A kind of recombinant bee venom polypeptide and its application

一种蜂毒、序列的技术，应用在应用、重组DNA技术、肽等方向，能够解决易降解、蜂毒肽性质不稳定、限制蜂毒肽等问题

Active Publication Date: 2021-10-08
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 in their lifetime, 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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  • A kind of recombinant bee venom polypeptide and its application
  • A kind of recombinant bee venom polypeptide and its application
  • A kind of recombinant bee venom polypeptide and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: A specific preparation method of recombinant bee venom polypeptide comprises the following steps:

[0029] 1. Synthesize the nucleotide sequence encoded by the recombinant bee venom polypeptide gene: react the nucleotide with the protected active group connected to the solid phase carrier CPG in advance with trichloroacetic acid to remove its 5'-hydroxyl protective group DMT , obtain free 5'-hydroxyl, synthesize DNA raw material phosphoramidite to protect nucleic acid monomer, mix with activator tetrazolium to obtain nucleoside phosphorous acid activated intermediate, activate its 3' end, and 5'-hydroxyl is still DMT protection, condensation reaction with free 5'-hydroxyl in the solution, followed by capping reaction, there may be very few 5'-hydroxyl groups in the condensation reaction that did not participate in the reaction, and subsequent reactions were terminated with acetic anhydride and 1-methylimidazole , and finally under the action of the oxidan...

Embodiment 2

[0033] Example 2: Effects of Recombinant Bee Venom Polypeptides on the Expression of Cervical Cancer Cell Lines and HPV16 / 18 E6 and E7

[0034] (1) Growth inhibition test of recombinant bee venom polypeptide cervical cancer cell line

[0035] a. Select Hela (HPV18 positive) and Caski (HPV16 positive) cells in the logarithmic growth phase and digest them with trypsin to make a cell suspension, count on a cell counting plate, and dilute the cells 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).

[0036] b. Four concentration gradient groups of 0ug / ml, 20ug / ml, 40ug / ml, and 80ug / ml, with 3 replicate wells 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 a new medium containing 10% CCK-8 solution.

[0037] c. After ...

Embodiment 3

[0055] Example 3: Recombinant bee venom polypeptide inhibits cervical cancer and HPV16 / 18 E6, E7 expression in vivo test on tumor-bearing nude mice

[0056] (1) Establishment and grouping of tumor-bearing nude mouse models

[0057] 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 medium, and the cell concentration was adjusted to 10^7 cells / ml with serum-free medium. Under aseptic conditions, 0.2 ml of 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.

[0058] b. About 4 days after the inoculation, nodules appeared under the skin of nude mice or until the tumor tissue grew to about 150mm 3 , perform random grouping. Volume V=long diameter*short diameter*longitudinal diameter

[005...

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PUM

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Abstract

The invention relates to a recombinant bee venom polypeptide and its application. The amino acid sequence of the recombinant bee venom polypeptide is SEQ ID No.1. A recombinant bee venom fusion gene is constructed by connecting the UA8 short peptide with the melittin gene to form a recombinant bee venom fusion gene. The eukaryotic expression of the toxic polypeptide fusion gene carries U-melittin-pPICZα, which is transferred to Pichia pastoris to induce the expression of recombinant melittin and then expressed and purified. Compared with melittin, the present invention has stronger anti-tumor activity and smaller Hemolytic toxicity, used in the prevention and treatment of cervical related diseases and cervical cancer caused by HPV virus.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant bee venom polypeptide and its 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 cases in the world. The pathogen...

Claims

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Application Information

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Patent Type & Authority Patents(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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