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Novel recombinant bee venom polypeptide as well as preparation method and application thereof

A new type of bee venom technology, applied in the field of novel recombinant bee venom polypeptide and its preparation, can solve the problems of restricting melittin, malignant transformation of cells, affecting the efficacy of melittin, etc.

Active Publication Date: 2018-10-09
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

70-80% of women have been infected with HPV all their lives, and 10% of them face the risk of persistent HPV infection. Although high-risk HPV infection can lead to high-grade CIN and cervical cancer, most of them are transient subtypes. Clinical changes are due to the fact that the free HPV genome poses little threat to the stability of the host genome. Once the HPV DNA integrates into the host genome, it will lead to a series of abnormal gene structures and functions, and eventually lead to malignant transformation of cells
[0005] As a non-specific small molecule polypeptide, melittin currently has the following problems in its application: 1. The molecular weight of phospholipase A2 and melittin is close and has high allergenicity. The existing traditional purification technology cannot effectively separate the two. Thereby the application of melittin is limited; two, melittin is extremely unstable in nature, and it is easy to degrade after entering the blood, thereby affecting the exertion of melittin drug effect; three, melittin, as a strong basic peptide, can be passed through Cleavage of cell membranes has extremely strong hemolytic side effects, and severe hemolysis and even lethal effects after intravenous injection have become the main reason for limiting its development
There are no similar reports on the application of melittin in the treatment of anti-HPV virus and cervical cancer. Compared with the current clinically used anti-virus and anti-tumor drugs, melittin has the advantages of relatively small molecular weight and low immunogenicity.

Method used

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  • Novel recombinant bee venom polypeptide as well as preparation method and application thereof
  • Novel recombinant bee venom polypeptide as well as preparation method and application thereof
  • Novel recombinant bee venom polypeptide as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: A specific preparation method of a novel recombinant bee venom polypeptide comprises the following steps:

[0030] 1. Synthesize the nucleotide sequence encoded by the novel recombinant bee venom polypeptide gene: react the nucleotide with the protected active group pre-connected on the solid phase carrier CPG with trichloroacetic acid to remove the protection of its 5'-hydroxyl group Group DMT, obtain free 5'-hydroxyl, synthesize DNA raw material phosphoramidite to protect nucleic acid monomer, mix with activator tetrazolium, obtain nucleoside phosphorous acid activated intermediate, activate 3' end, 5'- The hydroxyl group is still protected by DMT, and it undergoes a condensation reaction with the free 5'-hydroxyl group in the solution, followed by a capping reaction. In the condensation reaction, there may be very few 5'-hydroxyl groups that do not participate in the reaction. Use acetic anhydride and 1-methylimidazole Terminate the subsequent reaction,...

Embodiment 2

[0034] Example 2: Effect of novel recombinant bee venom polypeptide on cervical cancer cell lines and expression of HPV16 / 18E6 and E7

[0035] (1) Growth inhibition test of novel recombinant bee venom polypeptide cervical cancer cell line

[0036] a. Hela (HPV18 positive) and Caski (HPV16 positive) cells in the logarithmic growth phase were selected and digested with trypsin to make a cell suspension, counted on a cell counting plate, and diluted to a density of 4*104 / ml. After fully mixing the diluted cells, inoculate them in a well plate, and add 100ul to each well (add along the side wall, do not shake).

[0037] b. There are four concentration gradient groups of 0ug / ml, 20ug / ml, 40ug / ml, and 80ug / ml, and three replicate holes are set for each concentration. After the cells adhered to the wall for 12 hours, different concentrations of drugs were added to act for 24 hours and 48 hours. Discard the medium containing the drug, and add new medium containing 10% CCK-8 solution...

Embodiment 3

[0056] Example 3: Novel recombinant mee venom polypeptide inhibits cervical cancer and HPV16 / 18E6, E7 expression in vivo test on tumor-bearing nude mice

[0057] (1) Establishment and grouping of tumor-bearing nude mouse models

[0058] a. Balb / c-nu nude mice, female, age 6-8 weeks, body weight 18-22g, raised in SPF environment, 60 in total. HeLa and Caski cells in the logarithmic growth phase were digested with 0.25% trypsin, centrifuged and washed twice with serum-free culture medium, and the cell concentration was adjusted to 10^7 cells / ml with serum-free culture medium. Under sterile conditions, 0.2 ml of the cell suspension was extracted with a syringe with a No. 6 needle and inoculated subcutaneously in the right axilla of nude mice sterilized with 75% alcohol.

[0059] b. About 4 days after the inoculation, nodules appeared under the skin of the nude mice or until the tumor tissue grew to about 150mm 3 , perform random grouping. Volume V = long diameter * short diame...

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Abstract

The invention relates to a novel recombinant bee venom polypeptide. An amino acid sequence of the novel recombinant bee venom polypeptide is shown in SEQ ID No.1; a nucleotide sequence encoding a novel recombinant bee venom protein is shown in SEQ ID No.2; a preparation method comprises the following steps: connecting an UA8 short peptide with a melittin gene to form a recombinant bee venom fusiongene, constructing an eukaryotic expression vector U-melittin-pPICZalpha of the recombinant bee venom polypeptide fusion gene, and transferring into Pichia pastoris to induce the expression and the purification of the recombinant bee venom protein. The novel recombinant bee venom polypeptide is stronger in antitumor activity and less in hemolytic toxicity with compared with melittin, and is applied to the prevention and the treatment of cervical-related diseases and cervical cancer caused by an HPV virus.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a novel recombinant bee venom polypeptide and its preparation method and application. Background technique [0002] Cervical cancer is one of the three major malignant tumors of the female reproductive system. Its morbidity and fatality rates rank second and third respectively among female malignant tumors in developing countries, and the trend of rejuvenation is significant, seriously endangering women's physical and mental health . According to incomplete statistics, in 2012, there were 528,000 new cases of cervical cancer and 266,000 deaths in the world. The incidence rate of cervical cancer in East Asian population was 7.9 / 100,000. Due to the large population base in my country, cervical cancer screening and prevention measures have not been fully promoted. In recent years, the number of cases has increased year by year, accounting for 1 / 3 of the total number of case...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/81A61K38/17A61P35/00A61P31/20
CPCA61K38/00A61P31/20A61P35/00C07K14/43572C12N15/815Y02P20/55
Inventor 许天敏
Owner JILIN UNIV
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