CN107722115A

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

Info

Publication number: CN107722115A
Application number: CN201711223623.5A
Authority: CN
Inventors: 许天敏
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2017-11-29
Filing date: 2017-11-29
Publication date: 2018-02-23
Also published as: CN108623668A; CN108623668B

Abstract

The present invention relates to a kind of new restructuring bee venom peptide, amino acid sequence is SEQ ID No.1, the nucleotides sequence of encoding novel restructuring bee venom protein is classified as SEQ ID No.2, preparation method is that UA8 small peptides are connected with Melittin gene to be connected into restructuring bee venom fusion, structure restructuring bee venom peptide fusion eukaryotic expression carries U melittin pPICZ α, it is transferred to Pichia pastoris induced expression restructuring bee venom protein and is expressed and purified, the present invention has stronger antitumor activity and less hemolytic toxicity relative to melittin, prevention and treatment applied to the cervical carcinoma caused by HPV viruse.

Description

A kind of new restructuring bee venom peptide and its preparation method and application

Technical field

The invention belongs to biological technical field, and in particular to a kind of new restructuring bee venom peptide and preparation method thereof and should With.

Background technology

Cervical carcinoma is one of three big malignant tumour of female reproductive system, and its incidence of disease and fatal rate are in developing country women Second and the 3rd are occupied in malignant tumour respectively, and rejuvenation situation is notable, has seriously endangered the physical and mental health of women.According to not Count completely, the whole world new hair cervical carcinoma 528000 in 2012, lethal 266000, the cervical cancer pathogenesis rate of East Asia crowd is 7.9/10 ten thousand.Because China human mortality radix is big, cervical carcinoma screening, prophylactico-therapeutic measures are not yet popularized in an all-round way, and number of the infected is year by year in recent years Improve, account for the 1/3 of whole world morbidity sum.The pathogenic factors of cervical carcinoma is complicated, contact it is the closest be high-risk-type human papilloma The persistent infection of virus.In almost all of aggressive cervical carcinoma and 95% Cervical intraepitheliaI neoplasia, carcinoma in situ pathological tissue It can detect that HPV.70~80% women infected HPV in all one's life, wherein 10% women faces continuation HPV infection Precarious position, although high-risk HPV infection can cause the generation of high-level CIN and cervical carcinoma, but majority faces in transient Asia Bed changes, and is little because of stability threat of the free HPV genomes to host genome, once HPV DNA are integrated into place Key-gene group, will cause series of genes structure and dysfunction, finally make cell that vicious transformation occur.E6 and E7 is main Carcinogenic protein, plays important carcinogenesis after HPV is integrated into host genome, and E6 can be adjusted by approach such as ubiquitinations The cancer suppressing action of p53 albumen is influenceed, E7 can make Cycle Regulation out of control after being combined with pRb.Therefore early stage effectively suppress HPV and Virus protein E6, E7 expression turn into the effective way of prevention cervical carcinoma.

One of medicine the most frequently used in treatment of human cervical cancer cis-platinum at present, it is mainly to be acted on base on DNA, DNA is combined Cross key is formed, is no longer replicated so as to destroy DNA function, can suppress the synthesis of RNA and protein during high concentration, it is main secondary to make Improve its side effect with for digestive tract reaction, Toxicity of Kidney, bone marrow suppression and auditory nerve toxicity, the current effective ways that lack.It is dry It is the immune substance discharged after cell is infected by the virus to disturb plain (Interferon).Induced by material beyond virus and virus Interferon be referred to as I type and interferon type Ⅱ.Interferon is that one kind has broad anti-viral activity on allogenic cell Albumen, playing again by the regulation and control of cellular genome for its activity, is related to the synthesis of RNA and protein.Have been used for clinic Interferon have three classes：Interferon-' alpha ' is interferon caused by virus induction leucocyte, and β is that virus induction fibroblast produces Interferon, γ is interferon caused by virus induction lymphoid cell.Adverse reaction is mainly heating, tired, myalgia, headache Etc. influenza-like symptom.Next to that slight bone marrow suppression, aminopherase, serum creatinine rise.But interferon is used to treat in clinic HPV infection is ineffective, and HPV infection can not be controlled effectively, and a kind of medicine that can effectively treat HPV viruse of exploitation will The blank in the field can be filled up.

Melittin accounts for the 45%~50% of bee venom dry weight, is the main component of bee venom.Research find bee venom have it is antitumor, Antiviral, antibacterium, anti-inflammatory, decompression, analgesia, the alleviation critical function such as rheumatism and anti-freezing.Wherein, melittin it is antitumor, Antibiosis and antiviral functions receive much concern in recent years.The unique effect of melittin is by its structures shape：Melittin is as a kind of molecule The polypeptide chain for 2840 is measured, N- ends (1-20aa) is hydrophobicity, and C- ends (21-26aa) is hydrophily in specific PH and ion concentration When, self crosslinking of melittin is in alpha-helix tetramer structure, the feature with film combination peptide and memebrane protein transbilayer helix, therefore both It is water-soluble, cells play cytotoxic activity is can dissolve again.In addition, because bee venom peptide molecular weight is relatively small, in the prior art, honeybee The clinical practice of poison mainly has following several：(1) connective tissue disease, such as rheumatic and rheumatoid arthritis；(2) neuritis And neuralgia, such as treat sciatica, trigeminal neuralgia, occipital neuralgia, dorsal nerve root inflammation；(3) angiocardiopathy is such as high Blood pressure atherosclerosis, venous thronbosis, Buerger's disease and thromboendarteritis；(4) allergic disease, such as Bronchial astehma.Compared with the antineoplastic of current clinical practice, also with the lower slightly advantage of immunogenicity.Oophoroma, In the research of the kinds of tumor such as liver cancer, stomach cancer, breast cancer, melittin is to growth of tumour cell inhibitory action, direct dissolution It is confirmed.But application of the melittin in anti-HPV viruse and treatment of human cervical cancer there is no report.According to the morbidity of cervical carcinoma Mechanism, a variety for the treatment of characteristics such as antiviral, antitumor of melittin are by suitable for the treatment of HPV infection, and then cervical carcinoma Preventing and treating purpose.

Problems be present in its application at present as a kind of non-specific micromolecule polypeptide in melittin：First, phosphatidase A2 is close with bee venom peptide molecular weight and has high sensitization, and both can not be realized and efficiently separated by existing traditional purification technique, from And limit the application of melittin；2nd, bee venom peptide nature is extremely unstable, enter it is degradable after blood, so as to have impact on melittin drug effect Performance；3rd, melittin has extremely strong haemolysis side effect, vein note as a kind of strong basicity peptide by cell lysis film Penetrating rear serious haemocylolysis even lethal effect turns into the main reason for limiting its development.The development resistance of Gonna breakthrough melittin Hinder, first, using the advanced biomaterial coating transport melittin such as micro-nano, to improve its stability and targeting specific；Second, Utilize the structure of technique for gene engineering work directional transformation melittin；Third, changing formulation of the melittin as medicine, given from vein Medicine switchs to local application.Application of the bee venom protein in anti-HPV viruse and treatment of human cervical cancer has no identical report, with current clinic Antiviral, the antineoplastic of application are compared, and melittin has that molecular weight is relatively small, the advantages such as immunogenicity is low.UA8 fragments The small peptide being made up of 8 amino acid sequences, melittin is coupled with by transforming its structure to mitigate the haemolysis of melittin The toxic side effects such as toxicity.

The content of the invention

It is an object of the invention to provide a kind of new restructuring bee venom peptide, there is stronger antitumor work relative to melittin Property and less hemolytic toxicity, prevention and treatment applied to the cervical carcinoma caused by HPV viruse.

The amino acid sequence of new restructuring bee venom peptide of the present invention is SEQ ID No.1.

The nucleotides sequence for encoding above-mentioned new restructuring bee venom protein is classified as SEQ ID No.2.

It is a further object to provide more than new restructuring bee venom peptide preparation method, by UA8 small peptides with Melittin gene, which is connected, is connected into restructuring bee venom fusion, and structure restructuring bee venom peptide fusion eukaryotic expression carries U- Melittin-pPICZ α, it is transferred to Pichia pastoris induced expression restructuring bee venom protein and is expressed and purified, specifically include as follows Step：

1. synthesizing new recombinates the nucleotide sequence of bee venom peptide gene code：It will be connected in advance on solid phase carrier CPG The protected nucleotides of active group and trichloroacetic acid react, slough the blocking group DMT of its 5 '-hydroxyl, obtain free 5'- hydroxyls, synthetic DNA raw material phosphoramidite protection nucleic acid monomer, mix with activator tetrazole, obtain nucleosides phosphorous acid activation Intermediate, activate its 3' end, 5'- hydroxyls are still protected by DMT, and condensation reaction occurs for the 5'- hydroxyls with dissociating in solution, connects Get off to carry out band cap to react, there may be only a few 5'- hydroxyls not participate in reaction in condensation reaction, with acetic anhydride and 1- methyl miaows Azoles terminates subsequent reactions, and finally in the presence of oxidant iodine, phosphorous acyl form is changed into more stable phosphotriester, repeat with Upper step is connected until required all bases, is finally purified to obtain the nucleotides of new restructuring bee venom protein coding Sequence：5'-gcc taa agc tgc ttg tcc aag agt atg aaa ttc ttag tca acg ttg ccc ttg ttt tta tgg tcg tgt aca ttt ctt aca tct atg cg g ccc ctg aac cgg aac cgg cac cag agc cag agg cgg agg cag acg cgga ggc gat ccg gaa gcg gga att gga gca gtt ctg aag gta tta acc aca gga ttg ccc gcc ctc ata agt tgg att aaa cgt aag agg caa cag ggt tag-3'；

2. structure restructuring bee venom fusion carrier for expression of eukaryon skeleton U-melittin-pPICZ α, with SacII digestions position Point ccgcgg, SacII cohesive end are added in 5' ends, and EcoRI terminator codon and cohesive end are added to 3'；Afterwards Reacted as follows, 6 μ l 0.5mmol NaCl and the mixing of 25 μm of ol fusions, mixture reacted in 80 DEG C 3min then by Room temperature is gradually down to, pPICZ α C plasmids are sheared by SacII and EcoRI, and the DNA fragmentation for preparing completion is inserted into pPICZ α C carriers, Recombinant plasmid is transferred to Escherichia coli XL-Blue cell, the clone for carrying recombinant plasmid passes through 25 μ g/ml's Bleomycin resistance screens, and 0.8% agarose gel electrophoresis, gel extraction PCR primer, takes 20~25 μ g expression vector skeletons U- Melittin-pPICZ α are after SacI enzymic digestions, with phenol/chloroform and with ethanol precipitation, the recombinant expression plasmid of linearisation Dissolved with 10 μ l ultra-pure waters rearmounted standby on ice；

3. establish the expression system of new restructuring bee venom peptide：From picking on the YPD feminine gender culture plates of pichia pastoris X-33 Single bacterium colony, it is inoculated in 5ml YPD culture mediums, 250rpm, 30 DEG C of concussion and cultivates 8 hours, competent yeast is prepared with conventional Cell；Then the 80 above-mentioned competence bacterias of μ l are taken, are mixed with the recombinant expression plasmid of obtained linearisation in 20~25 μ g steps 2, Move into 0.2cm electricity conversion cups and carry out electric conversion；The bacterium solution after 50~100 μ l conversions is taken to be coated on containing Zeocin (100 μ g/ml) YPD flat boards on, 30 DEG C of incubator cultures 2~3 days, observe the upgrowth situation of transformant；Then PCR method, screening conversion are used Saccharomycete；After bacteria recovered by centrifugation, with glass bead method extraction pastoris genomic dna go forward side by side performing PCR identification, amplified production carry out 1.0% agarose gel electrophoresis；

4. the expression of new restructuring bee venom peptide：The clone that qualification result is positive in step 3 is taken to be inoculated in 10ml BMGY, PH is 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 Cell precipitations, 30 DEG C of concussion and cultivates, induced expression is resuspended for 6.0 in (10ml) BMMY, pH；In Induction Process, supplement within every 24 hours Methanol supplements sterilizing ultra-pure water to final concentration 0.5%, zymotic fluid cumulative volume is kept constant；In culture the 0th, 24th, 48,72,96,120,144 and 168 hours equi-time points respectively take 0.5ml zymotic fluids, centrifuging and taking supernatant.

It is a still further object of the present invention to provide application of the new restructuring bee venom peptide in uterine neck cancer drug is treated, especially It is related to by suppressing high-risk HPV 16, the expression of 18 type virus proteins is treated and prevented and treated in anti-HPV and drawn by HPV infection Application in the cervical carcinoma risen.

The protein component that is mainly characterized by of new restructuring bee venom peptide of the present invention includes ornamental equivalent and melittin Bulk composition, produced using pichia yeast expression system, make it have stronger antitumor activity and less non-specific raw Thing toxicity.For the bottleneck problem in current cervical carcinoma preventing and treating process and the blank in melittin research, the present invention is according to bee venom Peptide is antiviral, the extensive biological characteristics such as antitumor, 8 amino acid short peptides of addition melittin primary structure is transformed with Enhancing specificity mitigates hemolytic toxicity, expresses restructuring melittin by Pichia yeast expression system, it has further been found that novel heavy Group bee venom peptide has the expression for suppressing two kinds of cervical carcinoma high-risk viral E6/E7 albumen of HPV16, HPV18 and suppression cervical carcinoma thin Intracellular growth, promote its apoptosis etc. to act on, reach the effect of anti-HPV viruse, it is can apply to the palace caused by HPV viruse The prevention and treatment of neck cancer.Under each group administration concentration, suppression that melittin and new restructuring bee venom peptide increase tumor volume Make is strengthened with dose dependent, and wherein the new restructuring bee venom peptide of 8mg/kg melittins and 80mg/kg increases to tumor volume Long inhibitory action is substantially better than cis-platinum, but senior middle school's low dose group corresponding to two kinds of polypeptides maintains an equal level to the inhibiting rate of kind of knurl, without bright Significant difference is different.Melittin and new restructuring bee venom peptide are compared as follows table to hela and caski tumor weights inhibiting rate (%) 1。

Table 1

Brief description of the drawings

Fig. 1：The new restructuring bee venom peptide effect Hela, Caski of different quality concentration (0,10,20,40,80ug/ml) 24h, 48h, it increases with the increase of concentration and action time Hela, Caski growth inhibition effects；To Hela, Caski IC50 when acting on 24h is respectively 49.83ug/ml, 32.76ug/ml；

Fig. 2：Hela cells are after the new restructuring bee venom protein of 4 concentration is handled 24 hours, with new restructuring bee venom Peptide concentration is raised, and cell quantity is reduced, and gap increase, volume-diminished, form shrinkage is rounded；

Fig. 3：Caski cells are after the new restructuring bee venom peptide of 4 concentration is handled 24 hours, as concentration raises, carefully Born of the same parents' quantity is reduced, and gap increase, volume-diminished, form shrinkage is rounded；

Fig. 4：Hela cells are after the new restructuring bee venom peptide of 4 concentration is handled 24 hours, as concentration raises, HPV18E7, HPV18E6 expression quantity decline；

Fig. 5：Caski cells are after the new restructuring bee venom peptide of 4 concentration is handled 24 hours, as concentration raises, HPV16E7, HPV16E6 expression quantity decline；

Fig. 6：Hela cells are by new restructuring bee venom peptide processing 24h, with the increase of drug concentration, early apoptosis and Middle and advanced stage apoptosis increases, and adds as leading with middle and advanced stage increasing apoptosis；

Fig. 7：Caski cells are by new restructuring bee venom peptide processing 24h, with the increase of drug concentration, early apoptosis And middle and advanced stage apoptosis increases, and add as leading with middle and advanced stage increasing apoptosis；

Fig. 8：Under each group administration concentration, cervical carcinoma tumor volume increases and increased with the time.To tumor volume The inhibitory action of increase strengthens in dose dependent, and the inhibitory action that wherein 80mg/kg increases to tumor volume is substantially better than suitable Platinum；

Fig. 9：The expression of Hela cervical carcinoma tumor bodies E6, E7 after the processing of each administration group：With dense Degree rise, HPV16E7, HPV16E6 expression quantity decline, and E6 is particularly evident, and the expression quantity of 80mg/kg administration groups E6, E7 is obvious Less than interferon and cis-platinum group；

Figure 10：The expression of Caski cervical carcinoma tumor bodies E6, E7 after the processing of each administration group：With Concentration raises, and HPV16E7, HPV16E6 expression quantity decline, and E6 is particularly evident, and the expression quantity of 80mg/kg administration groups E6, E7 is bright It is aobvious to be less than interferon and cis-platinum group.

Embodiment

Below by the preferred forms of the embodiment description present invention.These embodiments are further intended to citing and illustrate this Invention, rather than it limit the invention in any way the scope of accompanying claims.

Embodiment 1：A kind of specific preparation method of new restructuring bee venom peptide comprises the following steps：

1. synthesizing new recombinates the nucleotide sequence of bee venom peptide gene code：It will be connected in advance on solid phase carrier CPG The protected nucleotides of active group and trichloroacetic acid react, slough the blocking group DMT of its 5 '-hydroxyl, obtain free 5'- hydroxyls, synthetic DNA raw material phosphoramidite protection nucleic acid monomer, mix with activator tetrazole, obtain nucleosides phosphorous acid activation Intermediate, activate its 3' end, 5'- hydroxyls are still protected by DMT, and condensation reaction occurs for the 5'- hydroxyls with dissociating in solution, connects Get off to carry out band cap to react, there may be only a few 5'- hydroxyls not participate in reaction in condensation reaction, with acetic anhydride and 1- methyl miaows Azoles terminates subsequent reactions, and finally in the presence of oxidant iodine, phosphorous acyl form is changed into more stable phosphotriester, repeat with Upper step is connected until required all bases, and the color that TCA processing stages can be observed in building-up process judges synthesis Efficiency, finally purified to obtain the nucleotide sequence of new restructuring bee venom protein coding：5'-gcc taa agc tgc ttg tcc aag agt atg aaa ttc ttag tca acg ttg ccc ttg ttt tta tgg tcg tgt aca ttt ctt aca tct atg cg g ccc ctg aac cgg aac cgg cac cag agc cag agg cgg agg cag acg cgga ggc gat ccg gaa gcg gga att gga gca gtt ctg aag gta tta acc aca gga ttg ccc gcc ctc ata agt tgg att aaa cgt aag agg caa cag ggt tag-3'。

2. structure restructuring bee venom fusion carrier for expression of eukaryon skeleton U-melittin-pPICZ α, with SacII digestions position Point ccgcgg, in order to which the restructuring bee venom peptide DNA that will have been synthesized inserts pPICZ α C plasmids, SacII cohesive end is added in 5' ends, EcoRI terminator codon and cohesive end are added to 3'；The yield synonym of increase recombinant polypeptide is determined The codon of yield is replaced, and reaction condition is as follows, and 6 μ l 0.5mmol NaCl, 25 μm of ol single stranded DNAs mixing, mixture is in 80 DEG C Reaction 3min is then gradually decreased to room temperature .pPICZ α C plasmids and sheared by SacII and EcoRI, and the DNA fragmentation for preparing completion is inserted Enter pPICZ α C carriers, recombinant plasmid is transferred to Escherichia coli XL-Blue cell, carry the clone of recombinant plasmid By bleomycin (25 μ g/ml) resistance screening, PCR reaction systems are established in 0.2ml EP pipes in following ratio：Recombinate matter Grain 100ng；10 × LA PCR buffer solutions 10 μ l, dNTPs (each 2.5mmol/L) 8 μ l, μ l (the 20pmol/ μ of primer 1 containing Mg2+ L), the μ l of TaKaRa LA Taq (5U/ μ l) 1, sterilizing ultra-pure water carry out cyclic amplification to the μ l of final volume 100 in PCR instrument；Amplification Program is：94 DEG C 4 minutes；94 DEG C 30 seconds, 50 DEG C 30 seconds, 70 DEG C 1 minute, totally 30 circulations, then 70 DEG C 8 minutes；0.8% fine jade Sepharose electrophoresis, gel extraction PCR primer, 20~25 μ g expression vector u-melittin-pPICZ α is taken through SacI enzymic digestions Afterwards, it is with phenol/chloroform and rearmounted standby on ice with the dissolving of 10 μ l ultra-pure waters with ethanol precipitation, the recombinant expression plasmid of linearisation With；- GGT TCT CGA the TGG of 5 '-GTT CCA TCG AAC TGT GAC CGA TGC TG-3 ' of sequence 5 ' of described primer TGG TGA GTT TCC AT-3′；

Embodiment 2：The influence that new restructuring bee venom peptide is expressed cervical cancer tumer line and HPV16/18E6, E7

(1) growth inhibition test of new restructuring bee venom peptide cervical cancer tumer line

A. select the cell Hela (HPV18 is positive) and Caski (HPV16 is positive) of exponential phase to be digested with pancreatin, be made Cell suspension, cell counting count board are counted, and cell is diluted into density 4*104/ml.After the fully mixed hook of the cell diluted, connect Kind adds 100ul (being added along side wall, do not rock) per hole in orifice plate.

B.0ug/ml, tetra- concentration gradient groups of 20ug/ml, 40ug/ml, 80ug/ml, each concentration set 3 multiple holes.Treat After the kind hour cell of plate 12 is adherent, various concentrations medicine is added, is acted on 24 hours and 48 hours.Drug containing training base is abandoned, is added new The culture medium of the solution containing 10%CCK-8.

C.37 after DEG C culture 1-4 hours, 450nm OD values are determined with ELIASA.

D. 24 hours and 48 hours various concentrations inhibiting rate and IC50 to Hela and Caski cells are calculated.As a result such as Fig. 1 It is shown.

E. observation adds Hela and Caski morphological changes after various concentrations under inverted microscope.Such as Fig. 2 and Fig. 3 institutes Show.

(2) inhibitory action of the new restructuring bee venom peptide to HPV18E6, E7 and HPV16E6, E7 protein expressions

Each group cell after drug-treated is digested through pancreatin, collects cell, PBS is washed 1 time.

A. lysate splits and (contains PMSF).It is subsequently placed on ice.

B. after cracking 30min, you can moved to lysate in 1.5ml centrifuge tubes with pipettor, then at 4 DEG C 12000rpm centrifuges 5min, takes supernatant to be sub-packed in 0.5ml centrifuge tubes and is placed in -20 DEG C of preservations.

C.BCA methods detect protein concentration, calculate applied sample amount.

D.SDS-PAGE electrophoresis：Concentrate glue 80V*30min, separation gel 120V*1h.

E. transferring film：300mA*30min.

F. close：5% skim milk closes 1h.

G. E6, E7, β-actin primary antibodies are incubated, 4 DEG C overnight.

H. secondary antibody, 45min are incubated.

I. develop the color, handle image, as shown in Figure 4 and Figure 5.

(3) new restructuring bee venom peptide promotes cervical cancer tumer line apoptotic effect

A. Hela the and Caski cells of exponential phase are digested with pancreatin, cell suspension is made.Cell counting count board meter Number, density 4*10 is diluted to by cell⁴/ ml, after the fully mixed Uniform of the cell diluted, 6 orifice plates are inoculated in, 2ml is added per hole. Inoculation adds various concentrations medicine after adherent, acts on 24 hours.

B. by different pharmaceutical concentration act on after cell culture fluid suck respectively it is standby in different centrifuge tubes, with without EDTA trypsin digestion cells simultaneously will digest lower different group cells and correspond to respectively and add in different centrifuge tubes, 5 points of 1000rpm centrifugations Supernatant is abandoned in clock, suction, adds the PBS of 1ml precoolings, and piping and druming is mixed, and 1000rpm is centrifuged 5 minutes, and supernatant is abandoned in suction.

C. washed again with PBS.Add 100ul Binding Buffer fully to mix in per solencyte, add 5ul Annexin V/FITC mix to be incubated 5 minutes after room temperature lucifuge, adds 10ul 20ug/ml phone the third ingot solution, and add 400ulPBS, flow cytometer detection its apoptosis is carried out at once, as a result as shown in Figure 6 and Figure 7.

Embodiment 3：New restructuring bee venom peptide suppresses in cervical carcinoma and HPV16/18E6, E7 expressional function tumor bearing nude mice body Experiment

(1) model of nude mice bearing tumor is established and is grouped

A.Balb/c-nu nude mices, female, in 6-8 weeks mouse age, body weight 18-22g, raise, 60 totally under SPF level environment.Take HeLa, Caski cell in exponential phase, with serum-free medium centrifuge washing 2 times after 0.25% Trypsin Induced, And it is 10^7/ml to adjust cell concentration with serum-free medium.Extracted under aseptic condition with the syringe of No. 6 syringe needles of band 0.2m1 cell suspension inoculations armpit on the right side of the nude mice through 75% alcohol disinfecting is subcutaneous.

B. nude mice by subcutaneous is vaccinated after about 4 days and tubercle occurs or to tumor tissue growth to about 150mm³When, carry out with Machine is grouped.Volume V=major diameter * minor axis * indulges footpath

C. it is grouped：Hela will be inoculated with, Caski mouse is respectively set to A, and B groups, every group sets following 6 groups, every group 5 respectively Only.

Blank control group：Physiological saline

Positive controls 1：Cis-platinum 2.5mg/kg

Positive controls 2：The U/kg of interferon alpha-2 500,000

New restructuring bee venom peptide low dose group：20mg/kg

New restructuring bee venom peptide middle dose group：40mg/kg

New restructuring bee venom peptide high dose group：80mg/kg

(2) tumor tissues size and tumor-like hyperplasia are measured

A. it is administered once according to the reagent and dosage multi-point injection of above-mentioned packet per 24h to tumor locus, continuous 20 days. Every 4 days of period measured the growing state of knurl body, calculated knurl volume, drew knurl body growth curve.

B. 24h after being discontinued, blood sampling, animal is put to death, takes out the heart, liver, kidney, lung, peeled off tumor mass, calculating of weighing, carry out follow-up Experiment.

C. tumor-like hyperplasia (%)=(average knurl weight of the average knurl weight/blank control group of 1- administration groups) * 100% is calculated

As a result illustrate：Tumor-like hyperplasia strengthens in dose dependent, and wherein 80mg/kg tumor-like hyperplasias are significantly greater than cis-platinum And interferon, as shown in Fig. 8 and table 2 and table 3.

Table 2

The tumor-like hyperplasia of Hela tumor-bearing mices

Table 3

The tumor-like hyperplasia of Caski tumor-bearing mices

(3) in tumor tissues HPV18, HPV16 E6, E7 expressing quantity measure

1/3 tissue block is taken from liquid nitrogen, is shredded tissue block with clean scissors.

A. lysate is split and (contains PMSF) in homogenizer, is homogenized.It is subsequently placed on ice.

C.BCA methods detect protein concentration, calculate applied sample amount.

E. transferring film：300mA*30min.

F. close：5% skim milk closes 1h.

G. E6, E7, β-actin primary antibodies are incubated, 4 DEG C overnight.

H. secondary antibody, 45min are incubated.

I. develop the color, processing image is as shown in Figure 9 and Figure 10.

Experiment is obtained as drawn a conclusion above：New restructuring bee venom peptide is used to suppress two kinds of cervical carcinomas of HPV16, HPV18 The expression of high-risk viral E6/E7 albumen and suppression cervical cancer cell growth, promote its apoptosis, reach the effect of anti-HPV viruse, make It can apply to the prevention of the cervical carcinoma caused by HPV viruse and treatment.It is thin that described high-risk HPV refers in particular to infection Hela The HPV18 of born of the same parents, the HPV16 for infecting caski cells.Wherein described high-risk HPV tumorigenesis albumen refers in particular to E6, E7 albumen.To HPV16, HPV18 of the new restructuring bee venom peptide of various dose is given to carry out in vitro study and grind in vivo with Western Blotting Detect tumorigenesis albumen E6, E7 expression quantity.Wherein described various dose is respectively negative control group, low dose group, middle dosage Group and high dose group.Wherein described in vitro study is cervical cancer tumer line Hela (HPV18), Caski (HPV16) cell is trained Support.Research object is cervical carcinoma model of nude mice bearing tumor inside wherein described.By using observation and CCK-8 methods under the microscope The inhibited proliferation of new restructuring bee venom peptide is detected, passes through the rush apoptosis of the new restructuring bee venom peptide of Flow cytometry Effect.With the increase of new restructuring bee venom peptide concentration, HPV18E6/E7 and HPV16E6/E7 expression quantity gradually reduces, explanation New restructuring bee venom peptide can effectively suppress the expression of high-risk HPV 18 and HPV16 tumorigenesis albumen, anti-so as to reach HPV18, HPV16 effect, further prevent cervical carcinoma.Research object is cervical carcinoma tumor bearing nude mice mould inside wherein described Type, the growth inhibition effect of new restructuring bee venom peptide is detected by tumor mass growth curve and tumor-like hyperplasia.This method is found New restructuring bee venom peptide can effectively suppress the propagation of cervical cancer cell, and promote its apoptosis, and further melittin has bright Aobvious antitumaous effect.

Embodiment 4：New restructuring bee venom peptide hemolytic test

The preparation of 2% red cell suspension takes 10~20ml of new fresh rabbit blood, is put into the conical flask for fill bead and shakes 10 minutes, or blood is stirred with glass bar, celloglobulin is removed, makes into defibrinated blood.Physiological water 100ml is added, is shaken up, 1000~1500r/min is centrifuged 15 minutes, removes supernatant, and the red blood cell of precipitation washs 2 as stated above with physiological saline again ~3 times, untill the not aobvious red of supernatant.With physiological saline gained red blood cell is made into 2% suspension, and (red blood cell 2ml, adds Physiological saline is to 100ml), it is for experiment.The clean teat glasses of 10ml 8 are taken, numbers, as shown in table 4, it is red to sequentially add 2% Cell liquid.0.9% sodium chloride solution or distilled water, after mixing, put in 37 ± 0.5 DEG C of water bath with thermostatic control and incubated immediately, see Examine and record the haemolysis situation of each pipe.Start after observation in 30 minutes 1 time, 1 hour, observed 1 time every 1 hour, it is general to see Examine 3 hours.Melittin and new restructuring bee venom peptide hemolytic test are more as shown in table 2.

Table 4

Note：- represent not haemolysis+expression part haemolysis ++ represent full haemolysis

The degree of hemolysis of melittin and new restructuring bee venom peptide is raised and raised with concentration, during corresponding two kinds of polypeptides are low High group is compared, and new restructuring bee venom protein significantly improves the hemolytic toxicity of melittin.

Prepare embodiment

It is a kind of to treat HPV and medicine that cervical carcinoma contains new restructuring bee venom peptide contains following weight Composition：New restructuring bee venom peptide 0.05-5%；Carbomer 0.1-1%；Triethanolamine 0.5-3% borneols 3-6%；Glucose 1- 5%；Glycerine 0.5-5%；Remaining is water；Described borneol is selected from natural borneol and/or synthetic borneol.Described pharmaceutical composition It is prepared into vagina administration preparation type.Described preparation includes suppository, ointment, capsule, effervescent tablet, gel, lotion, film Or foaming agent.

Prepare embodiment 1：

New restructuring bee venom peptide pessary, formula：New restructuring bee venom peptide 8g, gelatin 1000g, glycerine 1600g, Mannitol 6g, lactose 6g, citric acid 5g, glutaric acid 5g, sodium citrate 4g, beta-schardinger dextrin 6g.

Specifically preparation process is：

A. the preparation of matrix：1000g gelatin is weighed, the pure water for adding equivalent impregnates to put for 1 hour 80 DEG C are heated in water-bath Dissolving, it is a certain amount of (1600g) to add glycerine, stirring, boils off unnecessary moisture；

B. mannitol 6g, lactose 6g, citric acid 5g, glutaric acid 5g, sodium citrate 4g, beta-schardinger dextrin are added in matrix 6g, stir evenly, temperature control adds new restructuring bee venom peptide 8g at 37~40 DEG C, pours into and has sterilized at 37~40 DEG C after stirring evenly And scribble in the vaginal plug mould of lubricant；

C. usual room temperature is cooled to, temperature control prunes die orifice and overflow part, be stripped, sealing, obtain newly at 15~25 DEG C Type recombinates bee venom peptide suppository finished product；

Prepare embodiment 2：

New restructuring bee venom peptide vaginal ointment, formula：New restructuring bee venom peptide 6g, dodecyl sodium sulfate, which is modified, to be covered De- native 50g, atoleine 480g, glycerine 25g, isopropyl myristate 200g, Cyclomethicone 30g, stearyl alcohol 55g, borneol The specific preparation process of 10g, peppermint 2g is：

A. weigh Organic Montmorillonitewith Sodium Laurylsulfonate 50g and be dissolved in atoleine 480g, be placed in wet-milling point in colloid mill Dissipate 5 minutes, after fully dispersed, add glycerine 25g and continue to disperse 5 minutes, obtain Organic Montmorillonitewith Sodium Laurylsulfonate and disperse shape Into atoleine gel

B. 70 degrees Celsius are heated to, is then slowly added new restructuring bee venom peptide 6g thereto, is stirred in addition, so Addition is preheated to 55 degrees Celsius of isopropyl myristate 200g, Cyclomethicone 30g, stearyl alcohol 55g, borneol 10g, peppermint afterwards 2g, it is stirring while adding uniformly, it is to be mixed uniformly after, stop heating, continue stirring to room temperature, it is soft to produce new restructuring bee venom peptide Cream.

Prepare embodiment 3：

New restructuring bee venom peptide vagina effervescence, formula：New restructuring bee venom peptide 8g, 200 grams of mannitol, hydroxypropyl 30 grams of cellulose, 20 grams of sodium carboxymethyl starch, 180 grams of sodium acid carbonate, 200 grams of citric acid, 3 grams of magnesium stearate.It is specific to prepare step Suddenly it is：

A. the sodium acid carbonate and citric acid of formula ratio are pressed, is sufficiently mixed, crosses 80 mesh sieves.

B. plus appropriate ethanol solution, wet granular processed, excessively mixing, 20 mesh sieves, 42~45 DEG C of dryings are dried for 4~6 hours, Obtain effervescence granular.

C. by being formulated new restructuring bee venom peptide, it is diluted to debita spissitudo.

D. other auxiliary materials of formula ratio are taken, after being well mixed by coubling dilution, cross 80 mesh sieves；Add new restructuring bee venom more Peptide solution softwood, cross 20 mesh sieves, wet granular processed.

E.42~45 DEG C drying 4-6 hours；Moisture is surveyed, moisture it is more should to obtain new restructuring bee venom below 1% Peptide particles.

F. after effervescence granular is well mixed with new restructuring bee venom peptide particle, addition magnesium stearate, tabletting, packaging, Obtain new restructuring bee venom peptide effervescent tablet.

Prepare embodiment 4：

New restructuring bee venom peptide vaginal jellies：Formula：New restructuring bee venom peptide 12g, chlorhexidine acetate 1.6g, card ripple Nurse 10g, glycerine 120g, triethanolamine 4g, ethyl hydroxy benzoate 1g, remaining be purified water.Specific preparation process is as follows：

A. 1000g gels are prepared, take powder after new restructuring bee venom peptide 12g, chlorhexidine acetate 1.6g mixing by weight It is broken, it is standby to cross 100 mesh sieves；

B. take carbomer 10g, triethanolamine 4g and purified water standby by weight；Carbomer is uniformly spread on to purifying water meter Face, place 40-48 hours be fully swelled naturally after add triethanolamine and stir, adjust PH5-6, matrix be made；

C. it is uniformly mixed in the crushing medicine prepared in step a by weight addition glycerine 120g, ethyl hydroxy benzoate 1g It is standby to obtain mixture；

D. take the mixture in step c to be added gradually under stirring addition purified water in matrix made of step 2 to mix Uniformly 1000g gels are made in conjunction；

E. take the gel that step d is obtained new according to being obtained after the weight progress filling and package of 5 grams every of specification requirement Recombinate bee venom peptide vaginal jellies product.

Prepare embodiment 5：

New restructuring bee venom peptide lotion：Formula：New restructuring bee venom peptide 15g, Chinese knotweed 20g, sweet wormwood 20g, punching Lotus 20g, peppermint 5g, borneol 5g, nine inner bright 5g, water 1000ml.

The preparation method of above-mentioned gynecologic vaginal irrigation liquid is as follows：

Raw material are taken by weight ratio：Chinese knotweed, sweet wormwood, Herba Andrographitis, peppermint, borneol, nine inner bright；

Above-mentioned medicinal material is cleaned, chops up to be put into add water in flask and boils；

After its cooling after, with screen filtration twice after, collect filtrate；

By being formulated new restructuring bee venom peptide, debita spissitudo is diluted to.

New restructuring bee venom peptide solution is added into filtrate and adds benzoic acid anti-corrosion, bottling is standby.

Prepare embodiment 6：

New restructuring bee venom peptide albumen dressing, it is formulated (1000g)：Carbomer 10g, peppermint 5g, green-tea extract 20g, Glycerine 50g, nipagin esters 25g, Phenoxyethanol 20g, remaining is water.Comprise the following steps：

A. first glycerine, peppermint and green-tea extract are dissolved into pure water, then add carbomer, it is fully dissolved, Then the new restructuring bee venom peptide albumen crossed with 0.22 μm of membrane filtration is added, continues to stir, obtains homogeneous system.

B. well mixed Phenoxyethanol and nipagin esters are added, surplus is supplied with pure water, stirs.

C. obtain one liter of invisible film is finally adjusted into pH value to 4.5 to 6.5 with the NaOH solution that mass concentration is 20% Between, get product.

Clinical implementation is tested

This experiment is the new restructuring bee venom peptide suppository of the present invention, to not with the HPV persistent infections of high-level cervical lesionses The clinical observation of person's curative effect.

Research object selects：Detect positive, checked after 6 months still positive for HPV persistent infections (HPV-DNA)) and not companion High-level cervical lesionses, owner voluntarily enter group and sign informed consent form.

Inclusion criteria：

1 the past is without operation on cervix history

(row TCT checks that the patient for ASCUS and the above enters for TCT results for the high-level lesion of 2 exclusion uterine neck and canceration One walking vaginoscopy)

3 without new restructuring bee venom peptide contraindication

Antiviral drugs and immunomodulator are not used in 4 half a year

5 non-pregnancies, nursing period women

6 without acute inflammatory diseases such as acute vaginitis, acutes pelvitis of pelvic cavity

7 without irregular vagina bleeding

8 without immunologic hypofunction (malignant tumour, SLE, AIDS etc.)

9 have follow-up condition and partner treatment

10 all patients sign informed consent form, this clinical test of voluntary participation

Exclusion standard：

1 immunologic hypofunction person, after tumor post-operation or Radiotherapy chemotherapy, AIDS and systemic loupus erythematosus etc.；

2 gestation and women breast-feeding their children；

3 suffer from acute genital inflammation, such as gonorrhoea, mycoplasma or choamydiae infection；

Control group：Without using any pharmaceutical intervention, patient is practised contraception using sheath during observation；

Treatment group：Before sleeping after cleaning vulva, medicine is pushed into fornix portion using propeller, a 2g, once a day connected With being discontinued after 10 days, the next menstrual cycle repeats.Menstrual period disables, and next cycle the starts medication for 3-5 days after clean period, HPV is checked after 3 menstrual cycles of continuous use, and the HPV patients that turn out cloudy discontinue medication, and the HPV patients that do not turn out cloudy are further continued for using bee venom 3 menstrual cycles of peptide gel：Forbid hip bath and sexual life during medication, practised contraception in medication later six months using sheath.

Curative effect determination methods：

Turn out cloudy：HPV (-) during check；

Effectively：Quantitatively reduced in check HPV infection, but it is not fully erased

It is invalid：During check, HPV infection is quantitatively reduced or quantitatively increased.

As a result：Treat 3 Ge Yue treatment groups HPV clearance rates and effective percentage is higher than control group.After 6 months, treatment group HPV is clear Except rate and effective percentage are also high than control group.The effect of checking treatment group and control group after medication after 3 months and 6 months contrast is detailed It see the table below 5.

Table 5

Note：Clearance rate=N turns out cloudy/and N total numbers of persons × 100% is efficient=(N turn out cloudy+N effective)/N total number of persons × 100%

It these results suggest that the new restructuring bee venom peptide suppository of the present invention has significant curative effect to the treatment of people's HPV infection.

SEQUENCE LISTING

<110>Jilin University

<120>A kind of new restructuring bee venom peptide 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

A kind of 1. new restructuring bee venom peptide, it is characterised in that：Amino acid sequence is SEQ ID No.1.
2. the nucleotides sequence of coding new restructuring bee venom peptide as claimed in claim 1 is classified as SEQ ID No.2.
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. application of the new restructuring bee venom peptide in uterine neck cancer drug is treated as claimed in claim 1.
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)。