CN116063446A - Fusion protein for treating and preventing cancer and medical application thereof - Google Patents

Fusion protein for treating and preventing cancer and medical application thereof Download PDF

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CN116063446A
CN116063446A CN202111310832.XA CN202111310832A CN116063446A CN 116063446 A CN116063446 A CN 116063446A CN 202111310832 A CN202111310832 A CN 202111310832A CN 116063446 A CN116063446 A CN 116063446A
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蔡炯
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Yuanben Zhuhai Hengqin Biotechnology Co ltd
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Abstract

The invention discloses an optimized Maltose Binding Protein (MBP) and mucin (MUC 1), fusion proteins thereof, and encoding genes and application thereof. The invention also provides a nucleic acid molecule for encoding the fusion protein, a preparation method of the fusion protein, a recombinant vector containing the nucleic acid molecule and the fusion protein. The invention also discloses the application of the fusion protein in resisting tumors. The fusion protein can inhibit the growth of MUC1 positive tumor cells, especially colorectal cancer, and has wide application prospect.

Description

Fusion protein for treating and preventing cancer and medical application thereof
Technical Field
The invention relates to a fusion protein, in particular to a fusion protein containing human MUC1, a coding gene of the fusion protein, a preparation method of the fusion protein and medical application of the fusion protein.
Background
Colorectal cancer is the third leading cancer. Although surgery, radiation therapy, chemotherapy have standard treatment regimens for localized tumors, treatment of metastatic colorectal cancer is challenging. For decades, chemotherapy regimens for advanced colorectal cancer have been 5-fluorouracil based FOLFOX (oxaliplatin), FOLFIRI (ife Li Tikang), folfoxri (oxaliplatin ifer Li Tikang) regimens and bio-targeted therapies. Biological targeted therapies include VEGF blockade, EGFR blockade, and the like. However, the use of EGFR antibodies in patients with advanced tumors can lead to morbidity and overall patient benefit is not achieved. In summary, although chemotherapy plus biotargeting treatment resulted in an increase in overall survival in patients, the 5-year survival for colorectal cancer was only 14%, with an average lifetime of 30 months, and the therapeutic efficacy was still improved.
On the other hand, activation of the cancer immune system is another method of inhibiting colorectal cancer cell growth. In 2018, both antibodies, kedas and nivolumab, were approved for treatment of chemotherapy-resistant dMMR-MSI-H subtype colorectal cancer. Both antibodies, curida and nivolumab, were approved in 2020 for the treatment of inoperable, metastatic colorectal cancer of the dMMR-MSI-H subtype. During the initiation phase of T cell activation in lymphoid organs, CTLA-4 (cytotoxic lymphocyte antigen 4) binds to B7 protein on activated antigen presenting cells, thereby inhibiting T cell activation and reducing anti-tumor immune response. PD-1 interacts with PD-L1 and PD-L2 in peripheral tissues to limit the effector T cell activity of tumor microenvironment. Infiltration of T cells into tumor cells is a prognostic indicator, and the number and survival of tumor infiltrating T cells are improved, and low recurrence of cancer is also associated, and the monitoring of how tumors evade the immune system is gradually resolved. However, how cancer cells interact with the tumor microenvironment to reduce immune function, promote tumor growth is not fully understood, and in particular how to promote the change from cold to hot tumors is a current problem to be solved. For example, in the study of gastric, liver, colorectal cancer, it was found that tumor response rates are independent of biomarkers such as microsatellite highly unstable (MSI-H) or PD-L1 expression, and other immune escape mechanisms may exist.
The development of colon cancer is a complex process that accumulates genetic mutations, leading to heterogeneity in the therapeutic response. Only 5-15% of colorectal cancer patients have these predictive biomarkers of the dMMR-MSI-H subtype, while most patients cannot benefit from these treatments. Patients using antibodies help to reduce the risk of relapse, reduce the side effects of cytotoxic therapy, but are limited by individualised, co-morbid effects, immune microenvironment, intestinal microbial complications. Further, patients may suffer from various side effects such as fatigue, diarrhea, itching (pruritis), thyroid dysfunction, hepatitis, arthralgia (arthralgia), fever, rash (rash), etc. Combined immunospot monitoring therapy increases the chance of occurrence of grade 3-4 side effects.
For a long time, studies on adoptive cell therapy for colorectal cancer such as tumor-infiltrating lymphocyte Therapy (TIL) and cytokine-induced killer cell therapy (CIK) have been underway, but the effects are not clear, and may be related to less TIL for colorectal cancer and suppressed immune cells. Natural killer group 2D ligand (NKG 2D), epidermal growth factor receptor 2 (HER 2), mesothelin, guanylate cyclase, mucin 1, neoantigen, CEA-targeted CAR-T therapies are also in progress, expressed on cancer cells. The CAR-T treatment with CEA as a target achieves partial clinical effects, but is very expensive in face of the obstruction of physical barriers around cancer cells and immunosuppressive microenvironments.
For this reason, it is very important to develop less costly and better effective therapies, wherein therapeutic cancer vaccines are expected. At present, a relatively hot neoantigen (neoanti) vaccine needs high-throughput sequencing and bioinformatics screening, so that the time cost and the resource requirement are relatively high, and the vaccine is difficult to benefit patients. The invention discloses a gene optimization method of recombinant protein which can be used for preparing low-cost therapeutic cancer vaccine, is suitable for producing escherichia coli and inhibiting the growth of colon cancer cells.
Disclosure of Invention
In order to provide more effective biological agents for preventing and treating tumors, the invention provides an optimized MUC1-N, MBP and fusion proteins thereof, and medical application thereof.
The invention provides an optimized MUC1-N protein, which is characterized in that the nucleotide sequence is shown as SEQ ID NO. 3.
The invention also provides an optimised polynucleotide characterised in that it encodes a MUC1-N protein according to the invention. Preferably, the polynucleotide sequence is shown in SEQ ID NO. 3.
The invention provides an optimized MBP protein, which is characterized in that the nucleotide sequence is shown as SEQ ID NO. 6.
The invention also provides an optimized polynucleotide, characterized in that it encodes an MBP protein of the invention. Preferably, the polynucleotide sequence is shown in SEQ ID NO. 6.
The invention provides an optimized fusion protein, which is characterized in that the fusion protein comprises protein MBP and/or protein MUC1-N.
In one embodiment, the fusion protein is made up of the protein MBP gene and the protein MUC1-N gene in tandem.
Preferably, the fusion protein is formed by concatenating a maltose binding protein MBP gene and a mucin MUC1-N gene.
Furthermore, the nucleotide sequence of the maltose binding protein MBP gene is shown as SEQ ID NO.6, and the nucleotide sequence of the mucin MUC1-N gene is shown as SEQ ID NO. 3.
According to the invention, the amino acid sequence of the fusion protein is shown in SEQ ID NO. 7.
The present invention also provides a fusion protein comprising the MUC1-N protein of the present invention and/or the MBP protein of the present invention.
Preferably, the fusion protein is made up of the MUC1-N proteins of the invention and/or the MBP proteins of the invention in tandem.
The invention also provides an optimized polynucleotide encoding a fusion protein of the invention.
The invention also provides an optimized recombinant expression vector or host cell of the fusion protein, which comprises the sequence of the fusion protein or the polynucleotide sequence of the invention, preferably, the expression vector is a prokaryotic expression vector pET26b (+).
According to the invention, the fusion proteins comprise the protein MBP gene and/or the protein MUC1-N gene.
In one embodiment, the fusion protein is made up of the protein MBP gene and the protein MUC1-N gene in tandem.
Preferably, the fusion protein is formed by concatenating a maltose binding protein MBP gene and a mucin MUC1-N gene.
Furthermore, the nucleotide sequence of the maltose binding protein MBP gene is shown as SEQ ID NO.6, and the nucleotide sequence of the mucin MUC1-N gene is shown as SEQ ID NO. 3.
According to the invention, the amino acid sequence of the fusion protein is shown in SEQ ID NO. 7.
The present invention provides a pharmaceutical composition comprising the fusion protein of the present invention.
According to the invention, the fusion proteins comprise the protein MBP and/or the protein MUC1-N.
According to the invention, the fusion protein is formed by connecting a protein MBP gene and a protein MUC1-N gene in series.
Preferably, the fusion protein is formed by concatenating a maltose binding protein MBP gene and a mucin MUC1-N gene.
Furthermore, the nucleotide sequence of the maltose binding protein MBP gene is shown as SEQ ID NO.6, and the nucleotide sequence of the mucin MUC1-N gene is shown as SEQ ID NO. 3.
According to the invention, the amino acid sequence of the fusion protein is shown in SEQ ID NO. 7.
The present invention provides a vaccine comprising the fusion protein of the invention.
The invention also provides a preparation method of the fusion protein, which comprises the following steps:
(1) Amplifying MBP gene and MUC1-N gene;
(2) The gene sequences of MBP and Muc1-N genes are connected in series to obtain a fusion protein gene containing MBP-MUC1-N,
according to the present invention, the MBP gene is selected from maltose binding protein MBP genes; the protein MUC1-N gene is selected from mucin MUC1-N genes.
According to the invention, the nucleotide sequence of the maltose binding protein MBP gene is shown as SEQ ID NO.6, and the nucleotide sequence of the MUC1-N gene is shown as SEQ ID NO. 3.
According to the invention, the amino acid sequence of the fusion protein is shown in SEQ ID NO. 7.
More preferably, the above method further comprises inserting the MBP-MUC1-N fusion protein gene into an E.coli expression vector. Preferably, the expression vector is prokaryotic expression vector pET26b (+), the expression strain is escherichia coli BL21 (DE 3), and the affinity column is purified.
The invention also provides the application of the fusion protein in preparing antitumor drugs. Preferably, the tumor is a MUC1 positive tumor, more preferably, the tumor is colorectal cancer.
The invention also provides the application of the fusion protein in medicines for delaying the life span of tumor patients. Preferably, the tumor is a MUC1 positive tumor, more preferably, the tumor is colorectal cancer.
The invention also provides a method for preventing and/or treating tumors, which is characterized in that the fusion protein of the invention or the polynucleotide comprising the fusion protein is used.
Preferably, the tumor is a MUC1 positive tumor, more preferably, the tumor is colorectal cancer.
The invention also provides a method for delaying the longevity of a patient with a tumor, characterized in that the fusion protein of the invention or a polynucleotide comprising the fusion protein is used.
Preferably, the tumor is a MUC1 positive tumor, more preferably, the tumor is colorectal cancer.
The invention also provides application of the protein MBP gene and/or the protein MUC1-N gene in preparing medicaments for preventing and/or treating tumors, which is characterized in that the nucleotide sequence of the MUC1-N gene is shown as SEQ ID NO.3, and the MBP gene is shown as SEQ ID NO. 6.
According to the invention, the tumors include all MUC1 expressing tumors, including colorectal cancer expressing MUC 1.
The invention also provides application of the protein MBP gene and/or the protein MUC1-N gene in preparing medicaments for preventing and/or treating colorectal diseases, which is characterized in that the nucleotide sequence of the MUC1-N gene is shown as SEQ ID NO.3, and the nucleotide sequence of the MBP gene is shown as SEQ ID NO. 6.
The invention has the beneficial effects that:
1. the expression quantity of the optimized sequence MBP-Muc1-N is only 2% of the total protein, and the expression quantity of the optimized sequence MBP-Muc1-N of the invention is 51% of the total protein, and is increased by 25.5 times. After purification by an affinity column, the protein expressed by the non-optimized gene can be loaded and observed after 10 times concentration, the yield of the purified protein after concentration is 0.8mg of that of 100ml culture, and the yield of the optimized sequence MBP-Muc1-N is 9.6mg, and the yield is increased by 12 times. Greatly improves the expression quantity and the yield and greatly saves the production cost.
2. The fusion protein has remarkable inhibition effect on the growth of colon cancer cells, and prolongs the life span of cancer-bearing mice. The shortest survival time of the cancer mice is prolonged from 30 days to 50 days, namely, the survival time of the cancer mice is prolonged by 60 percent. Meanwhile, according to the specific embodiment, the drug can act after being injected for 6 days, and the highest inhibition rate reaches 48.7%.
Drawings
Fig. 1: gel diagram after protein expression and purification;
fig. 2: MBP-Muc1-N inhibits colorectal cancer cell proliferation experimental result;
FIG. 3 shows the experimental results of MBP-Muc1-N delaying the lifespan of colorectal cancer mice.
Detailed Description
Example 1: construction and expression of fusion proteins
1. Gene optimization
(1) Optimization of MUC1-N genes
According to the characteristics of the Muc1-N amino acid sequence SEQ ID NO.1, the invention carries out codon optimization to obtain SEQ ID NO.3, and the base sequence is optimized and modified by 28% through comparison;
①SEQ ID NO.1
Figure BDA0003338358010000051
(2) optimized pre-gene sequence SEQ ID NO.2
Figure BDA0003338358010000052
(3) Optimized gene sequence SEQ ID NO.3
Figure BDA0003338358010000053
Figure BDA0003338358010000061
(4) Optimization of 1 Pre-and post-Gene alignment (homology 72%, modification 28%)
Figure BDA0003338358010000062
(2) Optimization of MBP Gene
According to the characteristics of the MBP amino acid sequence SEQ ID NO.4, DNA software is utilized for codon optimization to obtain an optimized sequence SEQ ID NO.6, and the optimized sequence has 15% of base sequence change through comparison.
(1) Amino acid sequence SEQ ID NO.4
Figure BDA0003338358010000063
(2) Optimized pregenomic sequence SEQ ID NO.5
Figure BDA0003338358010000064
/>
Figure BDA0003338358010000071
(3) Optimized gene sequence SEQ ID NO.6
Figure BDA0003338358010000072
(4) Optimization of 1 Pre-and post-Gene comparison (homology 85%, modification 15%)
Figure BDA0003338358010000081
(3) Synthesis of MUC1-N fusion MBP optimized gene sequence
MBP and Muc1-N are sequentially expressed in series to obtain a fusion protein sequence SEQ ID NO.7, wherein the sequence is as follows:
KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGIEGRISGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAH
the preparation method comprises the following steps: tandem synthesis was performed based on the gene sequences of MBP and Muc1-N fusion proteins. For this purpose, the 1a_1, 1a_2, 1a_3, 1a_4, 1a_5, 1a_6, 1a_7, 1a_8, 1a_9, 1a_10, 1a_11, 1a_12, 1a_13, 1a_14, 1a_15, 1a_16, 1a_17, 1a_18, 1a_19, 1a_20, 1a_21, 1a_22, 1a_23, 1a_24, 1a_25, 1a_26, 1a_27, 1a_28, 1a_29, 1a_30 oligonucleotide sequences were synthesized, and then the 1b_1, 1b_2, 1b_3, 1b_4 sequences were synthesized, and gene amplification was performed using the 1-seq2, 1-R sequences to obtain the MUC1-N fusion MBP optimized gene sequences.
Figure BDA0003338358010000091
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Figure BDA0003338358010000101
EXAMPLE 2 protein purification, sample preparation procedure
The 5'PCR primer of the fusion gene is added with an NcoI restriction enzyme site, the 3' PCR primer is added with an EcoI restriction enzyme site, and the amplified gene is double-digested and inserted into a pET26b (+) escherichia coli expression vector which is also double-digested. Through screening and monoclonal selection of resistant bacterial culture plates, kanamycin-resistant medium was used for culture and IPTG induction of operon expression. The non-optimized sequence and the optimized sequence MBP-Muc1-N were each tested. The whole bacterial liquid obtained finally is pretreated with buffer solution containing SDS at 95 ℃ and analyzed by 5% -12% polyacrylamide gel electrophoresis.
As a result, as shown in FIG. 1, the expression level of the sequence MBP-Muc1-N before optimization was only 2% of the total protein, and the expression level of the sequence MBP-Muc1-N after optimization was 51% of the total protein, and was increased by 25.5 times. After purification by an affinity column, the protein expressed by the non-optimized gene can be loaded and observed after 10 times concentration, the yield of the purified protein after concentration is 0.8mg of that of 100ml culture, and the yield of the optimized sequence MBP-Muc1-N is 9.6mg, and the yield is increased by 12 times.
Example 3 Activity test of optimized fusion proteins
MBP-Muc1-N animal experiments
1. Material
Experimental reagent: recombining MBP-MUC1-N fusion proteins using the methods of the invention; MC38 colon cancer cells were purchased from national center for laboratory cell resources; physiological saline was injected and purchased from Beijing Tiantan biologicals Co.
Experimental animals: c57 BL/6J mice were purchased from Beijing Warcon Biotechnology Co.
2. Method of
MC38 colon cancer cells were cultured according to 1.0X10 6 The individual cells/arm were inoculated into the right armpit of C57BL/J mice, and after 7 days the tumor diameter reached 0.5cm, and the leg muscles were inoculated with the first needle MBP-Muc1-N. The tumors were measured 3 times per week by vaccinating 2 times per week 4, 6, 10, 12 days after the first MBP-Muc1-N injection.
3. Results
TABLE 1 tumor inhibiting effect of MBP-Muc1-N
Figure BDA0003338358010000111
As can be seen from FIG. 2, the tumor was significantly reduced after injection of MBP-Muc1-N. Especially, the tumor is extremely obviously reduced 6 days and 12 days after the first injection of MBP-Muc1-N. As can be seen from fig. 2 and table 1, the drug injection works 6 days later, and the highest inhibition rate reaches 48.7%. As can be seen from FIG. 3, the injection of MBP-Muc1-N prolonged survival of MC38 colon cancer mice compared to saline injected group controls. The shortest survival time of the cancer mice is prolonged from 30 days to 50 days, namely, the survival time of the cancer mice is prolonged by 60 percent.
4. Conclusion(s)
According to the invention, through an improved MBP-Muc1-N animal experiment, the improved fusion protein has a remarkable inhibition effect on the growth of colon cancer cells, the fusion protein acts after 6 days of drug injection, the highest inhibition rate reaches 48.7%, and the survival life of a cancer-bearing mouse is prolonged by 60%.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Yuan Ben (Zhuhai cross organ) biotechnology Co., ltd
<120> a fusion protein for treating and preventing cancer and medical use thereof
<130> 2021
<160> 7
<170> PatentIn version 3.5
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<213> unknown
<400> 4
Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys Gly
1 5 10 15
Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr Gly
20 25 30
Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe Pro
35 40 45
Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala His
50 55 60
Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr
65 70 75 80
Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala
85 90 95
Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala
100 105 110
Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr
115 120 125
Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys
130 135 140
Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu
145 150 155 160
Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr
165 170 175
Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly Leu
180 185 190
Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp Thr
195 200 205
Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala Met
210 215 220
Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys Val
225 230 235 240
Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys
245 250 255
Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn
260 265 270
Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu
275 280 285
Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala Leu
290 295 300
Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala Ala Thr
305 310 315 320
Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln Met
325 330 335
Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala Ser
340 345 350
Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn Ser
355 360 365
Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly Ile Glu
370 375 380
Gly Arg Ile Ser
385
<210> 5
<211> 1164
<212> DNA
<213> unknown
<400> 5
aaaatcgaag aaggtaaact ggtaatctgg attaacggcg ataaaggcta taacggtctc 60
gctgaagtcg gtaagaaatt cgagaaagat accggaatta aagtcaccgt tgagcatccg 120
gataaactgg aagagaaatt cccacaggtt gcggcaactg gcgatggccc tgacattatc 180
ttctgggcac acgaccgctt tggtggctac gctcaatctg gcctgttggc tgaaatcacc 240
ccggacaaag cgttccagga caagctgtat ccgtttacct gggatgccgt acgttacaac 300
ggcaagctga ttgcttaccc gatcgctgtt gaagcgttat cgctgattta taacaaagat 360
ctgctgccga acccgccaaa aacctgggaa gagatcccgg cgctggataa agaactgaaa 420
gcgaaaggta agagcgcgct gatgttcaac ctgcaagaac cgtacttcac ctggccgctg 480
attgctgctg acgggggtta tgcgttcaag tatgaaaacg gcaagtacga cattaaagac 540
gtgggcgtgg ataacgctgg cgcgaaagcg ggtctgacct tcctggttga cctgattaaa 600
aacaaacaca tgaatgcaga caccgattac tccatcgcag aagctgcctt taataaaggc 660
gaaacagcga tgaccatcaa cggcccgtgg gcatggtcca acatcgacac cagcaaagtg 720
aattatggtg taacggtact gccgaccttc aagggtcaac catccaaacc gttcgttggc 780
gtgctgagcg caggtattaa cgccgccagt ccgaacaaag agctggcaaa agagttcctc 840
gaaaactatc tgctgactga tgaaggtctg gaagcggtta ataaagacaa accgctgggt 900
gccgtagcgc tgaagtctta cgaggaagag ttggtgaaag atccgcgtat tgccgccact 960
atggaaaacg cccagaaagg tgaaatcatg ccgaacatcc cgcagatgtc cgctttctgg 1020
tatgccgtgc gtactgcggt gatcaacgcc gccagcggtc gtcagactgt cgatgaagcc 1080
ctgaaagacg cgcagactaa ttcgagctcg aacaacaaca acaataacaa taacaacaac 1140
ctcgggatcg agggaaggat ttca 1164
<210> 6
<211> 1164
<212> DNA
<213> Synthesis
<400> 6
aaaatcgaag aaggcaaact ggtgatctgg atcaacggtg ataagggtta taacggtctg 60
gcggaagtag gcaagaaatt cgaaaaagac accggtatca aagttaccgt tgaacatcca 120
gacaaactgg aagaaaaatt ccctcaggtg gcggctaccg gcgacggccc tgatatcatt 180
ttctgggcac atgatcgttt tggcggttac gcgcagtctg gcctgctggc agaaatcacg 240
ccggataagg cgttccagga caaactgtac ccttttacct gggacgcggt gcgttacaac 300
ggcaaactga tcgcttaccc gatcgcagtg gaagctctgt ccctgatcta caataaggac 360
ctgctgccga acccgcctaa aacgtgggaa gaaatcccgg ccctggacaa agaactgaaa 420
gcaaaaggta agagcgctct gatgttcaat ctgcaggaac cgtacttcac ttggccgctg 480
atcgcagctg acggcggtta tgcgtttaaa tacgaaaacg gtaaatatga cattaaggac 540
gtcggcgttg ataacgccgg cgccaaagcg ggcctgacct ttctggtcga cctgatcaaa 600
aacaaacaca tgaacgctga caccgattat tctattgcgg aggcggcttt taacaagggc 660
gagaccgcaa tgaccatcaa cggtccgtgg gcttggtcta acatcgacac ctccaaagta 720
aattacggtg ttaccgtcct gccgaccttc aaaggtcaac cgagcaaacc gttcgtgggc 780
gtgctgtccg caggtatcaa cgctgcctcc ccaaacaaag agctggccaa agagttcctg 840
gaaaactatc tgctgaccga cgaaggcctg gaagctgtta ataaagacaa accgctgggt 900
gctgttgcac tgaaatccta tgaagaagaa ctggtcaaag atccgcgtat tgccgccact 960
atggagaacg cgcagaaagg tgaaatcatg ccgaacatcc cgcaaatgtc cgctttttgg 1020
tacgcggtgc gtaccgctgt aattaacgcg gcgtccggtc gtcagactgt cgatgaagcg 1080
ctgaaagatg ctcagactaa ctctagctct aacaataaca ataataacaa caacaacaat 1140
ctgggtattg aaggtcgcat ctct 1164
<210> 7
<211> 528
<212> PRT
<213> Synthesis
<400> 7
Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys Gly
1 5 10 15
Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr Gly
20 25 30
Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe Pro
35 40 45
Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala His
50 55 60
Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr
65 70 75 80
Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala
85 90 95
Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala
100 105 110
Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr
115 120 125
Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys
130 135 140
Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu
145 150 155 160
Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr
165 170 175
Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly Leu
180 185 190
Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp Thr
195 200 205
Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala Met
210 215 220
Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys Val
225 230 235 240
Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys
245 250 255
Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn
260 265 270
Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu
275 280 285
Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala Leu
290 295 300
Lys Ser Tyr Glu Glu Glu Leu Val Lys Asp Pro Arg Ile Ala Ala Thr
305 310 315 320
Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln Met
325 330 335
Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala Ser
340 345 350
Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn Ser
355 360 365
Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly Ile Glu
370 375 380
Gly Arg Ile Ser Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
385 390 395 400
Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
405 410 415
Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
420 425 430
Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
435 440 445
Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
450 455 460
Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
465 470 475 480
Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
485 490 495
Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
500 505 510
Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
515 520 525

Claims (13)

1. A MUC1-N protein is characterized in that the nucleotide sequence of the protein is shown as SEQ ID NO. 3.
2. A polynucleotide encoding the protein of claim 1. Preferably, the polynucleotide sequence is shown in SEQ ID NO. 3.
3. An MBP protein is characterized in that the nucleotide sequence of the protein is shown as SEQ ID NO. 6.
4. A polynucleotide encoding the protein of claim 3. Preferably, the polynucleotide sequence is shown in SEQ ID NO. 6.
5. A fusion protein comprising the protein MBP and/or the protein MUC1-N.
Preferably, the fusion protein is formed by concatenating a protein MBP gene and a protein MUC1-N gene.
Also preferably, the fusion protein gene is formed by concatenating a maltose binding protein MBP gene and mucin MUC1-N genes.
More preferably, the nucleotide sequence of the maltose binding protein MBP gene is shown as SEQ ID NO.3, and the nucleotide sequence of the mucin MUC1-N gene is shown as SEQ ID NO. 6.
More preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO. 7.
6. A fusion protein comprising the MUC1-N protein of claim 1 and/or the MBP protein of claim 3.
Preferably, the fusion protein is made up of the MUC1-N protein of claim 1 and/or the MBP protein of claim 3 in tandem.
7. A polynucleotide encoding the fusion protein of claim 5 or 6.
8. A recombinant expression vector or host cell expressing the fusion protein of claim 5 or 6, comprising the sequence of the fusion protein of claim 5 or 6 or the polynucleotide of claim 7, preferably the expression vector is a prokaryotic expression vector pET26b (+).
9. A method for producing the fusion protein according to claim 5 or 6, comprising the steps of:
(1) Amplifying MBP gene and MUC1-N gene;
(2) The gene sequences of MBP and Muc1-N genes are connected in series to obtain a fusion protein gene containing MBP-MUC1-N,
preferably, the MBP gene is selected from the group consisting of maltose binding protein MBP genes; the protein MUC1-N gene is selected from mucin MUC1-N genes.
Preferably, the nucleotide sequence of the maltose binding protein MBP gene is shown as SEQ ID NO.3, and the nucleotide sequence of the MUC1-N gene is shown as SEQ ID NO. 6.
Preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO. 7.
Preferably, the MBP-MUC1-N fusion protein gene is inserted into an escherichia coli expression vector, preferably, the expression vector is a prokaryotic expression vector pET26b (+), and the expression strain is escherichia coli BL21 (DE 3).
10. A pharmaceutical composition comprising the sequence of the fusion protein of claim 5 or 6 or the polynucleotide of claim 7.
11. Use of the fusion protein of claim 5 or 6 or the polynucleotide of claim 7 for the preparation of a medicament for preventing and/or treating a tumor, and/or for delaying the lifetime of a tumor patient.
Preferably, the tumor is a MUC1 positive tumor, more preferably, the tumor is colorectal cancer.
12. The use of the protein MBP gene as claimed in claim 1 and/or the protein MUC1-N gene as claimed in claim 3 in the preparation of medicaments for preventing and/or treating tumors, wherein the nucleotide sequence of the MUC1-N gene is shown as SEQ ID NO.3, and the MBP gene is shown as SEQ ID NO. 6.
Preferably, the neoplasm includes all neoplasms expressing MUC1, including colorectal cancer expressing MUC 1.
13. The use of the protein MBP gene as claimed in claim 1 and/or the protein MUC1-N gene as claimed in claim 3 in the preparation of a medicament for preventing and/or treating colorectal cancer, wherein the nucleotide sequence of the MUC1-N gene is shown as SEQ ID NO.3, and the nucleotide sequence of the MBP gene is shown as SEQ ID NO. 6.
CN202111310832.XA 2021-11-04 2021-11-04 Fusion protein for treating and preventing cancer and medical application thereof Pending CN116063446A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111310832.XA CN116063446A (en) 2021-11-04 2021-11-04 Fusion protein for treating and preventing cancer and medical application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111310832.XA CN116063446A (en) 2021-11-04 2021-11-04 Fusion protein for treating and preventing cancer and medical application thereof

Publications (1)

Publication Number Publication Date
CN116063446A true CN116063446A (en) 2023-05-05

Family

ID=86175732

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111310832.XA Pending CN116063446A (en) 2021-11-04 2021-11-04 Fusion protein for treating and preventing cancer and medical application thereof

Country Status (1)

Country Link
CN (1) CN116063446A (en)

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