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Gene of streptokinase, recombination protein and preparation method

A thrombolytic enzyme and gene technology, applied in the field of preparation of the source strain of the thrombolytic enzyme gene, the thrombolytic enzyme gene and the protein produced by its expression, can solve the problem of high cost, high treatment cost, and increased burden on patients and society and other issues to achieve the effect of low cost, cost reduction and broad application prospects

Inactive Publication Date: 2005-01-12
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] (4) Due to the high cost of some approved thrombolytic drugs, the cost of treatment is also high, which increases the burden on patients and society

Method used

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  • Gene of streptokinase, recombination protein and preparation method
  • Gene of streptokinase, recombination protein and preparation method
  • Gene of streptokinase, recombination protein and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0122] Example 1 Preparation process of Bacillus subtilis QK02

[0123] (1) Take rice leaves and heat-treat them at 85°C for 10 minutes, wrap the sterilized soybeans with them, cultivate them at 42°C for 24 hours, and then use the culture medium (soybean extract contains 0.5% salt, 0.5% Na 2 HPO 4 , 2% liquefied starch) soak the soybeans that have been cultivated, and make the nutrient solution of soaked soybeans for 10 -2 -10 -6 Dilute, spread on a nutrient agar plate, grow colonies on the plate after inverting at 37°C.

[0124] (2) The obtained colonies were respectively inoculated in 5 ml of culture solution and cultured at 37° C. and 250 rpm for 24 hours, and then centrifuged at 6000 rpm for 5 minutes. Take 20 μl of supernatant to measure thrombolytic enzyme activity on a fibrin plate, and then select the strain with the highest enzyme activity.

Embodiment 2

[0125] Example 2 PCR amplification and cloning and sequence determination of gene qk encoding thrombolytic enzyme QK

[0126] (1) Genomic DNA extraction of Bacillus subtilis QK02 was performed according to a conventional method (Saito, H. et al, 1963, Biochim. Biophys. Acta 72: 619-629).

[0127] (2) The primers for PCR amplification were designed according to the N-terminal 20 amino acids of thrombolyticase QK (AQSVPYGISQIKAPALHSQG, sequenced at Hunan Normal University) and the C-terminal sequence of the Bacillus subtilis aprN gene. The size of the amplified fragment was 828bp.

[0128] Primer P8: 5’CGC CTG CAG ATG GCG CAA TCT GTT CCT TAT GGC ATT

[0129] Primer P9: 5’GCG GAA TTC TTA CTA TTA TTG TGC AGC TGC TTG

[0130] The above primers were synthesized by Shanghai Jikang Bioengineering Company. In the 50 μl PCR amplification reaction system: 9 μl Bacillus subtilis genomic DNA as a template; the total concentration of each primer is 0.8 pmol / μl; dNTP 1 mM; 10×PCR buffer 5...

Embodiment 3

[0133] Construction and expression of the recombinant expression plasmid of the gene qk of embodiment 3 encoding thrombolytic enzyme QK

[0134] (1) Use PstI and EcoRI to excise and recover the qk gene from the pBS-qk plasmid, and add T to the pRSET A plasmid DNA that has been digested and recovered by PstI / EcoRI 4 DNA ligase was ligated at 18°C ​​for 10 hours, and the ligation product was transformed into CaCl 2 E.coli BL21 (DE3) competent cells prepared by the method were spread on plates to screen recombinants, plasmids were extracted by rapid method, and positive recombinants were further identified by PstI / EcoRI double enzyme digestion. See Figure 2 for the specific process.

[0135] (2) The expression vector pRSET is controlled by the T7 promoter, and the expression plasmid constructed with this vector can be induced and expressed by adding IPTG at 37°C or at room temperature. Pick a single positive colony and inoculate it in 2ml of LB medium, place it on a shaker at 3...

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Abstract

This invention discloses a thrombolytic zyme gene, a recombination protein and its preparation method relating to a thrombus dissolving material, relating to a source strain of thrombus dissolving gene and two reorganized strains containing said gene. Study shows that the zyme is a new plasmin and its vitality is 41000IUmg computed by the fibrin panel method having the function of anti-damage and suppressing protein oxidation arisen from beam, NaNO2 and H2O2.

Description

technical field [0001] The present invention relates to a thrombus-dissolving substance; in particular, it relates to a thrombolytic enzyme gene, a protein produced by its expression, and a preparation method; the present invention also relates to a source strain of a thrombolytic enzyme gene and two recombinant strains containing the gene strain. Background technique [0002] The morbidity and mortality of thromboembolic diseases are very high, which seriously threatens human life and health. There are about 15 million patients in the world. At present, the incidence of blood embolism diseases caused by various reasons, such as cerebral thrombosis, myocardial infarction, coronary heart disease, limb vascular embolism, etc., is on the rise. In my country alone, the annual cost of prevention and rehabilitation is as high as hundreds of billions of yuan. Such diseases seriously endanger the quality of life of patients and increase the burden on patients' families and society. ...

Claims

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Application Information

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IPC IPC(8): C07H21/04C12N15/11C12N15/57
Inventor 齐义鹏高柱虎阎俊鹏
Owner WUHAN UNIV
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