CN107722115A - A kind of new restructuring bee venom peptide and its preparation method and application - Google Patents

Abstract

The invention relates to a novel recombinant melittin polypeptide, the amino acid sequence is SEQ ID No.1, the nucleotide sequence encoding the novel recombinant melittin is SEQ ID No.2, and the preparation method is to combine the UA8 short peptide with the melittin gene Connected into a recombinant melittin fusion gene, construct a recombinant melittin polypeptide fusion gene for eukaryotic expression carrying U-melittin-pPICZα, transfer to Pichia pastoris to induce expression of recombinant melittin and perform expression and purification. The present invention is relative to melittin It has stronger anti-tumor activity and less hemolytic toxicity, and is used in the prevention and treatment of cervical cancer caused by HPV virus.

Description

A novel recombinant bee venom polypeptide and its preparation method and application

technical field

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

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 pathogenesis of cervical cancer is complex, and the most closely related is the persistent infection of high-risk HPV. HPV can be detected in almost all invasive cervical cancers and 95% of cervical intraepithelial neoplasia and carcinoma in situ. 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. The 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 cause a series of abnormal gene structures and functions, and eventually lead to malignant transformation of cells. E6 and E7 are the main oncoproteins, which play an important role in carcinogenesis after HPV is integrated into the host genome. E6 can regulate and affect the tumor suppressor effect of p53 protein through ubiquitination and other pathways. The combination of E7 and pRb can make the cell cycle regulation out of control . Therefore, early effective inhibition of HPV and viral protein E6, E7 expression has become an effective way to prevent cervical cancer.

At present, cisplatin, one of the most commonly used drugs in the treatment of cervical cancer, mainly interacts with the bases on the DNA chain, and DNA combines to form cross bonds, thereby destroying the function of DNA and no longer replicating. When it is high in concentration, it will inhibit the synthesis of RNA and protein. The main side effects are gastrointestinal reactions, nephrotoxicity, bone marrow suppression and auditory nerve toxicity, and currently there is no effective way to improve the side effects. Interferon (Interferon) is an immune substance released after cells are infected by viruses. Interferons induced by viruses and substances other than viruses are called type I and type II interferons, respectively. Interferon is a kind of protein with broad-spectrum antiviral activity on the same kind of cells, and its activity is regulated and controlled by the cell genome, involving the synthesis of RNA and protein. There are three types of interferons that have been used clinically: interferon α is the interferon produced by the virus-induced leukocytes, β is the interferon produced by the virus-induced fibroblasts, and γ is the interferon produced by the virus-induced lymphoid cells. Adverse reactions are mainly flu-like symptoms such as fever, fatigue, myalgia, and headache. Followed by mild bone marrow suppression, aminotransferase, serum creatinine increased. However, the clinical effect of interferon in treating HPV infection is not good, and HPV infection cannot be effectively controlled. The development of a drug that can effectively treat HPV virus will fill the gap in this field.

Melittin accounts for 45% to 50% of the dry weight of bee venom and is the main component of bee venom. Studies have found that bee venom has important functions such as anti-tumor, anti-virus, anti-bacteria, anti-inflammation, lowering blood pressure, analgesia, relieving rheumatism and anticoagulation. Among them, the antitumor, antibacterial and antiviral effects of melittin have attracted much attention in recent years. The unique role of melittin is determined by its structure: as a polypeptide chain with a molecular weight of 2840, the N-terminal (1-20aa) is hydrophobic, and the C-terminal (21-26aa) is hydrophilic. At pH and ion concentration, melittin self-crosslinks to form an α-helical tetramer structure, which has the characteristics of membrane-bound peptides and membrane protein transmembrane helices, so it is soluble in water and can dissolve cells to exert cytotoxic activity. In addition, because the molecular weight of melittin is relatively small, in the prior art, the clinical application of melittin mainly contains the following types: (1) connective tissue diseases, such as rheumatism and rheumatoid arthritis; (2) neuritis and Neuralgia, such as the treatment of sciatica, trigeminal neuralgia, occipital neuralgia, dorsal radiculitis, etc.; (3) cardiovascular diseases, such as hypertension, atherosclerosis, venous thrombosis, thromboangiitis obliterans and arterial Endometritis; (4) allergic diseases, such as bronchial asthma. Compared with the anti-tumor drugs currently used in clinical practice, it also has the advantage of slightly lower immunogenicity. In the research of ovarian cancer, liver cancer, gastric cancer, breast cancer and other common tumors, melittin's growth inhibitory effect and direct dissolution effect on tumor cells have been confirmed. But the application of melittin in the treatment of anti-HPV virus and cervical cancer has not been reported yet. According to the pathogenesis of cervical cancer, the antiviral, antitumor and other therapeutic properties of melittin will be applicable to the treatment of HPV infection, so as to achieve the purpose of prevention and treatment of cervical cancer.

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 a strong hemolytic side effect, and severe hemolysis or even lethal effect after intravenous injection has become the main reason for limiting its development. To break through the obstacles to the development of melittin, one is to use advanced biological materials such as micro-nano to coat and transport melittin to improve its stability and targeting specificity; the other is to use genetic engineering technology to modify the structure of melittin The third is to change the dosage form of melittin as a drug, from intravenous administration to topical administration. 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. The UA8 fragment is a short peptide composed of 8 amino acid sequences, which can be connected to melittin to reduce the hemolytic toxicity and other side effects of melittin by modifying its structure.

Contents of the invention

The purpose of the present invention is to provide a novel recombinant melittin polypeptide, which has stronger anti-tumor activity and less hemolytic toxicity than melittin, and is applied to the prevention and treatment of cervical cancer caused by HPV virus.

The amino acid sequence of the novel recombinant bee venom polypeptide of the present invention is SEQ ID No.1.

The nucleotide sequence encoding the novel recombinant melittin is SEQ ID No.2.

Another object of the present invention is to provide the preparation method of the above novel recombinant melittin polypeptide, link the UA8 short peptide and the melittin gene to form a recombinant melittin fusion gene, and construct the eukaryotic expression of the recombinant melittin polypeptide fusion gene containing U- melittin-pPICZα, transformed into Pichia pastoris to induce the expression of recombinant melittin, express and purify, specifically including the following steps:

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, and finally under the action of the oxidant iodine, the phosphorous acid form is converted into a more stable phosphotriester, repeat the above steps until all the required bases are connected, and finally purify to obtain the new recombinant melittin encoded Nucleotide sequence: 5'-gcc taa agc tgc ttg tcc aag agt atg aaa ttc ttag tca acg ttg ccc ttgttt tta tgg tcg tgt aca ttt ctt aca tct atg cg g ccc ctg aac cgg aac cgg caccag agc cag agg cgg agg cag acg cgga ggc gat ccg gaa gcg gga att gga gca gttctg aag gta tta acc aca gga ttg ccc gcc ctc ata agt tgg att aaa cgt aag aggcaa cag ggt tag-3';

2. Construct the recombinant bee venom fusion gene eukaryotic expression vector backbone U-melittin-pPICZα, with the SacII restriction site ccgcgg, the sticky end of SacII is added to the 5' end, and the stop codon and sticky end of EcoRI are added to the 3' end '; Afterwards, the following reaction was carried out, 6μl 0.5mmol NaCl and 25μmol fusion gene were mixed, the mixture was reacted at 80°C for 3min and then gradually lowered to room temperature, the pPICZαC plasmid was cut by SacII and EcoRI, the prepared DNA fragment was inserted into the pPICZαC vector, and the recombinant The plasmid was transferred into Escherichia coli XL-Blue cells, and the clones carrying the recombinant plasmid were screened for 25 μg/ml bleomycin resistance, electrophoresed on 0.8% agarose gel, and the PCR product was recovered by cutting the gel, and 20-25 μg of the expression vector backbone was taken U-melittin-pPICZα was digested with SacI, extracted with phenol/chloroform and precipitated with ethanol, and the linearized recombinant expression plasmid was dissolved in 10 μl ultrapure water and kept on ice for later use;

3. Establish the expression system of the new recombinant melittin polypeptide: Pick a single colony from the YPD negative culture plate of Pichia pastoris X-33, inoculate it in 5ml YPD medium, culture it with shaking at 250rpm, 30°C for 8 hours, and prepare it by conventional method. Yeast competent cells; then take 80 μl of the above competent bacteria, mix them with 20-25 μg of the linearized recombinant expression plasmid prepared in step 2, and transfer them into a 0.2 cm electroporation cup for electrotransformation; take 50-100 μl of transformed bacteria The solution was spread on the YPD plate containing Zeocin (100 μg/ml), and cultured in a 30°C incubator for 2 to 3 days to observe the growth status of the transformant; then use the PCR method to screen the transformed yeast; The yeast genomic DNA was extracted by the glass bead method and identified by PCR, and the amplified product was subjected to 1.0% agarose gel electrophoresis;

4. Expression of new recombinant bee venom polypeptide: Inoculate the positive clones identified in step 3 in 10ml of BMGY medium with a pH of 6.0, culture with shaking at 30°C for 24 hours, and collect the cells when the OD600 reaches 2.0 to 6.0. Volume (10ml) BMMY, pH 6.0 resuspended cell pellet, shake culture at 30°C, and induce expression; during the induction process, supplement methanol once every 24 hours to a final concentration of 0.5%, and supplement sterilized ultrapure water at the same time to make the fermentation broth total The volume remained constant; 0.5 ml of fermentation broth was taken at the time points of 0, 24, 48, 72, 96, 120, 144 and 168 hours of cultivation, and the supernatant was obtained by centrifugation.

Another object of the present invention is to provide the application of novel recombinant bee venom polypeptide in the treatment of cervical cancer drugs, especially related to the anti-HPV treatment and prevention and treatment of cervical cancer caused by HPV infection by inhibiting the expression of high-risk HPV16 and 18 virus proteins. application in cancer.

The main feature of the new recombinant melittin polypeptide of the present invention is that the protein components include modified components and the main component of melittin, and are produced using the Pichia pastoris expression system, so that it has stronger anti-tumor activity and less non-specificity Biological toxicity. Aiming at the bottleneck problem in the current cervical cancer prevention and treatment process and the blank in melittin research, the present invention adds 8 amino acid short peptides to improve the primary structure of melittin according to its extensive biological properties such as anti-virus and anti-tumor. It was modified to enhance specificity and reduce hemolytic toxicity. The recombinant melittin was expressed through the expression system of Pichia pastoris. It was further found that the new recombinant melittin could inhibit the expression of E6/E7 proteins of two high-risk cervical cancer viruses, HPV16 and HPV18, and inhibit cervical cancer. Cancer cells grow, promote their apoptosis, etc., to achieve the effect of anti-HPV virus, so that it can be applied to the prevention and treatment of cervical cancer caused by HPV virus. At the concentration of each group, the inhibitory effect of melittin and new recombinant melittin on tumor volume increase was enhanced in a dose-dependent manner. The inhibitory effect on body volume growth was significantly better than that of cisplatin, but the inhibitory rates of the high, middle and low dose groups corresponding to the two peptides on tumor growth were the same, with no significant difference. Table 1 below compares the inhibition rate (%) of melittin and the novel recombinant melittin polypeptide on the tumor weight of hela and caski tumor-bearing mice.

Table 1

Description of drawings

Figure 1: Different mass concentrations (0, 10, 20, 40, 80ug/ml) of the new recombinant bee venom polypeptide act on Hela and Caski for 24h and 48h, and its growth inhibitory effect on Hela and Caski increases with the increase of concentration and action time; The IC50 for Hela and Caski for 24 hours were 49.83ug/ml and 32.76ug/ml respectively;

Figure 2: After Hela cells were treated with 4 concentrations of the new recombinant melittin for 24 hours, as the concentration of the new recombinant melittin peptide increased, the number of cells decreased, the gap increased, the volume decreased, and the shape shrunk and rounded;

Figure 3: After Caski cells were treated with 4 concentrations of new recombinant mee venom polypeptide for 24 hours, as the concentration increased, the number of cells decreased, the gap increased, the volume decreased, and the shape shrunk and rounded;

Figure 4: After Hela cells were treated with 4 concentrations of new recombinant mee venom polypeptide for 24 hours, the expression of HPV18E7 and HPV18E6 decreased as the concentration increased;

Figure 5: After Caski cells were treated with 4 concentrations of novel recombinant mee venom polypeptide for 24 hours, the expression of HPV16E7 and HPV16E6 decreased as the concentration increased;

Figure 6: Hela cells were treated with the new recombinant bee venom polypeptide for 24 hours. With the increase of drug concentration, both early apoptosis and middle and late apoptosis increased, and the increase of middle and late apoptosis was the main one;

Figure 7: Caski cells were treated with the new recombinant bee venom polypeptide for 24 hours. With the increase of drug concentration, both early apoptosis and middle and late apoptosis increased, and the increase of middle and late apoptosis was the main one;

Figure 8: Under the administration concentrations of each group, the tumor volume of cervical cancer tumor-bearing mice increases with time. The inhibitory effect on tumor volume increase was enhanced in a dose-dependent manner, and the inhibitory effect on tumor volume increase at 80 mg/kg was significantly better than that of cisplatin;

Figure 9: The expression of E6 and E7 in the tumor body of Hela cervical cancer tumor-bearing mice treated with each administration group: with the increase of concentration, the expression of HPV16E7 and HPV16E6 decreased, especially E6, and 80mg/kg administration The expression levels of group E6 and E7 were significantly lower than those of interferon and cisplatin group;

Figure 10: The expression of E6 and E7 in Caski cervical cancer tumor-bearing mice treated by each administration group: with the increase of concentration, the expression of HPV16E7 and HPV16E6 decreased, especially E6, and 80mg/kg administration The expression levels of group E6 and E7 were significantly lower than those of interferon and cisplatin group.

detailed description

The best mode of carrying out the invention is described below with the aid of examples. These examples are intended to further illustrate the invention and not to limit the scope of the appended claims of the invention in any way.

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

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, and finally under the action of the oxidant iodine, the phosphorous acid form is converted into a more stable phosphotriester, repeat the above steps until all the required bases are connected, and the color judgment of the TCA treatment stage can be observed during the synthesis process Synthetic efficiency, finally purified to obtain the nucleotide sequence encoded by the novel recombinant melittin: 5'-gcc taa agc tgc ttgtcc aag agt atg aaa ttc ttag tca acg ttg ccc ttg ttt tta tgg tcg tgt aca tttctt aca tct atg cg g ccc ctg aac cgg aac cgg cac cag agc cag agg cgg agg cagacg cgga ggc gat ccg gaa gcg gga att gga gca gtt ctg aag gta tta acc aca ggattg ccc gcc ctc ata agt tgg att aaa cgt aag agg caa cag ggt tag-3' .

2. Construct the recombinant melittin fusion gene eukaryotic expression vector backbone U-melittin-pPICZα, with the SacII restriction site ccgcgg, in order to insert the synthesized recombinant melitin polypeptide DNA into the pPICZαC plasmid, the cohesive end of SacII was added to the 5' The stop codon and cohesive end of EcoRI were added to the 3'; the synonymous codons to increase the yield of recombinant polypeptides were replaced by codons that determine the yield. The reaction conditions were as follows: 6μl 0.5mmol NaCl, 25μmol single-stranded DNA mixed, the mixture was in React at 80°C for 3 minutes and then gradually lower to room temperature. The pPICZαC plasmid was cut by SacII and EcoRI, and the prepared DNA fragment was inserted into the pPICZαC vector, and the recombinant plasmid was transferred into Escherichia coli XL-Blue cells. For mycin (25 μg/ml) resistance screening, a PCR reaction system was established in a 0.2ml EP tube according to the following ratio: 100ng of recombinant plasmid; 10μl of 10×LA PCR buffer containing Mg2+, 8μl of dNTPs (each 2.5mmol/L), Primer 1 μl (20 pmol/μl), TaKaRa LA Taq (5U/μl) 1 μl, sterilized ultrapure water to a final volume of 100 μl, and perform cycle amplification on a PCR machine; the amplification program is: 94°C for 4 minutes; 94°C for 30 seconds, 50°C for 30 seconds, 70°C for 1 minute, a total of 30 cycles, and then 70°C for 8 minutes; 0.8% agarose gel electrophoresis, gel cutting to recover PCR products, take 20-25 μg expression vector u-melittin-pPICZα and pass SacI After enzyme digestion, extracted with phenol/chloroform and precipitated with ethanol, the linearized recombinant expression plasmid was dissolved in 10 μl ultrapure water and kept on ice for later use; the sequence of the primer was 5′-GTT CCA TCG AAC TGT GAC CGA TGC TG -3'5'-GGT TCT CGA TGGTGG TGA GTT TCC AT-3';

3. Establish the expression system of the new recombinant melittin polypeptide: Pick a single colony from the YPD negative culture plate of Pichia pastoris X-33, inoculate it in 5ml YPD medium, culture it with shaking at 250rpm, 30°C for 8 hours, and prepare it by conventional method. Yeast competent cells; then take 80 μl of the above competent bacteria, mix them with 20-25 μg of the linearized recombinant expression plasmid prepared in step 2, and transfer them into a 0.2 cm electroporation cup for electrotransformation; take 50-100 μl of transformed bacteria The solution was spread on the YPD plate containing Zeocin (100 μg/ml), and cultured in a 30°C incubator for 2 to 3 days to observe the growth status of the transformant; then use the PCR method to screen the transformed yeast; The yeast genomic DNA was extracted by the glass bead method and identified by PCR, and the amplified product was subjected to 1.0% agarose gel electrophoresis;

4. Expression of new recombinant bee venom polypeptide: Inoculate the positive clones identified in step 3 in 10ml of BMGY medium with a pH of 6.0, culture with shaking at 30°C for 24 hours, and collect the cells when the OD600 reaches 2.0 to 6.0. Volume (10ml) BMMY, pH 6.0 resuspended cell pellet, shake culture at 30°C, and induce expression; during the induction process, supplement methanol once every 24 hours to a final concentration of 0.5%, and supplement sterilized ultrapure water at the same time to make the fermentation broth total The volume remained constant; 0.5 ml of fermentation broth was taken at the time points of 0, 24, 48, 72, 96, 120, 144 and 168 hours of cultivation, and the supernatant was obtained by centrifugation.

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

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

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).

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.

c. After incubating at 37°C for 1-4 hours, measure the OD value at 450nm with a microplate reader.

d. Calculate the inhibition rate and IC50 of different concentrations on Hela and Caski cells at 24 hours and 48 hours. The result is shown in Figure 1.

e. Observe the morphological changes of Hela and Caski after adding different concentrations under an inverted microscope. As shown in Figure 2 and Figure 3.

(2) Inhibitory effect of novel recombinant bee venom polypeptide on HPV18E6, E7 and HPV16E6, E7 protein expression

After drug treatment, the cells in each group were digested with trypsin, and the cells were collected and washed once with PBS.

a. Lysis solution (containing PMSF). Then place on ice.

b. After 30 minutes of lysis, use a pipette to transfer the lysate to a 1.5ml centrifuge tube, then centrifuge at 12000 rpm for 5 minutes at 4°C, take the supernatant and put it in a 0.5ml centrifuge tube and store at -20°C.

c. The BCA method was used to detect the protein concentration and calculate the loading amount.

d. SDS-PAGE electrophoresis: stacking gel 80V*30min, separating gel 120V*1h.

e. Transfer film: 300mA*30min.

f. Blocking: 5% skimmed milk for blocking for 1 hour.

g. Incubate E6, E7, β-actin primary antibodies overnight at 4°C.

h. Incubate the secondary antibody for 45min.

i. Color development, image processing, as shown in Figure 4 and Figure 5.

(3) Novel recombinant bee venom polypeptide promotes apoptosis of cervical cancer cell lines

a. Hela and Caski cells in the logarithmic growth phase were digested with trypsin to make cell suspension. Count on a cell counting plate, dilute the cells to a density of 4*10 ⁴ /ml, mix the diluted cells thoroughly, inoculate in a 6-well plate, and add 2ml to each well. After being inoculated to adhere to the wall, drugs of different concentrations were added to act for 24 hours.

b. Suck the cell culture fluids treated with different drug concentrations into different centrifuge tubes for later use, digest the cells with EDTA-free trypsin and add the digested cells of different groups into different centrifuge tubes, centrifuge at 1000rpm for 5 minutes, Aspirate and discard the supernatant, add 1ml of pre-cooled PBS, mix by pipetting, centrifuge at 1000rpm for 5 minutes, and aspirate and discard the supernatant.

c. Wash again with PBS. Add 100ul of Binding Buffer to each tube of cells and mix well, add 5ul of Annexin V/FITC and mix well, then incubate at room temperature in the dark for 5 minutes, add 10ul of 20ug/ml phone propidium solution, and add 400ul of PBS, and immediately perform flow detection. Apoptosis, the results are shown in Figure 6 and Figure 7.

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

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

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.

b. About 4 days after the nude mice were inoculated with subcutaneous nodules or when the tumor tissue grew to about 150 mm ³ , they were randomly divided into groups. Volume V = long diameter * short diameter * longitudinal diameter

c. Grouping: Set the rats inoculated with Hela and Caski into groups A and B respectively, and set up the following 6 groups for each group, with 5 rats in each group.

Blank control group: normal saline

Positive control group 1: cisplatin 2.5mg/kg

Positive control group 2: interferon 2.5 million U/kg

Low-dose group of new recombinant bee venom polypeptide: 20mg/kg

Medium dose group of new recombinant bee venom polypeptide: 40mg/kg

High-dose group of new recombinant bee venom polypeptide: 80mg/kg

(2) Measurement of tumor tissue size and tumor weight inhibition rate

a. Inject the reagents and doses according to the above groups into the tumor site at multiple points, and administer once every 24 hours for 20 consecutive days. During the period, the growth of the tumor was measured every 4 days, the volume of the tumor was calculated, and the growth curve of the tumor was drawn.

b. 24 hours after drug withdrawal, blood was collected, the animals were sacrificed, the heart, liver, kidney, and lung were taken out, the tumor mass was peeled off, weighed and calculated, and the follow-up experiment was carried out.

c. Calculation of tumor weight inhibition rate (%)=(1-average tumor weight of administration group/average tumor weight of blank control group)*100%

The results showed that the tumor weight inhibition rate was enhanced in a dose-dependent manner, and the tumor weight inhibition rate at 80 mg/kg was significantly higher than that of cisplatin and interferon, as shown in Figure 8 and Table 2 and Table 3.

Table 2

Tumor Weight Inhibition Rate of Hela Tumor-bearing Mice

table 3

Tumor weight inhibition rate of Caski tumor-bearing mice

(3) Determination of E6 and E7 protein expression levels of HPV18 and HPV16 in tumor tissue

Take 1/3 of the tissue block from liquid nitrogen and cut the tissue block into pieces with clean scissors.

a. The lysate (containing PMSF) is placed in a homogenizer for homogenization. Then place on ice.

b. After 30 minutes of lysis, use a pipette to transfer the lysate to a 1.5ml centrifuge tube, then centrifuge at 12000 rpm for 5 minutes at 4°C, take the supernatant and put it in a 0.5ml centrifuge tube and store at -20°C.

c. The BCA method was used to detect the protein concentration and calculate the loading amount.

d. SDS-PAGE electrophoresis: stacking gel 80V*30min, separating gel 120V*1h.

e. Transfer film: 300mA*30min.

f. Blocking: 5% skimmed milk for blocking for 1 hour.

g. Incubate E6, E7, β-actin primary antibodies overnight at 4°C.

h. Incubate the secondary antibody for 45min.

i. Color development, the processed images are shown in Figure 9 and Figure 10 .

The above experiments lead to the following conclusions: the new recombinant bee venom polypeptide is used to inhibit the expression of E6/E7 proteins of two high-risk cervical cancer viruses, HPV16 and HPV18, inhibit the growth of cervical cancer cells, promote their apoptosis, and achieve the effect of resisting HPV viruses. It can be applied to the prevention and treatment of cervical cancer caused by HPV virus. The high-risk HPV specifically refers to HPV18 infecting Hela cells and HPV16 infecting caski cells. The high-risk HPV tumorigenic proteins mentioned herein specifically refer to E6 and E7 proteins. In vitro and in vivo studies were conducted on HPV16 and HPV18 given different doses of the new recombinant mee venom polypeptide, and Western Blotting was used to detect the expression of tumorigenic proteins E6 and E7. Wherein said different doses are negative control group, low dose group, middle dose group and high dose group respectively. Wherein said in vitro research is cervical cancer cell line Hela (HPV18), Caski (HPV16) cell culture. The in vivo research object mentioned herein is a cervical cancer tumor-bearing nude mouse model. The proliferation inhibitory effect of the novel recombinant bee venom polypeptide was detected by observation under a microscope and the CCK-8 method, and the apoptosis-promoting effect of the new recombinant bee venom polypeptide was detected by flow cytometry. With the increase of the concentration of the new recombinant bee venom peptide, the expression of HPV18E6/E7 and HPV16E6/E7 gradually decreased, indicating that the new recombinant bee venom peptide can effectively inhibit the expression of high-risk HPV 18 and HPV16 tumorigenic proteins, thereby achieving anti-HPV18, HPV16 The effect of further preventing cervical cancer. The in vivo research object is a nude mouse model of cervical cancer, and the growth inhibitory effect of the new recombinant bee venom polypeptide is detected by the growth curve of the tumor mass and the inhibition rate of the tumor weight. The method finds that the novel recombinant melittin polypeptide can effectively inhibit the proliferation of cervical cancer cells and promote its apoptosis, and furthermore, the melittin has obvious anticancer effect.

Embodiment 4: Novel recombinant bee venom polypeptide hemolysis test

Preparation of 2% erythrocyte suspension Take 10-20ml of fresh rabbit blood, put it into a Erlenmeyer flask filled with glass beads and shake it for 10 minutes, or stir the blood with a glass rod to remove fibrous protein and make it into defibrinated blood. Add 100ml of physiological water, shake well, centrifuge at 1000-1500r/min for 15 minutes, remove the supernatant, and wash the precipitated red blood cells with normal saline for 2-3 times according to the above method until the supernatant does not appear red. The obtained erythrocytes were mixed with physiological saline to form a 2% suspension (2 ml of erythrocytes, added with physiological saline to make 100 ml) for the test. Take 8 10ml clean glass test tubes, numbered, as shown in Table 4, and add 2% red blood cell solution in turn. 0.9% sodium chloride solution or distilled water, after mixing, immediately place in a constant temperature water bath at 37±0.5°C for incubation, observe and record the hemolysis of each tube. At the beginning, observe once every 30 minutes, and then observe once every hour after 1 hour, and generally observe for 3 hours. Table 2 shows the comparison of melittin and the new recombinant melittin polypeptide hemolysis test.

Table 4

Note: - means no hemolysis + means partial hemolysis ++ means full hemolysis

The degree of hemolysis of melittin and the new recombinant melittin polypeptide increased with the increase of the concentration, corresponding to the comparison of the low, middle and high groups of the two polypeptides, the new recombinant melittin significantly improved the hemolytic toxicity of melittin.

Preparation Example

A drug containing novel recombinant bee venom polypeptide for treating human papillomavirus and cervical cancer contains the following components by weight: 0.05-5% of novel recombinant bee venom polypeptide; 0.1-1% of carbomer; 0.5-3% of triethanolamine borneol 3 -6%; glucose 1-5%; glycerin 0.5-5%; the rest is water; the borneol is selected from natural borneol and/or synthetic borneol. The pharmaceutical composition is prepared into vaginal administration formulation. The preparations include suppository, ointment, capsule, effervescent tablet, gel, lotion, film or foam.

Preparation Example 1:

New recombinant bee venom polypeptide vaginal suppository, formula: new recombinant bee venom polypeptide 8g, gelatin 1000g, glycerin 1600g, mannitol 6g, lactose 6g, citric acid 5g, glutaric acid 5g, sodium citrate 4g, β-cyclodextrin 6g.

Concrete preparation steps are:

a. Preparation of matrix: weigh 1000g of gelatin, add an equal amount of pure water to soak for 1 hour, place in a water bath and heat to 80°C to dissolve, then add a certain amount of glycerin (1600g), stir, and evaporate excess water;

b. Add 6g of mannitol, 6g of lactose, 5g of citric acid, 5g of glutaric acid, 4g of sodium citrate, and 6g of β-cyclodextrin into the matrix, stir well, control the temperature at 37-40°C, and add new recombinant honey Toxic peptide 8g, stir well and pour it into the vaginal suppository mold which has been sterilized and coated with lubricant at 37-40°C;

c. Cool to normal room temperature, the temperature is controlled at 15-25°C, cut off the overflow part of the mold mouth, demould, seal, and obtain the finished product of the novel recombinant bee venom polypeptide suppository;

Preparation Example 2:

New recombinant bee venom polypeptide vaginal ointment, formula: new recombinant bee venom polypeptide 6g, sodium dodecyl sulfate modified montmorillonite 50g, liquid paraffin 480g, glycerin 25g, isopropyl myristate 200g, cyclomethicone Ketone 30g, stearyl alcohol 55g, borneol 10g, peppermint 2g The specific preparation steps are:

a. Weigh 50 g of sodium dodecylsulfonate modified montmorillonite and dissolve it in 480 g of liquid paraffin, place it in a colloid mill for wet grinding and disperse for 5 minutes, after fully dispersing, add 25 g of glycerin and continue dispersing for 5 minutes to obtain dodecane Liquid paraffin gel formed by dispersion of sodium sulfonate modified montmorillonite

b. Heating to 70 degrees Celsius, then slowly adding 6 g of the new recombinant bee venom polypeptide to it, stirring while adding, and then adding 200 g of isopropyl myristate, 30 g of cyclomethicone, and stearyl alcohol preheated to 55 degrees Celsius 55g, borneol 10g, peppermint 2g, and stir evenly while adding, after mixing evenly, stop heating, continue to stir to room temperature, obtains novel recombinant bee venom polypeptide ointment.

Preparation Example 3:

New recombinant bee venom polypeptide vaginal effervescent tablet, formula: new recombinant bee venom polypeptide 8g, mannitol 200 grams, hydroxypropyl cellulose 30 grams, carboxymethyl starch sodium 20 grams, sodium bicarbonate 180 grams, citric acid 200 grams , 3 grams of magnesium stearate. Concrete preparation steps are:

a. Mix the sodium bicarbonate and citric acid according to the formula amount thoroughly, and pass through an 80-mesh sieve.

b. Add an appropriate amount of ethanol solution, make wet granules, mix well, pass through a 20-mesh sieve, and dry at 42-45°C for 4-6 hours to obtain effervescent granules.

c. Dilute the new recombinant bee venom polypeptide to an appropriate concentration according to the formula.

d. Take other excipients in the formula amount, mix them uniformly by doubling dilution method, and pass through an 80-mesh sieve; add new recombinant bee venom polypeptide solution to make soft materials, pass through a 20-mesh sieve, and make wet granules.

e. Dry at 42-45°C for 4-6 hours; measure the moisture content, the moisture content should be below 1%, and obtain the new recombinant bee venom polypeptide particles.

f. After uniformly mixing the effervescent granules and the new recombinant bee venom polypeptide granules, adding magnesium stearate, tableting and packaging to obtain the new recombinant bee venom polypeptide effervescent tablets.

Preparation Example 4:

New Recombinant Bee Venom Peptide Vaginal Gel: Formula: New Recombinant Bee Venom Peptide 12g, Chlorhexidine Acetate 1.6g, Carbomer 10g, Glycerin 120g, Triethanolamine 4g, Ethylparaben 1g, and the rest is purified water. Concrete preparation steps are as follows:

a. Prepare 1000g of gel, take 12g of new recombinant bee venom polypeptide and 1.6g of chlorhexidine acetate according to the weight ratio, mix them and pulverize them, and pass through a 100-mesh sieve for later use;

b. Take 10g of carbomer, 4g of triethanolamine and purified water according to the weight ratio; sprinkle the carbomer evenly on the surface of the purified water, let it sit for 40-48 hours to fully swell naturally, then add triethanolamine and stir evenly, and adjust the pH to 5-6 , made into matrix;

c. Add 120 g of glycerin and 1 g of ethylparaben by weight to the pulverized medicine prepared in step a, stir and mix evenly to obtain a mixture for subsequent use;

d. Take the mixture in step c and gradually add it to the matrix made in step 2 under stirring, add purified water and mix evenly to make 1000g of gel;

e. The gel obtained in step d is filled and packaged according to the weight required by the specifications of 5 grams per tube to obtain a new recombinant bee venom polypeptide vaginal gel product.

Preparation Example 5:

New recombinant bee venom polypeptide lotion: Formula: 15g new recombinant bee venom polypeptide, 20g tanmu, 20g Artemisia annua, 20g Andrographis paniculata, 5g mint, 5g borneol, 5g Jiuli Ming, 1000ml water.

The preparation method of above-mentioned gynecological vaginal douche is as follows:

Raw materials are taken according to weight ratio: mother of charcoal, Artemisia annua, Andrographis paniculata, mint, borneol, Jiuli Ming;

Wash and chop the above medicinal materials into a flask and add water until boiled;

After it cools, filter twice with a sieve, and collect the filtrate;

According to the formula, the new recombinant bee venom polypeptide is diluted to an appropriate concentration.

Add the new recombinant bee venom polypeptide solution to the filtrate, add benzoic acid for preservation, and bottle it for later use.

Preparation Example 6:

New recombinant bee venom polypeptide protein dressing, formula (1000g): Carbomer 10g, peppermint 5g, green tea extract 20g, glycerin 50g, paraben 25g, phenoxyethanol 20g, the rest is water. Include the following steps:

a. Dissolve glycerin, peppermint and green tea extracts in pure water first, then add carbomer to make it fully dissolved, then add the new recombinant mee venom protein filtered through a 0.22μm membrane, and continue stirring to obtain a uniform system.

b. Add the well-mixed phenoxyethanol and paraben, make up the balance with pure water, and stir well.

c. Finally, adjust the pH value of the obtained one-liter invisible film to between 4.5 and 6.5 with a NaOH solution having a mass concentration of 20%, to obtain the finished product.

Clinical Implementation Trial

This experiment is a clinical observation of the curative effect of the novel recombinant bee venom polypeptide suppository of the present invention on persistent HPV infection patients without high-grade cervical lesions.

Selection of research subjects: For persistent HPV infection (HPV-DNA) test positive, reexamination after 6 months is still positive) and without high-grade cervical lesions, all people are voluntarily enrolled and signed an informed consent.

standard constrain:

1 No previous history of cervical surgery

2Exclude high-grade cervical lesions and cancers (TCT examination, and colposcopy for patients with TCT results of ASCUS and above)

3 No new recombinant bee venom polypeptide contraindications

4 No use of antiviral drugs and immunomodulatory preparations within half a year

5 Non-pregnant and lactating women

6 No acute inflammatory diseases such as acute vaginitis and acute pelvic inflammatory disease

7 No irregular vaginal bleeding

8 No immunocompromised (malignant tumor, SLE, AIDS, etc.)

9 Have follow-up conditions and cooperate with treatment

10 All patients signed the informed consent and voluntarily participated in this clinical trial

Exclusion criteria:

1 Immunocompromised patients, such as postoperative tumors or after radiotherapy and chemotherapy, AIDS and systemic lupus erythematosus, etc.;

2 Pregnant and lactating women;

3 Suffering from acute reproductive tract inflammation, such as gonorrhea, mycoplasma or chlamydia infection;

Control group: without any drug intervention, patients used condoms for contraception during the observation period;

Treatment group: After cleaning the vulva before going to bed, push the drug to the fornix with a propeller, 2g at a time, once a day for 10 days, then stop the drug, and repeat in the next menstrual cycle. Stop using it during menstrual period, and start taking medicine from the 3rd to 5th day after menstruation is clean in the next cycle. After 3 consecutive menstrual cycles, recheck HPV, stop taking medicine if HPV turns negative, and continue to use melittin gel if HPV does not turn negative 3 menstrual cycles: Sitz baths and sexual life are prohibited during the medication period, and condoms should be used for contraception within half a year after medication.

Judgment method of curative effect:

Negative: HPV(-) at reexamination;

Effective: Quantitative reduction of HPV infection after reexamination, but not completely cleared

Invalid: During reexamination, HPV infection quantitatively decreased or quantitatively increased.

Results: After 3 months of treatment, the HPV clearance rate and effective rate of the treatment group were higher than those of the control group. After 6 months, the HPV clearance rate and effective rate in the treatment group were also higher than those in the control group. The curative effect comparison between the treatment group and the control group after 3 months and 6 months after the treatment is detailed in Table 5 below.

table 5

Note: Clearance rate = N turned negative/N total number of people × 100% Effective rate = (N turned negative + N effective)/N total number of people × 100%

The above results show that the novel recombinant bee venom polypeptide suppository of the present invention has significant curative effect on the treatment of human HPV infection.

SEQUENCE LISTING

<110> Jilin University

<120> A Novel Recombinant Bee Venom Polypeptide and Its Preparation Method and Application

<130> 2017

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 34

<212> PRT

<213> Artificial sequence

<400> 1

Lys Pro Ser Ser Pro Pro Glu Glu Gly Ile Gly Ala Val Leu Lys Val

1 5 10 15

Leu Thr Thr Gly Leu Pro Ala Leu Ile Ser Trp Ile Lys Arg Lys Arg

20 25 30

Gln Gln

<210> 2

<211> 236

<212>DNA

<213> Artificial sequence

<400> 2

gcctaaagct gcttgtccaa gagtatgaaa ttcttagtca acgttgccct tgtttttatg 60

gtcgtgtaca tttcttacat ctatgcggcc cctgaaccgg aaccggcacc agagccagag 120

gcggaggcag acgcggaggc gatccggaag cgggaattgg agcagttctg aaggtattaa 180

ccacaggatt gcccgccctc ataagttgga ttaaacgtaa gaggcaacag ggttag 236

<210> 3

<211> 52

<212>DNA

<213> Artificial sequence

<400> 3

gttccatcga actgtgaccg atgctgggtt ctcgatggtg gtgagtttcc at 52

Claims (5)

1. A kind of 1. new restructuring bee venom peptide, it is characterised in that：Amino acid sequence is SEQ ID No.1.
2. 2. the nucleotides sequence of coding new restructuring bee venom peptide as claimed in claim 1 is classified as SEQ ID No.2.
3. 3. the preparation method of new restructuring bee venom peptide as claimed in claim 1, it is characterised in that specifically include following step Suddenly：

   One, synthesis SEQ ID No.2：By the protected nucleotides of active group being connected in advance on solid phase carrier CPG and three Chloroacetate reaction, the blocking group DMT of its 5 '-hydroxyl is sloughed, obtain free 5'- hydroxyls, synthetic DNA raw material phosphoramidite is protected Nucleic acid monomer is protected, is mixed with activator tetrazole, obtains nucleosides phosphorous acid activated intermediate, activates its 3' end, 5'- hydroxyls are still So protected by DMT, condensation reaction occurs for the 5'- hydroxyls with dissociating in solution, is reacted followed by band cap, can in condensation reaction There can be only a few 5'- hydroxyls not participate in reaction, subsequent reactions be terminated with acetic anhydride and 1- methylimidazoles, finally in oxidant iodine In the presence of, phosphorous acyl form is changed into more stable phosphotriester, repeats above step until required all bases are connect Up, finally purified to obtain the nucleotide sequence of new restructuring bee venom protein coding；

   Two, structure restructuring bee venom fusion carrier for expression of eukaryon skeleton U-melittin-pPICZ α, with SacII restriction enzyme sites Ccgcgg, SacII cohesive end are added in 5' ends, and EcoRI terminator codon and cohesive end are added to 3'；It is laggard Obtained fusion mixing, mixture are anti-in 80 DEG C in the following reaction of row, 6 μ l 0.5mmol NaCl and 25 μm of ol step 1 3min is answered then to be gradually decreased to room temperature, pPICZ α C plasmids are sheared by SacII and EcoRI, and the DNA fragmentation for preparing completion is inserted PPICZ α C carriers, recombinant plasmid are transferred to Escherichia coli XL-Blue cell, and the clone for carrying recombinant plasmid is led to 25 μ g/ml bleomycin resistance screening is crossed, 0.8% agarose gel electrophoresis, gel extraction PCR primer, takes 20~25 μ g tables Up to carrier framework U-melittin-pPICZ α after SacI enzymic digestions, with phenol/chloroform and with ethanol precipitation, linearisation Recombinant expression plasmid dissolving is rearmounted standby on ice；

   Three, establish the expression system of new restructuring bee venom peptide：It is single from picking on the YPD feminine gender culture plates of pichia pastoris X-33 Bacterium colony, it is inoculated in 5ml YPD culture mediums, 250rpm, 30 DEG C of concussion and cultivates 8 hours, competent yeast cells is prepared with conventional； Then the 80 above-mentioned competence bacterias of μ l are taken, are carried out after being mixed with the recombinant expression plasmid of obtained linearisation in 20~25 μ g step 2 Electricity conversion；The bacterium solution after 50~100 μ l conversions is taken to be coated on the YPD flat boards containing Zeocin (100 μ g/ml), 30 DEG C of incubators Culture 2~3 days；Then transformed yeast bacterium is screened with PCR method；After bacteria recovered by centrifugation, yeast genes are extracted with glass bead method Group DNA go forward side by side performing PCR identification, amplified production carry out 1.0% agarose gel electrophoresis；

   The expression of the new restructuring bee venom peptides of four,：The clone that qualification result is positive in step 3 is taken to be inoculated in 10ml BMGY, pH For in 6.0 culture mediums, 30 DEG C of concussion and cultivates 24 hours, cell is collected when reaching 2.0~6.0 to OD600, with isometric (10ml) Cell precipitations, 30 DEG C of concussion and cultivates, induced expression is resuspended for 6.0 in BMMY, pH；In Induction Process, first of every 24 hours supplements Alcohol supplements sterilizing ultra-pure water to final concentration 0.5%, zymotic fluid cumulative volume is kept constant；In culture the 0th, 24,48, 72nd, 96,120,144 and 168 hours equi-time points respectively take 0.5ml zymotic fluids, centrifuging and taking supernatant.
4. 4. application of the new restructuring bee venom peptide in uterine neck cancer drug is treated as claimed in claim 1.
5. 5. application as claimed in claim 4, it is characterised in that：Described cervical carcinoma comes from cervical cancer tumer line Hela (HPV18)、Caski(HPV16)。