Method for preparing bitter melon polypeptide with function of reducing blood sugar bioactivity by gene engineering and application

A biologically active, bitter gourd polypeptide technology, applied in the field of genetic engineering, to achieve the effect of simple purification process, lower blood sugar level, and easy expansion of cultivation

Inactive Publication Date: 2009-09-30
SHANGHAI UNIV OF T C M
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These oral chemical hypoglycemic drugs can cause drug resistance, a series of toxic and side effects such as lactic acidosis and liver damage, and the use of insulin injection therapy often causes a lot of inconvenience to patients. New therapeutic drug that can be used safely for a long time

Method used

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  • Method for preparing bitter melon polypeptide with function of reducing blood sugar bioactivity by gene engineering and application
  • Method for preparing bitter melon polypeptide with function of reducing blood sugar bioactivity by gene engineering and application
  • Method for preparing bitter melon polypeptide with function of reducing blood sugar bioactivity by gene engineering and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Fragment of Nucleotide Sequence of Bitter Melon Polypeptide KGP with Hypoglycemic Biological Activity Obtained by PCR

[0037]According to the published amino acid sequence of the bitter melon hypoglycemic activity polypeptide KGP such as SEQ ID: 3 polypeptide sequence, according to the codon preference of bioengineering bacteria, the nucleotide sequence with enzyme cleavage sites at both ends is designed and artificially chemically synthesized. The sequence is used as a template, and primers are synthesized with 15-35bp fragments (including restriction sites) at both ends of the nucleotide sequence. The PCR reaction system is as follows:

[0038] wxya 2 O 18.9 μl

[0039] PCR buffer (10X) 2.5μl

[0040] MgCl 2 (25mM) 1.5μl

[0041] dNTP mix (10mM) 0.5μl

[0042] Primer 1 (10μM) 0.5μl

[0043] Primer 2 (10μM) 0.5μl

[0044] Taq DNA polymerase (5u / μL) 0.3μl

[0045] Template 03μl

[0046] Total 25ul

[0047] The PCR cycling conditions were as follows:

[004...

Embodiment 2

[0054] Construction of expression vector (pET32a-KGP / pQE8-KGP)

[0055] Digest the PCR product of the target gene and the expression vector pET32a / pQE8 with the correct identified sequence respectively with the corresponding enzyme cutting sites. The enzyme cutting system is as follows:

[0056] Recovered PCR product 43μl

[0057] 10× buffer 5μl

[0058] Enzyme 1 1 μl

[0059] Enzyme 2 1 μl

[0060] Total 50ul

[0061] Digest at 37°C for 1-4h and purify the digested product with a gel extraction kit.

[0062] The method of extracting the expression plasmid is as follows:

[0063] (1) Pick a single bacterium colony from the plate, transfer to a plate containing 5mlLB (Amp + ) in a test tube of liquid medium, cultured with vigorous shaking at 37°C for 16-20 hours, and the speed of the shaker was 250rpm;

[0064] (2) Take the bacterial solution and centrifuge at 12000rpm for 30s at 4°C in a centrifuge tube, discard the supernatant, and dry the precipitate;

[0065] (3) ...

Embodiment 3

[0090] Expression of target protein of bitter gourd polypeptide KGP with hypoglycemic activity

[0091] The recombinant plasmid pET32a-KGP / pQE8-KGP was extracted, and the recombinant plasmid was transformed into the corresponding expression strain BL21 / M15. Randomly pick out a single colony that grows, and culture it in LB+Amp / Kan (100mg / ml) liquid medium at 37°C overnight, draw 50μl, then boil for 5min, centrifuge at 2000rpm for 1min, take the Qing Dynasty was used as a template for PCR amplification reaction to detect recombinants.

[0092] The PCR-positive recombinant bacteria were inoculated in 1.5 ml of LB+Amp / Kan (100 mg / ml) liquid medium. An additional culture was inoculated as an uninduced control. Incubate overnight at 37°C with shaking. Then take 500 μl from the overnight culture and inoculate it into 100ml preheated LB culture solution (adding antibiotics), shake and culture at 37°C until the absorbance is OD 600 0.3-0.9. Add IPTG to the bacterial solution, div...

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Abstract

The invention provides a method for preparing bitter melon polypeptide with function of reducing blood sugar bioactivity by gene engineering and application. The method comprises construction, expression, purification and the like of an expression vector. The method can directionally, stably and effectively express the functional polypeptide with biological activity, and is favorable for industrialized production. Proved by animal experiments, the polypeptide prepared by the method can remarkably reduce the blood sugar value of a diabetes pattern animal, and can be used for preparing medicaments for preventing and treating diabetes.

Description

technical field [0001] The invention relates to genetic engineering, in particular to a genetic engineering preparation method of bitter gourd polypeptide KGP with hypoglycemic biological activity and its application in the preparation of anti-diabetic drugs. Background technique [0002] Diabetes is one of the most threatening diseases to human beings in modern society, and the number of patients is increasing year by year. At present, in addition to intravenous insulin, four types of oral hypoglycemic drugs are mainly used clinically, which are sulfonylureas, biguanides, glucosidase inhibitors and thiazolidinones. These oral chemical hypoglycemic drugs can cause drug resistance, a series of toxic and side effects such as lactic acidosis and liver damage, and the use of insulin injection therapy often causes a lot of inconvenience to patients. New therapeutic drugs that can be used safely for a long time. [0003] Bitter melon is a traditional plant medicinal material in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/70C12N15/75C12N15/81C07K14/415A61P3/10
Inventor 王富军胡之璧刘思秀付中平周吉燕
Owner SHANGHAI UNIV OF T C M
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