (Hitach Centrifuge 20PR-52D), absorbed the upper aqueous phase, mixed with an equal volume of isopropanol, and frozen at -20°C for at least 1 hour. Centrifuge at 10,000 g for 20 min to collect the precipitate and dissolve it i...

Embodiment 2

[0032] Embodiment 2 bioactivity assay:

[0033] 1. Qualitative determination of plasmin activity: fibrinogen is transformed into fibrin under the action of thrombin, and a milky white clot is formed in the test tube. Add a sample containing plasmin, and plasmin acts on fibrin to transform it into Soluble small molecular peptides and amino acids, the clot dissolves and is in a liquid state, and the time required for clot dissolution is related to the activity of plasmin. According to the 1995 edition of the second part of the Pharmacopoeia of the People's Republic of China, with a slight improvement, 150ul of 6.5mg / ml Fg was added to the test tube, and then 400ul of GBSB buffer (gelatin-barbital-Nacl buffer) and 100ul of the sample to be tested were placed at a constant temperature of 37°C In the water bath, add 3u / ml T 100ul and 1.5u / ml Pg 100ul after 3 minutes, mix as soon as possible, continue to keep warm at 37°C, and observe the different degrees of dissolution at differen...

Embodiment 3

[0039] Embodiment 3 Amino Acid Composition Determination

[0040] Add a certain amount of sample to a small test tube, add 1ml of 6mol / L HCl and a drop of mercaptoethanol, and seal the tube under reduced pressure. Hydrolyze in an oven at 105-110°C for 24 hours, open the sealed tube, extract HCl gas, and measure amino acid composition on an amino acid automatic analyzer L-8500.

[0041] The amino acid composition of this snake venom fibrinolytic enzyme is compared with other homologous proteins, see Figure 4 . The amino acid sequence of the snake venom fibrinolytic enzyme obtained by the present invention is consistent with the measured value.

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Abstract

Plasmin is obtained from agkistrodon blomhoffii ussurensis by isolation and purification. Ten amino acid sequence at N terminal of the plasmin are measured, according to which forward primer is designed; and reverse primer is designed according to a region, wherein the region is much higher similar to the non-coding region at 3' terminal of the snake venom plasmin of other vipers. Total RNA is extracted from a poison gland; gene is amplified from the RNA by PCR. The result of complete sequence determination after cloning indicates that c DNA of the maturation protein codes 708 nucleotides, that is, 236 amino acids. The successful clone of the gene and the determination of the gene sequence create favorable conditions for the development of gene engineering products.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a gene sequence encoding snake venom protein plasmin and its amino acid sequence. Background technique [0002] Plasmin refers to a proteolytic enzyme that can specifically degrade fibrin gel, and is an important component of the fibrinolytic system. The coagulation and fibrinolysis systems in the body are interdependent and closely linked. Once the body produces a blood coagulation reaction, it also activates the fibrinolytic system almost at the same time, so that the excess thrombus in the body is removed, and the level of fibrinogen in the body is reduced through the negative feedback effect, thereby avoiding excessive aggregation of fibrin. The distribution of snake venom fibrinolytic enzyme is very extensive, and it has been reported that almost all rattlesnake venoms have fibrinolytic components. Fuffado et al. have detected 9 kinds of Agkistrodon venoms and found that they ...

Claims

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Application Information

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IPC IPC(8): C12N15/57C12N9/68C12N15/11
Inventor 罗云波黄昆仑许文涛马骉张雅楠宋梦薇王云鹏
Owner CHINA AGRI UNIV
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