CN110327299A - Cationic polymer nanoparticle/hIL-22BP gene composite of anti-curing cancers and its preparation method and application - Google Patents
Cationic polymer nanoparticle/hIL-22BP gene composite of anti-curing cancers and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to the cationic polymer of anti-curing cancers nanoparticle/hIL-22BP gene composites and its preparation method and application, belong to field of biological pharmacy.The present invention provides the cationic polymer of anti-curing cancers nanoparticle/hIL-22BP gene composites, it is that cationic polymer nanoparticle and the recombinant vector containing hIL-22BP encoding gene act on compound obtain by charge adsorption.Compound of the present invention can weaken the growing multiplication signal of tumour cell by direct inducing apoptosis of tumour cell and blocking IL-22/IL-22R1 transmembrane signal, realize the inhibition to tumor cell proliferation, achieve the effect that treat tumour.Zoopery proves that compound of the present invention has the function of inhibiting the growth of the kinds of tumors such as lung cancer, oophoroma, liver cancer, colon cancer, and it is a kind of cancer treatment drugs having a extensive future that average tumor inhibiting rate, which is more than 60%,.
Description
Technical field
The present invention relates to the cationic polymer of anti-curing cancers nanoparticle/hIL-22BP gene composites and preparation method thereof
And purposes, belong to field of biological pharmacy.
Background technique
Gene therapy is one of the important means of tumor biotherapy, and multiple gene therapy products are controlled in the clinic of tumour
It plays a role in treatment.As the important component of gene therapy medicament, the therapeutic genes for oncotherapy pass through inhibition
Tumor cell proliferation promotes the mechanism such as apoptosis of tumor cells and regulation antineoplastic immune, realizes antineoplaston effect.Into one
Step is found and gene of the exploitation with good therapeutic effect is therapy of tumor important topic still to be solved.
Interleukins is generally acknowledged one of inherent immunity and the adaptation most important regulatory factor of immune correlated disease.It is white thin
Born of the same parents' interleukin -22 (Interleukin-22, IL-22) is mainly by adaptive immunity cell (CD4 positive T cell, CD8 positive T cell
Deng) and inherent immunity cell (LTi cell, NK cell etc.) secretion, belong to IL-10 cytokine family member.Since 2000
Since IL-22 is found, have been found to play the weight including anti-inflammatory and immunological regulation in the endothelial cell of numerous tissues
It acts on.IL-22 on the one hand can promote tissue proliferation and regeneration and adjust host Mucosa Barrier defence, on the other hand
It may cause the inflammatory reaction of Histopathologic.As one of the inflammatory cytokine in tumor microenvironment, IL-22 is including knot
The generations of the malignant tumours such as intestinal cancer, liver cancer, gastric cancer, lung cancer, cancer of pancreas, oral squamous cell carcinoma, malignant tumour of skin, development,
It is played an important role in proliferation, apoptosis and invasion transfer.For example, studies have found that, IL-22 and its transmembrane receptor IL-
22R1 high expression in colon cancer tissue, and expression quantity is positively correlated with gross tumor volume size；IL-22/IL-22R1 can be by swashing
STAT3 signal path living inhibits the apoptosis of tumour cell and promotes the growth and transfer of tumour；In addition, IL-22 can also be by solid
There is the expression of lymphocyte to promote the occurrence and development of colon cancer.Therefore, IL-22 is one of important molecule target spot of oncotherapy.
IL-22 binding protein (IL-22 binding protein, IL-22BP) is IL-22 another in addition to IL-22R1
Receptor (IL-22R2).This receptor is present in extracellularly in a manner of soluble protein, can be free with IL-22R1 competitive binding
The IL-22 factor, and block the related biological activities of IL-22, be IL- to prevent the signal transduction in cell factor downstream
22 soluble antagonist.High specific and high-affinity are combined with due to IL-22BP and IL-22, therefore IL-22BP is in vivo
The bioactivity of IL-22 can effectively be adjusted.In addition, the expression of IL-22BP in vivo and IL-22/IL-22R1 function are in height
Correlation, the meeting great expression when the latter's functional imbalance, to play control and balanced action.Therefore, IL-22BP is adjusting IL-
The inflammation and immune response aspect of 22 inductions play a significant role.Since IL-22/IL-22R1 can be logical by activation STAT3 signal
The modes such as road inhibit the apoptosis of tumour cell and promote the generation, growth and transfer of tumour, promote generation and the hair of malignant tumour
Exhibition.In addition, expression of the IL-22BP in cell can also direct inducing apoptosis of tumour cell, to inhibit the increasing of tumour cell
It grows.Therefore, it by promoting tumour cell to the secreting, expressing of soluble IL-22BP receptor, improves to the extracellular free IL-22 factor
Neutralization, block or reduce IL-22 with the IL-22R1 transmembrane receptor on tumor cell membrane in conjunction with, and then reduction tumour it is thin
The growing multiplication signal that born of the same parents are regulated and controled by IL-22, and inducing apoptosis of tumour cell killing simultaneously is, it can be achieved that tumor cell proliferation
Inhibition achievees the effect that treat tumour.
Summary of the invention
The purpose of the present invention is to provide the cationic polymer of anti-curing cancers nanoparticle/hIL-22BP gene composite and
Preparation method and use.
The present invention provides the cationic polymer of anti-curing cancers nanoparticle/hIL-22BP gene composite, it be sun from
Sub- polymer nanoparticle and recombinant vector containing hIL-22BP encoding gene by charge adsorption act on it is compound obtain,
In, cationic polymer nanoparticle: the mass ratio of the recombinant vector containing hIL-22BP encoding gene is (3~30): 1, institute
The nucleotide sequence of hIL-22BP encoding gene is stated as shown in SEQ ID NO.1, the cationic polymer nanoparticle is with sun
What cationic lipid and polymer material were prepared as a raw material.
Further, the compound method is by cationic polymer nanoparticle and containing hIL-22BP encoding gene
Recombinant vector is scattered in aqueous solution, is sufficiently mixed uniformly to get compound.
Preferably, solution left standstill 5~30 minutes after mixing.
Preferably, solution left standstill 15 minutes after mixing.
Preferably, in being stored at room temperature.
Further, the aqueous solution is a kind of in 5% glucose solution, physiological saline, ultrapure water or deionized water
Or it is two or more.
Further, the cationic polymer nanoparticle: the quality of the recombinant vector containing hIL-22BP encoding gene
Ratio is 10:1 or 30:1.
Further, the cationic polymer nanoparticle is prepared with film hydration method.
Preferably, the cationic polymer nanoparticle is prepared with following methods: by cation lipid and polymer
Material co-dissolve in solvent, remove solvent after form a film, take the film out and be dissolved in aqueous solution, fully shake to get.
Preferably, the solvent is methylene chloride.
Preferably, solvent is removed by rotary evaporation.
Preferably, the aqueous solution it is a kind of in 5% glucose solution, physiological saline, ultrapure water or deionized water or
It is two or more.
Further, the cation lipid is or mixtures thereof DOTAP, DC-Cholesteral.
Preferably, the cation lipid is DOTAP.
Further, the polymer material in mPEG-PCL, mPEG-PLA, mPEG-PLGA one or two with
On.
Preferably, the polymer material is mPEG-PCL.
Preferably, the molecular weight of mPEG-PCL is 4000~10000Da.
Preferably, the molecular weight of mPEG-PCL is 4000Da, and wherein mPEG segment is 2000Da, and PCL segment is 2000Da.
Further, the cationic polymer nanoparticle is prepared as a raw material with DOTAP and mPEG-PCL,
The mass ratio of middle DOTAP:mPEG-PCL is 1:(1~20).
Preferably, the mass ratio of DOTAP:mPEG-PCL is 1:9.
Further, the carrier is plasmid vector.
Preferably, the carrier is pVAX1.
Further, the recombinant vector containing hIL-22BP encoding gene, nucleotide sequence such as SEQ ID NO.4
It is shown.
Further, the recombinant vector containing hIL-22BP encoding gene is prepared by the following method to obtain: using Hind
III and Xbal I digestion with restriction enzyme carrier recycles carrier segments, handles and return with identical digestion with restriction enzyme
The hIL-22BP encoding gene segment of receipts be attached reaction to get.
The present invention provides the compound preparation methods: encoding by cationic polymer nanoparticle and containing hIL-22BP
The recombinant vector of gene by charge adsorption act on it is compound to get.
The present invention provides the compounds to prepare the purposes in anticancer drug.
Preferably, the drug is the drug of inhibitor against colon carcinoma cells, oophoroma, lung cancer and/or liver cancer.
The present invention provides the pharmaceutical compositions of anticancer, it is using the compound as active constituent, and addition pharmaceutically may be used
The preparation that the auxiliary material of receiving or complementary ingredient are prepared.
Further, the preparation is ejection preparation or oral preparation.
Further, the preparation is the preparation of inhibitor against colon carcinoma cells, oophoroma, lung cancer and/or liver cancer.
In the present invention, the encoded protein amino acid sequence of hIL-22BP encoding gene (SEQ ID No.5,
263aa) are as follows:
MMPKHCFLGFLISFFLTGVAGTQSTHESLKPQRVQFQSRNFHNILQWQPGRALTGNSSVYFVQYKIMF
SCSMKSSHQKPSGCWQHISCNFPGCRTLAKYGQRQWKNKEDCWGTQELSCDLTSETSDIQEPYYGRVRAASAGSYS
EWSMTPRFTPWWETKIDPPVMNITQVNGSLLVILHAPNLPYRYQKEKNVSIEDYYELLYRVFIINNSLEKEQKVYE
GAHRAVEIEALTPHSSYCVVAEIYQPMLDRRSQRSEERCVEIP。
The present invention provides a kind of novel cation nanometer grain/hIL-22BP gene composite, which can pass through
Direct inducing apoptosis of tumour cell and the growing multiplication for blocking IL-22/IL-22R1 transmembrane signal and then reduction tumour cell
Signal realizes the inhibition to tumor cell proliferation, achievees the effect that treat tumour.Zoopery proves that the present invention is compound
Object has the function of the kinds of tumors growth such as inhibition lung cancer, oophoroma, liver cancer, colon cancer, and average tumor inhibiting rate is more than 60%,
It is a kind of cancer treatment drugs having a extensive future.
Detailed description of the invention
Fig. 1 is pVAX1 plasmid vector construct schematic diagram in embodiment 1；
Fig. 2 is pVAX1-hIL-22BP plasmid construct schematic diagram in embodiment 1；
Fig. 3 is that the agarose that pVAX1-hIL-22BP plasmid is identified with Hind III and XbaI double digestion in embodiment 1 is solidifying
Gel electrophoresis spectrogram；
Fig. 4 is colon carcinoma animal model therapeutic effect figure in test example 1；
Fig. 5 is ovary carcinoma animal model therapeutic effect figure in test example 2；
Fig. 6 is lung cancer animal models therapeutic effect figure in test example 3；
Fig. 7 is liver cancer animal model therapeutic effect figure in test example 4.
Specific embodiment
The present invention provides the cationic polymer of anti-curing cancers nanoparticle/hIL-22BP gene composite, it be sun from
Sub- polymer nanoparticle and recombinant vector containing hIL-22BP encoding gene by charge adsorption act on it is compound obtain,
In, cationic polymer nanoparticle: the mass ratio of the recombinant vector containing hIL-22BP encoding gene is (3~30): 1, institute
The nucleotide sequence of hIL-22BP encoding gene is stated as shown in SEQ ID NO.1, the cationic polymer nanoparticle is with sun
What cationic lipid and polymer material were prepared as a raw material.
The present invention is the following discovery based on inventor and completes:
Based on the IL-22 factor that IL-22BP can dissociate with IL-22R1 competitive binding, so that IL-22 be blocked to promote tumour
Expression of the related biological activities and IL-22BP of growth in cell can directly swell in inducing apoptosis of tumour cell, inhibition
The characteristic of tumor cell proliferation, the present invention, which attempts building, to be used for cancer by the genomic medicine of high expression IL-22BP in tumor tissues
The treatment of disease.For this purpose, inventor selects cationic polymer nanoparticle as gene delivery system, there is low toxicity, load and hold
Amount is big, prepares the advantages such as convenient.
Cationic polymer nanoparticle/hIL-22BP gene composite difficult point is prepared to be to improve its transduction efficiency.And
Inventor has found that the ingredient proportion of nanoparticle and gene is non-for expression of the hIL-22BP in tumor tissues by investigating repeatedly
Chang Guanjian.Specifically, nanoparticle: the recombinant vector mass ratio containing hIL-22BP encoding gene is in (3~30): 1 range
Interior, hIL-22BP genophore could be efficiently directed into cell by obtained compound, could obtain ideal hIL-22BP
Gene transcription level plays apparent therapeutic effect to tumour with the human il-22 BP albumen for ensuring to give expression to.Above-mentioned investigation result
Details are as follows:
The influence of 1 nanoparticle of table and gene ingredient proportion for transduction efficiency
Note: recombinant vector involved by upper table, nanoparticle are prepared referring to the method for embodiment 1,2, differ only in throwing
Material is than different.
The detection method of channel genes efficiency is as follows: blowing and beating the cell after transfection out of hole with liquid-transfering gun, collects
In streaming pipe；And cleaned again with 1mlPBS solution, remaining cell is also collected in streaming pipe together, with 1500rpm from
Heart 3min abandons supernatant, and 1mlPBS is added and is resuspended.Repeated washing is twice, finally heavy according to the appropriate PBS of how much additions of cell precipitation
Flow cytomery transfection efficiency is used after outstanding cell precipitation.The mRNA level in-site detection method of IL-22BP intracellular is as follows: transfection
After 48 hours, cell total rna is extracted using Trizol method, and be cDNA by obtained RNA reverse transcription.Then pass through qPCR method point
Analyse mRNA level in-site.
The present invention can also have following additional technical feature:
According to some embodiments of the present invention, the compound method is by cationic polymer nanoparticle and to contain hIL-
The recombinant vector of 22BP encoding gene is scattered in aqueous solution, is sufficiently mixed uniformly to get compound.Wherein, the aqueous solution
One or more, most preferably ultrapure water preferably in 5% glucose solution, physiological saline, ultrapure water or deionized water
Or deionized water.Compared to the solvent environment of other types, the stable composite being prepared using ultrapure water or deionized water
Property is best, to the importing efficiency optimization of hIL-22BP genophore.Details are as follows for above-mentioned investigation result:
Influence of the 2 solvent type of table to complex stabilities transduction efficiency
Note: recombinant vector involved by upper table, nanoparticle are prepared referring to the method for embodiment 1,2, are differed only in molten
Agent type is different.
The detection method of channel genes efficiency is as follows: blowing and beating the cell after transfection out of hole with liquid-transfering gun, collects
In streaming pipe；And cleaned again with 1mlPBS solution, remaining cell is also collected in streaming pipe together, with 1500rpm from
Heart 3min abandons supernatant, and 1mlPBS is added and is resuspended.Repeated washing is twice, finally heavy according to the appropriate PBS of how much additions of cell precipitation
Flow cytomery transfection efficiency is used after outstanding cell precipitation.Use the particle size dispersion feelings of Malvern ParticleSizer analysis compound
Condition and PDI value.
According to some embodiments of the present invention, the cationic polymer nanoparticle is with DOTAP and mPEG-PCL for original
What material was prepared.Compared to the cation lipid or other polymeric materials of other types such as DC-cholesterol, PEI
Material such as mPEG-PLA, mPEG-PLGA, preferred cationic polymer nanoparticle have stability is high, particle diameter distribution uniformly and
Partial size is small, is conducive to the advantage of gene delivery importing.
According to some embodiments of the present invention, the mass ratio of DOTAP:mPEG-PCL is 1:(1~20).Thus it is prepared into
It is good to stability, and the cationic polymer nanoparticle of small toxicity.
According to some embodiments of the present invention, the molecular weight of mPEG-PCL is 4000~10000Da, most preferably
4000Da, wherein mPEG segment is 2000Da, and PCL segment is 2000Da.Thus the cationic polymer nanoparticle being prepared
Particle diameter distribution is smaller.
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Particular technique or item are not specified in embodiment
Part, it described technology or conditions or is carried out according to the literature in the art according to product description.Agents useful for same or instrument
Production firm person is not specified in device, and being can be with conventional products that are commercially available.
The building of 1 hIL-22BP gene expression plasmid of embodiment
The acquisition of 1.hIL-22BP gene
1) according to US National Biotechnology Information center NCBI (National Center for Biotechnology
Information) the coded sequence synthesis of hIL-22BP (GeneID:116379, NM_052962) contains overall length in database
The cDNA plasmid of hIL-22BP gene order.The coded sequence (SEQ ID NO.1,792bp) of hIL-22BP are as follows:
ATGATGCCTAAACATTGCTTTCTAGGCTTCCTCATCAGTTTCTTCCTTACTGGTGTAGCAGGAACTCA
GTCAACGCATGAGTCTCTGAAGCCTCAGAGGGTACAATTTCAGTCCCGAAATTTTCACAACATTTTGCAATGGCAG
CCTGGGAGGGCACTTACTGGCAACAGCAGTGTCTATTTTGTGCAGTACAAAATCATGTTCTCATGCAGCATGAAAA
GCTCTCACCAGAAGCCAAGTGGATGCTGGCAGCACATTTCTTGTAACTTCCCAGGCTGCAGAACATTGGCTAAATA
TGGACAGAGACAATGGAAAAATAAAGAAGACTGTTGGGGTACTCAAGAACTCTCTTGTGACCTTACCAGTGAAACC
TCAGACATACAGGAACCTTATTACGGGAGGGTGAGGGCGGCCTCGGCTGGGAGCTACTCAGAATGGAGCATGACGC
CGCGGTTCACTCCCTGGTGGGAAACAAAAATAGATCCTCCAGTCATGAATATAACCCAAGTCAATGGCTCTTTGTT
GGTAATTCTCCATGCTCCAAATTTACCATATAGATACCAAAAGGAAAAAAATGTATCTATAGAAGATTACTATGAA
CTACTATACCGAGTTTTTATAATTAACAATTCACTAGAAAAGGAGCAAAAGGTTTATGAAGGGGCTCACAGAGCGG
TTGAAATTGAAGCTCTAACACCACACTCCAGCTACTGTGTAGTGGCTGAAATATATCAGCCCATGTTAGACAGAAG
AAGTCAGAGAAGTGAAGAGAGATGTGTGGAAATTCCATGA。
2) corresponding primer is designed and synthesized
Upstream primer: 5 '-GGGAAAGCTTATGATGCCTAAACATTGCTTTC-3 ' (SEQ ID NO.2), design introduce
Hind III digestion site；
Downstream primer 5 '-GGGATCTAGATCATGGAATTTCCACACATCTC-3 ' (SEQ ID NO.3), design introduce
Xbal I restriction enzyme site；
The DNA fragmentation of hIL-22BP is obtained by PCR amplification, agarose gel electrophoresis recycles the segment, obtains hIL-
22BP genetic fragment.
The clone of 2.pVAX1-hIL-22BP plasmid constructs
1) Hind III and Xbal I restriction enzyme are used, in 37 ° of lower double digestion eukaryotic expression vector pVAX1s, fine jade
Sepharose electrophoresis recycles the pVAX1 carrier segments of linearisation of corresponding size, and is handled with the same terms digestion and passes through fine jade
The hIL-22BP genetic fragment of sepharose electrophoresis recycling is attached reaction 2 hours, connection product conversion in 37 DEG C of water-baths
DH5a competent escherichia coli cell is simultaneously coated on the LB plate of resistance containing kanamycin.Extraction clone is chosen after 24 hours,
It is placed in 3mL LB culture medium containing kanamycin and shakes bacterium overnight incubation, next day extracts pVAX1-hIL-22BP plasmid and recombinates matter
Grain.The structure of pVAX1 plasmid vector is as shown in Figure 1, the structure of pVAX1-hIL-22BP plasmid is as shown in Figure 2.
2) pVAX1-hIL-22BP plasmid is subjected to digestion identification with Hind III and Xbal I restriction enzyme respectively,
Agarose gel electrophoretogram is shown in Fig. 3, and wherein M indicates DNAMarker, and 1 indicates pVAX1-hIL-22BP plasmid, and 2 indicate
The double digestion result of pVAX1-hIL-22BP.Fig. 3 shows successful connection.Constructed pVAX1-hIL-22BP carrier overall length
3717bp, complete sequence (SEQ ID NO.4,3717bp) are as follows:
GACTCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAAT
CAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGG
CTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTC
CATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTA
CGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCC
TACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGT
GGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAA
ATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGA
GGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCAC
TATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTATGATGCCTAAACATTGCTTTCTAGGCTTCCTCAT
CAGTTTCTTCCTTACTGGTGTAGCAGGAACTCAGTCAACGCATGAGTCTCTGAAGCCTCAGAGGGTACAATTTCAG
TCCCGAAATTTTCACAACATTTTGCAATGGCAGCCTGGGAGGGCACTTACTGGCAACAGCAGTGTCTATTTTGTGC
AGTACAAAATCATGTTCTCATGCAGCATGAAAAGCTCTCACCAGAAGCCAAGTGGATGCTGGCAGCACATTTCTTG
TAACTTCCCAGGCTGCAGAACATTGGCTAAATATGGACAGAGACAATGGAAAAATAAAGAAGACTGTTGGGGTACT
CAAGAACTCTCTTGTGACCTTACCAGTGAAACCTCAGACATACAGGAACCTTATTACGGGAGGGTGAGGGCGGCCT
CGGCTGGGAGCTACTCAGAATGGAGCATGACGCCGCGGTTCACTCCCTGGTGGGAAACAAAAATAGATCCTCCAGT
CATGAATATAACCCAAGTCAATGGCTCTTTGTTGGTAATTCTCCATGCTCCAAATTTACCATATAGATACCAAAAG
GAAAAAAATGTATCTATAGAAGATTACTATGAACTACTATACCGAGTTTTTATAATTAACAATTCACTAGAAAAGG
AGCAAAAGGTTTATGAAGGGGCTCACAGAGCGGTTGAAATTGAAGCTCTAACACCACACTCCAGCTACTGTGTAGT
GGCTGAAATATATCAGCCCATGTTAGACAGAAGAAGTCAGAGAAGTGAAGAGAGATGTGTGGAAATTCCATGATCT
AGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCC
CCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTG
TCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGC
AGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCC
AGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTCGCCGCCAAGGATCTGA
TGGCGCAGGGGATCAAGCTCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACG
CAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGC
CGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAA
CTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCA
CTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGC
CGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCAC
CAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAG
AGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGCGAGGATCTCGTCGT
GACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGG
CTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGG
CTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGA
GTTCTTCTGAATTATTAACGCTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACAC
CGCATACAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATAT
GTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATAGCACGTGCTAAAACTTCATTTTTAATTTAAA
AGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGT
CAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAA
AAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTC
AGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCAC
CGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTT
GGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTG
GAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAA
AGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTG
GTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGG
AGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGGCTTTTGCTGGCCTTTTGCTCACATGTTC
TT。
The preparation of the DMP cation nanometer grain/hIL-22BP gene composite of the present invention of embodiment 2
1, the preparation of DOTAP-mPEG-PCL polymer nanoparticle solution
By cation lipid (2,3- dioleoyl-propyl)-trimethylamine (1,2-dioleoyl-3-
Trimethylammonium-propane, DOTAP) and methyl polyethylene glycol-polycaprolactone (mPEG-PCL) high molecular polymer
(molecular weight 4000Da, PEG-PCL=2000Da-2000Da) is mixed by the mass ratio of 1:9, and by mixture dichloro
Methane is dissolved, and is placed on Rotary Evaporators to form a film after 60 DEG C of revolving 30min.Formed film be dissolved in after taking out from
In sub- water, 5min then is shaken under 60 DEG C of water bath conditions, obtains the DMP cationic polymer nanoparticle solution of certain concentration.
2, hIL-22BP gene expression plasmid is prepared
PVAX1-hIL-22BP plasmid is converted into DH5 α competent escherichia coli cell and is coated on containing kanamycin anti-
On the LB plate of property.Extraction clone is chosen after 24 hours, is placed in 3mL LB culture medium containing kanamycin and is shaken bacterium overnight incubation,
Next day extracts pVAX1-hIL-22BP plasmid recombinant plasmid.Purified plasmid can meet wanting for inside and outside experiment after testing
It asks.
3, DMP cation nanometer grain/hIL-22BP Plasmid DNA gene composite preparation
In DMP cation nanometer grain: DNA with the ratio of 10:1 (w/w) by nanoparticle solution (be scattered in deionized water,
Concentration is 5mg/mL) it is added in pVAX1-IL-22BP solution (being scattered in deionized water, concentration 1mg/mL), Zhi Houli
Quarter is blown and beaten with liquid-transfering gun and is mixed, then stands 15 minutes in room temperature environment to get DMP cation nanometer grain Plasmid DNA compound.
The preparation of the DCMP cation nanometer grain/hIL-22BP gene composite of the present invention of embodiment 3
1, the preparation of DC-Cholesterol-mPEG-PCL polymer nanoparticle solution
By 3 β of cation lipid-[N- (N', N'- dimethyl aminoethyl) amido formacyl] cholesterol (3 β-[N- (N, N-
Dimethylaminoethane)-carbamoyl] cholesterolhydrochloride, DC-Cholesteral) and methyl
Polyethylene glycol-polycaprolactone (mPEG-PCL) high molecular polymer (molecular weight 4000Da, PEG-PCL=2000Da-
It 2000Da) is mixed by the mass ratio of 1:9, and mixture is dissolved with methylene chloride, be placed on Rotary Evaporators
To form a film after 60 DEG C of revolving 30min.Formed film is dissolved in deionized water solution after taking out, then under 60 DEG C of water bath conditions
5min is shaken, the DCMP cationic polymer nanoparticle solution of certain concentration is obtained.
2, hIL-22BP gene expression plasmid is prepared
PVAX1-hIL-22BP plasmid is converted into DH5 α competent escherichia coli cell and is coated on containing kanamycin anti-
On the LB plate of property.Extraction clone is chosen after 24 hours, is placed in 3mL LB culture medium containing kanamycin and is shaken bacterium overnight incubation,
Next day extracts pVAX1-hIL-22BP plasmid recombinant plasmid.Purified plasmid can meet wanting for inside and outside experiment after testing
It asks.
3, DCMP cation nanometer grain/hIL-22BP Plasmid DNA gene composite preparation
In DCMP cation nanometer grain: DNA with the ratio of 10:1 (w/w) by nanoparticle solution (be scattered in deionized water,
Concentration is 5mg/mL) it is added in pVAX1-hIL-22BP solution (being scattered in deionized water, concentration 1mg/mL), Zhi Houli
Quarter is blown and beaten with liquid-transfering gun and is mixed, then stands 15 minutes in room temperature environment to get DCMP cation nanometer grain plasmid dna complex of the present invention
Close object.
The preparation of the DPA cation nanometer grain/hIL-22BP gene composite of the present invention of embodiment 4
1, the preparation of DOTAP-mPEG-PLA polymer nanoparticle solution
By cation lipid (2,3- dioleoyl-propyl)-trimethylamine (1,2-dioleoyl-3-
Trimethylammonium-propane, DOTAP) it is high with poly- (the ethylene oxide)-poly(D,L-lactide) (mPEG-PLA) of Alpha-Methyl
Molecularly Imprinted Polymer (molecular weight 4000Da, PEG-PLA=2000Da-2000Da) is mixed by the mass ratio of 1:9, and will be mixed
It closes object to be dissolved with methylene chloride, be placed on Rotary Evaporators to form a film after 60 DEG C of revolving 30min.After formed film takes out
It is dissolved in deionized water solution, then shakes 5min under 60 DEG C of water bath conditions, obtain the DPA cationic polymerization of certain concentration
Object nanoparticle solution.
2, hIL-22BP gene expression plasmid is prepared
PVAX1-hIL-22BP plasmid is converted into DH5 α competent escherichia coli cell and is coated on containing kanamycin anti-
On the LB plate of property.Extraction clone is chosen after 24 hours, is placed in 3mL LB culture medium containing kanamycin and is shaken bacterium overnight incubation,
Next day extracts pVAX1-hIL-22BP plasmid recombinant plasmid.Purified plasmid can meet wanting for inside and outside experiment after testing
It asks.
3, DPA cation nanometer grain/hIL-22BP Plasmid DNA gene composite preparation
In DPA cation nanometer grain: DNA with the ratio of 10:1 (w/w) by nanoparticle solution (be scattered in deionized water,
Concentration is 5mg/mL) it is added in pVAX1-hIL-22BP solution (being scattered in deionized water, concentration 1mg/mL), Zhi Houli
Quarter is blown and beaten with liquid-transfering gun and is mixed, then stands 15 minutes in room temperature environment to get DPA cation nanometer grain plasmid dna complex of the present invention
Close object.
The preparation of the DPGA cation nanometer grain/hIL-22BP gene composite of the present invention of embodiment 5
1, the preparation of DOTAP-mPEG-PLGA polymer nanoparticle solution
By cation lipid (2,3- dioleoyl-propyl)-trimethylamine (1,2-dioleoyl-3-
Trimethylammonium-propane, DOTAP) and methoxy poly (ethylene glycol)-poly (glycolide-lactide) (mPEG-PLGA) macromolecule
Polymer (molecular weight 4000Da, PEG-PLGA=2000Da-2000Da) is mixed by the mass ratio of 1:9, and will mixing
Object is dissolved with methylene chloride, is placed on Rotary Evaporators to form a film after 60 DEG C of revolving 30min.Formed film is molten after taking out
Then solution shakes 5min under 60 DEG C of water bath conditions in deionized water solution, obtain the DPGA cationic polymerization of certain concentration
Object nanoparticle solution.
2, hIL-22BP gene expression plasmid is prepared
PVAX1-hIL-22BP plasmid is converted into DH5 α competent escherichia coli cell and is coated on containing kanamycin anti-
On the LB plate of property.Extraction clone is chosen after 24 hours, is placed in 3mL LB culture medium containing kanamycin and is shaken bacterium overnight incubation,
Next day extracts pVAX1-hIL-22BP plasmid recombinant plasmid.Purified plasmid can meet wanting for inside and outside experiment after testing
It asks.
3, DPGA cation nanometer grain/hIL-22BP Plasmid DNA gene composite preparation
In DPGA cation nanometer grain: DNA with the ratio of 10:1 (w/w) by nanoparticle solution (be scattered in deionized water,
Concentration is 5mg/mL) it is added in pVAX1-hIL-22BP solution (being scattered in deionized water, concentration 1mg/mL), Zhi Houli
Quarter is blown and beaten with liquid-transfering gun and is mixed, then stands 15 minutes in room temperature environment to get DPGA cation nanometer grain plasmid dna complex of the present invention
Close object.
Beneficial effects of the present invention are proved below by way of test example.
1 subject cationic nanoparticle of test example/hIL-22BP gene composite inhibitor against colon carcinoma cells test
In order to study the cation nanometer grain/anti-tumor effect of hIL-22BP gene composite in vivo, in BalB/c-nu
Mouse (6-8 week old, female) subcutaneously establishes human colon carcinoma heterotopic transplantation tumor model.By the HCT116 human colon carcinoma of in vitro culture
It is cells trypsinised, and constant volume serum-free, antibiotic-free 1640 culture mediums in, in the subcutaneous vaccination of every mouse
1×107A cell after cell inoculation 7 days, starts that random GP TH (every group 5) are carried out as follows:
A) blank control group: 5% glucose solution；
B) empty plasmid control group: cation nanometer grain/pVAX gene composite is placed in 5% glucose solution；
C) IL-22BP treatment group: DMP cation nanometer grain/hIL-22BP gene composite is placed in 5% glucose solution
In.
Intratumor injection mode is taken to be treated, the method system that DMP cation nanometer grain/DNA compound presses embodiment 1,2
Standby, match as follows: Plasmid DNA: cation nanometer grain/DNA compound is diluted in by cation nanometer grain=1:10 (W/W)
In glucose solution, and adjust so that glucose final concentration of 5%.The volume injected of each every mouse is 100 μ L, wherein containing
50 μ g of 5 μ g of plasmid and cation nanometer grain.It is administered once daily, treats 7 times altogether.Treatment measures gross tumor volume after starting daily
Size.Animal was put to death and dissected in the 9th day after treatment end, separate subcutaneous tumor tissue and weighed.Tumor growth inhibition
With variance analysis, P < 0.05 item thinks statistically significant.Tumor weight, the tumor growth curve of the above groups of animals are shown in Fig. 4,
Wherein Fig. 4 a indicates that tumor growth curve, Fig. 4 b indicate average tumor weight.
From fig. 4, it can be seen that cation nanometer grain/hIL-22BP gene composite treatment group tumors slow growth, and it is right
Very fast according to group tumour growth, cation nanometer grain/hIL-22BP gene composite shows the extremely strong work for inhibiting tumour growth
With inhibitory rate is to 74.8% compared with blank control group.
The experimental results showed that, cation nanometer grain/hIL-22BP gene composite has significant anti-human colon cancer above
Effect.
2 subject cationic nanoparticle of test example/hIL-22BP gene composite ovarian cancer resistance test
In order to study the cation nanometer grain/anti-tumor effect of hIL-22BP gene composite in vivo, in BalB/c-nu
Mouse (6-8 week old, female) abdominal cavity establishes human ovarian cancer Metastasis tumor of abdomen model.By the SKOV3 human ovarian cancer of in vitro culture
It is cells trypsinised, and constant volume serum-free, antibiotic-free DMEM culture medium in, in the intraperitoneal inoculation of every mouse
1×107A cell after cell inoculation 7 days, starts that random GP TH (every group 5) are carried out as follows:
A) blank control group: 5% glucose solution；
B) empty plasmid control group: cation nanometer grain/pVAX gene composite is placed in 5% glucose solution；
C) IL-22BP treatment group: DMP cation nanometer grain/hIL-22BP gene composite is placed in 5% glucose solution
In.
Intraperitoneal injection mode is taken to be treated, the method that DMP cation nanometer grain/DNA compound presses embodiment 1,2
Preparation matches as follows: Plasmid DNA: cation nanometer grain=1:10 (W/W) dilutes cation nanometer grain/DNA compound
In glucose solution, and adjust so that glucose final concentration of 5%.The volume injected of each every mouse is 100 μ L, wherein
Containing 50 μ g of 5 μ g of plasmid and cation nanometer grain.It is administered once daily, treats 7 times altogether.Animal was put to death in the 7th day after treatment end
And it dissects, separate belly cavity tumor tissue and carries out weighing and tumor nodule counting.Tumor growth inhibition variance analysis, P < 0.05
Then think statistically significant.Tumor weight, the tumor nodule number of the above groups of animals are shown in Fig. 5, and wherein Fig. 5 a indicates average swollen
Tumor weight, Fig. 5 b indicate average tumor tubercle number.
From fig. 5, it can be seen that cation nanometer grain/hIL-22BP gene composite treatment group tumors slow growth, and it is right
Very fast according to group tumour growth, cation nanometer grain/hIL-22BP gene composite shows the extremely strong work for inhibiting tumour growth
With inhibitory rate is to 71.1% compared with blank control group.
The experimental results showed that, cation nanometer grain/hIL-22BP gene composite has significant anti-human ovarian carcinoma above
Effect.
3 subject cationic nanoparticle of test example/hIL-22BP gene composite anti-lung cancer test
In order to study the cation nanometer grain/anti-tumor effect of hIL-22BP gene composite in vivo, in BalB/c-nu
Mouse (6-8 week old, female) subcutaneously establishes human lung cancer heterotopic transplantation tumor model.The A549 human lung carcinoma cell of in vitro culture is used
Trypsin digestion, and constant volume serum-free, antibiotic-free DMEM culture medium in, in the subcutaneous vaccination 1 × 10 of every mouse7
A cell after cell inoculation 7 days, starts that random GP TH (every group 5) are carried out as follows:
A) blank control group: 5% glucose solution；
B) empty plasmid control group: cation nanometer grain/pVAX gene composite is placed in 5% glucose solution；
C) IL-22BP treatment group: DMP cation nanometer grain/hIL-22BP gene composite is placed in 5% glucose solution
In.
Intratumor injection mode is taken to be treated, the method system that DMP cation nanometer grain/DNA compound presses embodiment 1,2
Standby, match as follows: Plasmid DNA: cation nanometer grain/DNA compound is diluted in by cation nanometer grain=1:10 (W/W)
In glucose solution, and adjust so that glucose final concentration of 5%.The volume injected of each every mouse is 100 μ L, wherein containing
50 μ g of 5 μ g of plasmid and cation nanometer grain.It is administered once daily, treats 7 times altogether.Treatment measures gross tumor volume after starting daily
Size.Animal was put to death and dissected in the 9th day after treatment end, separate subcutaneous tumor tissue and weighed.Tumor growth inhibition
With variance analysis, P < 0.05 item thinks statistically significant.Tumor weight, the tumor growth curve of the above groups of animals are shown in Fig. 6,
Wherein Fig. 6 a indicates that tumor growth curve, Fig. 6 b indicate average tumor weight.
From fig. 6, it can be seen that cation nanometer grain/hIL-22BP gene composite treatment group tumors slow growth, and it is right
Very fast according to group tumour growth, cation nanometer grain/hIL-22BP gene composite shows the extremely strong work for inhibiting tumour growth
With inhibitory rate is to 66.87% compared with blank control group.
The experimental results showed that, cation nanometer grain/hIL-22BP gene composite has significant anti-human lung cancer above
Effect.
4 subject cationic nanoparticle of test example/hIL-22BP gene composite anti-liver cancer and anti-test
In order to study the cation nanometer grain/anti-tumor effect of hIL-22BP gene composite in vivo, in BalB/c-nu
Mouse (6-8 week old, female) subcutaneously establishes human liver cancer heterotopic transplantation tumor model.By the HepG2 human liver cancer cell of in vitro culture
With trypsin digestion, and constant volume serum-free, antibiotic-free DMEM culture medium in, every mouse subcutaneous vaccination 1 ×
107A cell after cell inoculation 7 days, starts that random GP TH (every group 5) are carried out as follows:
A) blank control group: 5% glucose solution；
B) empty plasmid control group: cation nanometer grain/pVAX gene composite is placed in 5% glucose solution；C)
IL-22BP treatment group: DMP cation nanometer grain/hIL-22BP gene composite is placed in 5% glucose solution.
Intratumor injection mode is taken to be treated, the method system that DMP cation nanometer grain/DNA compound presses embodiment 1,2
Standby, match as follows: Plasmid DNA: cation nanometer grain/DNA compound is diluted in by cation nanometer grain=1:10 (W/W)
In glucose solution, and adjust so that glucose final concentration of 5%.The volume injected of each every mouse is 100 μ L, wherein containing
50 μ g of 5 μ g of plasmid and cation nanometer grain.It is administered once daily, treats 7 times altogether.Treatment measures gross tumor volume after starting daily
Size.Animal was put to death and dissected in the 9th day after treatment end, separate subcutaneous tumor tissue and weighed.Tumor growth inhibition
With variance analysis, P < 0.05 item thinks statistically significant.Tumor weight, the tumor growth curve of the above groups of animals are shown in Fig. 7,
Wherein Fig. 7 a indicates that tumor growth curve, Fig. 7 b indicate average tumor weight.
From figure 7 it can be seen that cation nanometer grain/hIL-22BP gene composite treatment group tumors slow growth, and it is right
Very fast according to group tumour growth, cation nanometer grain/hIL-22BP gene composite shows the extremely strong work for inhibiting tumour growth
With inhibitory rate is to 63% compared with blank control group.
The experimental results showed that, cation nanometer grain/hIL-22BP gene composite has significant anti-human liver cancer above
Effect.
It should be noted that particular features, structures, materials, or characteristics described in this specification can any one or
It can be combined in any suitable manner in multiple embodiments.In addition, without conflicting with each other, those skilled in the art can incite somebody to action
The feature of difference embodiment described in this specification and different embodiments is combined.
Sequence table
<110>Sichuan University
<120>cationic polymer nanoparticle/hIL-22BP gene composite and its preparation method and application of anti-curing cancers
<130> A190607K
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 792
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
atgatgccta aacattgctt tctaggcttc ctcatcagtt tcttccttac tggtgtagca 60
ggaactcagt caacgcatga gtctctgaag cctcagaggg tacaatttca gtcccgaaat 120
tttcacaaca ttttgcaatg gcagcctggg agggcactta ctggcaacag cagtgtctat 180
tttgtgcagt acaaaatcat gttctcatgc agcatgaaaa gctctcacca gaagccaagt 240
ggatgctggc agcacatttc ttgtaacttc ccaggctgca gaacattggc taaatatgga 300
cagagacaat ggaaaaataa agaagactgt tggggtactc aagaactctc ttgtgacctt 360
accagtgaaa cctcagacat acaggaacct tattacggga gggtgagggc ggcctcggct 420
gggagctact cagaatggag catgacgccg cggttcactc cctggtggga aacaaaaata 480
gatcctccag tcatgaatat aacccaagtc aatggctctt tgttggtaat tctccatgct 540
ccaaatttac catatagata ccaaaaggaa aaaaatgtat ctatagaaga ttactatgaa 600
ctactatacc gagtttttat aattaacaat tcactagaaa aggagcaaaa ggtttatgaa 660
ggggctcaca gagcggttga aattgaagct ctaacaccac actccagcta ctgtgtagtg 720
gctgaaatat atcagcccat gttagacaga agaagtcaga gaagtgaaga gagatgtgtg 780
gaaattccat ga 792
<210> 2
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gggaaagctt atgatgccta aacattgctt tc 32
<210> 3
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gggatctaga tcatggaatt tccacacatc tc 32
<210> 4
<211> 3717
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gactcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60
atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120
acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180
aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240
ctatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300
ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360
atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420
gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 480
tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540
aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600
ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660
aattaatacg actcactata gggagaccca agctggctag cgtttaaact taagcttatg 720
atgcctaaac attgctttct aggcttcctc atcagtttct tccttactgg tgtagcagga 780
actcagtcaa cgcatgagtc tctgaagcct cagagggtac aatttcagtc ccgaaatttt 840
cacaacattt tgcaatggca gcctgggagg gcacttactg gcaacagcag tgtctatttt 900
gtgcagtaca aaatcatgtt ctcatgcagc atgaaaagct ctcaccagaa gccaagtgga 960
tgctggcagc acatttcttg taacttccca ggctgcagaa cattggctaa atatggacag 1020
agacaatgga aaaataaaga agactgttgg ggtactcaag aactctcttg tgaccttacc 1080
agtgaaacct cagacataca ggaaccttat tacgggaggg tgagggcggc ctcggctggg 1140
agctactcag aatggagcat gacgccgcgg ttcactccct ggtgggaaac aaaaatagat 1200
cctccagtca tgaatataac ccaagtcaat ggctctttgt tggtaattct ccatgctcca 1260
aatttaccat atagatacca aaaggaaaaa aatgtatcta tagaagatta ctatgaacta 1320
ctataccgag tttttataat taacaattca ctagaaaagg agcaaaaggt ttatgaaggg 1380
gctcacagag cggttgaaat tgaagctcta acaccacact ccagctactg tgtagtggct 1440
gaaatatatc agcccatgtt agacagaaga agtcagagaa gtgaagagag atgtgtggaa 1500
attccatgat ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt 1560
tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact 1620
cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat 1680
tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc 1740
aggcatgctg gggatgcggt gggctctatg gcttctactg ggcggtttta tggacagcaa 1800
gcgaaccgga attgccagct ggggcgccct ctggtaaggt tgggaagccc tgcaaagtaa 1860
actggatggc tttctcgccg ccaaggatct gatggcgcag gggatcaagc tctgatcaag 1920
agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg 1980
ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg 2040
atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc 2100
tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga 2160
cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc 2220
tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag 2280
tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat 2340
tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg 2400
tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca 2460
ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct 2520
tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg 2580
gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg 2640
gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc 2700
gcatcgcctt ctatcgcctt cttgacgagt tcttctgaat tattaacgct tacaatttcc 2760
tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata caggtggcac 2820
ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat 2880
gtatccgctc atgagacaat aaccctgata aatgcttcaa taatagcacg tgctaaaact 2940
tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 3000
cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 3060
ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 3120
accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 3180
cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca 3240
cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 3300
tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 3360
taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 3420
gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 3480
agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 3540
ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 3600
acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 3660
caacgcggcc tttttacggt tcctgggctt ttgctggcct tttgctcaca tgttctt 3717
<210> 5
<211> 263
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 5
Met Met Pro Lys His Cys Phe Leu Gly Phe Leu Ile Ser Phe Phe Leu
1 5 10 15
Thr Gly Val Ala Gly Thr Gln Ser Thr His Glu Ser Leu Lys Pro Gln
20 25 30
Arg Val Gln Phe Gln Ser Arg Asn Phe His Asn Ile Leu Gln Trp Gln
35 40 45
Pro Gly Arg Ala Leu Thr Gly Asn Ser Ser Val Tyr Phe Val Gln Tyr
50 55 60
Lys Ile Met Phe Ser Cys Ser Met Lys Ser Ser His Gln Lys Pro Ser
65 70 75 80
Gly Cys Trp Gln His Ile Ser Cys Asn Phe Pro Gly Cys Arg Thr Leu
85 90 95
Ala Lys Tyr Gly Gln Arg Gln Trp Lys Asn Lys Glu Asp Cys Trp Gly
100 105 110
Thr Gln Glu Leu Ser Cys Asp Leu Thr Ser Glu Thr Ser Asp Ile Gln
115 120 125
Glu Pro Tyr Tyr Gly Arg Val Arg Ala Ala Ser Ala Gly Ser Tyr Ser
130 135 140
Glu Trp Ser Met Thr Pro Arg Phe Thr Pro Trp Trp Glu Thr Lys Ile
145 150 155 160
Asp Pro Pro Val Met Asn Ile Thr Gln Val Asn Gly Ser Leu Leu Val
165 170 175
Ile Leu His Ala Pro Asn Leu Pro Tyr Arg Tyr Gln Lys Glu Lys Asn
180 185 190
Val Ser Ile Glu Asp Tyr Tyr Glu Leu Leu Tyr Arg Val Phe Ile Ile
195 200 205
Asn Asn Ser Leu Glu Lys Glu Gln Lys Val Tyr Glu Gly Ala His Arg
210 215 220
Ala Val Glu Ile Glu Ala Leu Thr Pro His Ser Ser Tyr Cys Val Val
225 230 235 240
Ala Glu Ile Tyr Gln Pro Met Leu Asp Arg Arg Ser Gln Arg Ser Glu
245 250 255
Glu Arg Cys Val Glu Ile Pro
260
Claims (16)
1. cationic polymer nanoparticle/hIL-22BP gene composite of anti-curing cancers, it is characterized in that: it is cationic polymerization
Object nanoparticle and recombinant vector containing hIL-22BP encoding gene act on compound obtain by charge adsorption, wherein sun from
Sub- polymer nanoparticle: the mass ratio of the recombinant vector containing hIL-22BP encoding gene is (3~30): 1, the hIL-
For the nucleotide sequence of 22BP encoding gene as shown in SEQ ID NO.1, the cationic polymer nanoparticle is with cationic lipid
What matter and polymer material were prepared as a raw material.
2. compound as described in claim 1, it is characterized in that: the compound method be by cationic polymer nanoparticle and
Recombinant vector containing hIL-22BP encoding gene is scattered in aqueous solution, is sufficiently mixed uniformly to get compound；Preferably,
Solution left standstill 5~30 minutes after mixing；Preferably, solution left standstill 15 minutes after mixing；Preferably, in being stored at room temperature.
3. compound as claimed in claim 2, it is characterized in that: the aqueous solution be selected from 5% glucose solution, physiological saline,
It is one or more kinds of in ultrapure water or deionized water.
4. compound as described in claim 1, it is characterized in that: the cationic polymer nanoparticle: being compiled containing hIL-22BP
The mass ratio of the recombinant vector of code gene is 10:1 or 30:1.
5. compound as described in claim 1, it is characterized in that: the cationic polymer nanoparticle is prepared with film hydration method
It obtains；Preferably, the cationic polymer nanoparticle is prepared with following methods: by cation lipid and polymer material
Co-dissolve in solvent, remove solvent after form a film, take the film out and be dissolved in aqueous solution, fully shake to get；Preferably,
The solvent is methylene chloride；Preferably, solvent is removed by rotary evaporation；Preferably, the aqueous solution is selected from 5% glucose
It is one or more kinds of in solution, physiological saline, ultrapure water or deionized water.
6. compound as claimed in claim 1 or 5, it is characterized in that: the cation lipid is DOTAP, DC-
Or mixtures thereof Cholesteral；Preferably, the cation lipid is DOTAP.
7. compound as claimed in claim 1 or 5, it is characterized in that: the polymer material is selected from mPEG-PCL, mPEG-
It is one or more kinds of in PLA, mPEG-PLGA；Preferably, the polymer material is mPEG-PCL；Preferably, mPEG-PCL
Molecular weight be 4000~10000Da；Preferably, the molecular weight of mPEG-PCL is 4000Da, and wherein mPEG segment is 2000Da,
PCL segment is 2000Da.
8. the compound as described in claim 1,5~7 any one, it is characterized in that: the cationic polymer nanoparticle is
It is prepared as a raw material with DOTAP and mPEG-PCL, wherein the mass ratio of DOTAP:mPEG-PCL is 1:(1~20)；It is excellent
Selection of land, the mass ratio of DOTAP:mPEG-PCL are 1:9.
9. compound as described in claim 1, it is characterized in that: the carrier is plasmid vector；Preferably, the carrier
For pVAX1.
10. the compound as described in claim 1 or 9, it is characterized in that: the recombination containing hIL-22BP encoding gene carries
Body, nucleotide sequence is as shown in SEQ ID NO.4.
11. the compound as described in claim 1 or 9, it is characterized in that: the recombinant vector containing hIL-22BP encoding gene
It is prepared by the following method to obtain: with Hind III and Xbal I digestion with restriction enzyme carrier, recycling carrier segments, and with phase
Handled with digestion with restriction enzyme and the hIL-22BP encoding gene segment that recycles be attached reaction to get.
12. compound preparation method described in claim 1~11 any one, it is characterized in that: by cationic polymer nanoparticle
With the recombinant vector containing hIL-22BP encoding gene by charge adsorption act on it is compound to get.
13. compound described in claim 1~11 any one is preparing the purposes in anticancer drug；Preferably, the medicine
Object is the drug of inhibitor against colon carcinoma cells, oophoroma, lung cancer and/or liver cancer.
14. the pharmaceutical composition of anticancer, it is characterized in that: it is with compound described in claim 1~11 any one for activity
The preparation that pharmaceutically acceptable auxiliary material or complementary ingredient are prepared is added in ingredient.
15. pharmaceutical composition as claimed in claim 14, it is characterized in that: the preparation is ejection preparation or oral preparation.
16. the pharmaceutical composition as described in claims 14 or 15, it is characterized in that: the preparation be inhibitor against colon carcinoma cells, oophoroma,
The preparation of lung cancer and/or liver cancer.
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| CN201910649942.5A CN110327299B (en) | 2019-07-18 | 2019-07-18 | Cationic polymer nanoparticle/hIL-22 BP gene compound for preventing and treating cancer and preparation method and application thereof |
| PCT/CN2020/102600 WO2021008600A1 (en) | 2019-07-18 | 2020-07-17 | Cationic polymer nanoparticle/hil-22bp gene compound for preventing and treating cancer and preparation method therefor and application thereof |
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| WO2021008600A1 (en) * | 2019-07-18 | 2021-01-21 | 四川大学 | Cationic polymer nanoparticle/hil-22bp gene compound for preventing and treating cancer and preparation method therefor and application thereof |
| CN112870373A (en) * | 2021-04-02 | 2021-06-01 | 四川大学 | Polypeptide polymer composite nanoparticle for mRNA delivery and preparation method and application thereof |
| CN112999360A (en) * | 2021-04-02 | 2021-06-22 | 四川大学 | Use of DMP nanoparticles in mRNA delivery |
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| CN110157733A (en) * | 2018-02-11 | 2019-08-23 | 四川大学 | Recombinate mIL-22BP carrier, liposome complex and its preparation method and application |
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