Fusion protein for treating and preventing cancer and medical use thereof

Abstract

The application discloses a maltose binding protein (MBP) and mucin (MUC1) and their fusion protein and an encoding gene and application thereof. The application also simultaneously provides a nucleic acid molecule for encoding the fusion protein, a preparation method of the fusion protein, and a recombinant vector and the fusion protein containing the nucleic acid molecule. The application also discloses the use of the fusion protein in anti-tumor. The fusion protein of the application can inhibit the growth of MUC1 positive tumor cells, especially colorectal cancer, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to a fusion protein, specifically a fusion protein containing human MUC1, the gene encoding the fusion protein, a method for preparing the fusion protein, and its medical uses. Background Technology

[0002] Colorectal cancer is the third leading cause of cancer death. While surgery, radiotherapy, and chemotherapy are standard treatments for localized tumors, treating metastatic colorectal cancer presents challenges. For decades, chemotherapy regimens for advanced colorectal cancer have included 5-fluorouracil-based regimens such as FOLFOX (oxaliplatin), FOLFIRI (irinotecan), and FOLFOXIRI (oxaliplatin-irinotecan), as well as targeted biological therapies. Targeted biological therapies include VEGF blockade and EGFR blockade. However, the use of EGFR antibodies in patients with advanced tumors can cause pathological changes, and overall, patients do not benefit. In conclusion, although chemotherapy combined with targeted biological therapy has improved overall patient survival, the 5-year survival rate for colorectal cancer is only 14%, with an average lifespan of just 30 months, indicating that treatment effectiveness needs further improvement.

[0003] On the other hand, activation of the cancer immune system is another method to inhibit the growth of colorectal cancer cells. In 2018, Keytruda and nivolumab were approved for the treatment of chemotherapy-resistant dMMR-MSI-H subtype colorectal cancer. In 2020, Keytruda and nivolumab were also approved for the treatment of unresectable dMMR-MSI-H subtype metastatic colorectal cancer. During the initiation phase of T cell activation in lymphoid organs, CTLA-4 (cytotoxic lymphocyte antigen 4) binds to the B7 protein on activated antigen-presenting cells, thereby inhibiting T cell activation and reducing the anti-tumor immune response. PD-1 interacts with PD-L1 and PD-L2 in peripheral tissues, limiting the activity of effector T cells in the tumor microenvironment. T cell infiltration in tumor cells is a marker of good prognosis; the number of tumor-infiltrating T cells is associated with improved survival and lower cancer recurrence rates. How tumors evade the immune system's surveillance is gradually being elucidated. However, how cancer cells interact with the tumor microenvironment to reduce immune function and promote tumor growth is not fully understood, especially how to promote the transformation from a "cold" tumor to a "hot" tumor, which is a problem that needs to be solved. For example, in studies of gastric cancer, liver cancer, and colorectal cancer, it has been found that tumor response rates do not depend on biomarkers such as microsatellite instability-high (MSI-H) or PD-L1 expression, suggesting the existence of other immune escape mechanisms.

[0004] The development of colorectal cancer is a complex process that accumulates genetic mutations, leading to heterogeneity in treatment responses. Only 5-15% of colorectal cancer patients possess predictive biomarkers such as the dMMR-MSI-H subtype, and the majority of patients do not benefit from these treatments. Antibody therapy can help reduce the risk of recurrence and the side effects of cytotoxic therapy, but its effectiveness is limited by individualization, comorbidities, the immune microenvironment, and the complexity of the gut microbiota. More troublesome for patients are the occurrence of various side effects, including fatigue, diarrhea, pruritus, thyroid dysfunction, hepatitis, arthralgia, fever, and rash. Combined immune point monitoring therapy increases the likelihood of grade 3-4 side effects.

[0005] For a long time, research on adoptive cell therapy for colorectal cancer, such as tumor-infiltrating lymphocyte therapy (TIL) and cytokine-induced killer cell therapy (CIK), has been ongoing, but the efficacy remains unclear, possibly due to the low availability of TILs in colorectal cancer and the suppression of immune cells. CAR-T therapy targeting natural killer cell 2D ligand (NKG2D), epidermal growth factor receptor 2 (HER2), mesothelin, guanylate cyclase, mucin 1, neoantigen, and CEA expressed on cancer cells is also progressing. Among these, CAR-T therapy targeting CEA has achieved some clinical efficacy, but it faces obstacles such as the physical barrier surrounding cancer cells, the immunosuppressive microenvironment, and is also very expensive.

[0006] Therefore, developing lower-cost and more effective treatments is crucial, with therapeutic cancer vaccines holding high promise. Currently, neoantigen vaccines, which are the subject of much research, require high-throughput sequencing and bioinformatics screening, resulting in significant time and resource demands, making it difficult to benefit patients. This invention discloses a gene optimization method for a recombinant protein suitable for E. coli production and inhibiting the growth of colon cancer cells, applicable to the preparation of inexpensive therapeutic cancer vaccines. Summary of the Invention

[0007] In order to provide more effective biological agents for tumor prevention and treatment, the present invention provides an optimized MUC1-N, MBP and its fusion protein, and their pharmaceutical uses.

[0008] The present invention provides an optimized MUC1-N protein, characterized in that its nucleotide sequence is shown in SEQ ID NO.3.

[0009] The present invention also provides an optimized polynucleotide, characterized in that it encodes the MUC1-N protein of the present invention. Preferably, the polynucleotide sequence is shown in SEQ ID NO.3.

[0010] The present invention provides an optimized MBP protein, characterized in that its nucleotide sequence is shown in SEQ ID NO.6.

[0011] The present invention also provides an optimized polynucleotide, characterized in that it encodes the MBP protein of the present invention. Preferably, the polynucleotide sequence is shown in SEQ ID NO.6.

[0012] The present invention provides an optimized fusion protein, characterized in that the fusion protein comprises protein MBP and / or protein MUC1-N.

[0013] In one specific embodiment, the fusion protein is composed of the MBP protein gene and the MUC1-N protein gene linked together.

[0014] Preferably, the fusion protein is composed of the maltose-binding protein MBP gene and the mucin MUC1-N gene linked together.

[0015] Furthermore, the nucleotide sequence of the maltose-binding protein MBP gene is shown in SEQ ID NO.6, and the nucleotide sequence of the mucin MUC1-N gene is shown in SEQ ID NO.3.

[0016] According to the present invention, the amino acid sequence of the fusion protein is shown in SEQ ID NO.7.

[0017] The present invention also provides a fusion protein, characterized in that the fusion protein contains the MUC1-N protein of the present invention and / or the MBP protein of the present invention.

[0018] Preferably, the fusion protein is formed by the tandem of the MUC1-N protein of the present invention and / or the MBP protein of the present invention.

[0019] The present invention also provides an optimized polynucleotide encoding the fusion protein of the present invention.

[0020] The present invention also provides an optimized recombinant expression vector or host cell for a fusion protein, comprising the sequence of the fusion protein described in the present invention or the polynucleotide sequence of the present invention. Preferably, the expression vector is a prokaryotic expression vector pET26b(+).

[0021] According to the present invention, the fusion protein includes the protein MBP gene and / or the protein MUC1-N gene.

[0022] In one specific embodiment, the fusion protein is composed of the MBP protein gene and the MUC1-N protein gene linked together.

[0023] Preferably, the fusion protein is composed of the maltose-binding protein MBP gene and the mucin MUC1-N gene linked together.

[0024] Furthermore, the nucleotide sequence of the maltose-binding protein MBP gene is shown in SEQ ID NO.6, and the nucleotide sequence of the mucin MUC1-N gene is shown in SEQ ID NO.3.

[0025] According to the present invention, the amino acid sequence of the fusion protein is shown in SEQ ID NO.7.

[0026] The present invention provides a pharmaceutical composition comprising the fusion protein of the present invention.

[0027] According to the present invention, the fusion protein includes protein MBP and / or protein MUC1-N.

[0028] According to the present invention, the fusion protein is composed of the protein MBP gene and the protein MUC1-N gene linked together.

[0029] Preferably, the fusion protein is composed of the maltose-binding protein MBP gene and the mucin MUC1-N gene linked together.

[0030] Furthermore, the nucleotide sequence of the maltose-binding protein MBP gene is shown in SEQ ID NO.6, and the nucleotide sequence of the mucin MUC1-N gene is shown in SEQ ID NO.3.

[0031] According to the present invention, the amino acid sequence of the fusion protein is shown in SEQ ID NO.7.

[0032] The present invention provides a vaccine comprising the fusion protein of the present invention.

[0033] This invention also provides a method for preparing a fusion protein, comprising the following steps:

[0034] (1) Amplify the MBP gene and the MUC1-N gene;

[0035] (2) The gene sequences of MBP and Muc1-N were tandemly linked to obtain a fusion protein gene containing MBP-MUC1-N.

[0036] According to the present invention, the MBP gene is selected from the maltose-binding protein MBP gene; the MUC1-N protein gene is selected from the mucin MUC1-N gene.

[0037] According to the present invention, the nucleotide sequence of the maltose-binding protein MBP gene is shown in SEQ ID NO.6, and the nucleotide sequence of the MUC1-N gene is shown in SEQ ID NO.3.

[0038] According to the present invention, the amino acid sequence of the fusion protein is shown in SEQ ID NO.7.

[0039] More preferably, the above method further includes inserting the MBP-MUC1-N fusion protein gene into an Escherichia coli expression vector. Preferably, the expression vector is a prokaryotic expression vector pET26b(+), the expression strain is Escherichia coli BL21(DE3), and the expression is purified using an affinity column.

[0040] The present invention also provides the use of the fusion protein in the preparation of antitumor drugs. Preferably, the tumor is a MUC1-positive tumor, more preferably, the tumor is colorectal cancer.

[0041] The present invention also provides the use of the fusion protein in a medicament for prolonging the life of cancer patients. Preferably, the tumor is a MUC1-positive tumor, more preferably, the tumor is colorectal cancer.

[0042] The present invention also provides a method for preventing and / or treating tumors, characterized in that it uses the fusion protein of the present invention or a polynucleotide containing the fusion protein.

[0043] Preferably, the tumor is a MUC1-positive tumor; more preferably, the tumor is colorectal cancer.

[0044] The present invention also provides a method for prolonging the life of cancer patients, characterized by using the fusion protein of the present invention or a polynucleotide containing the fusion protein.

[0045] Preferably, the tumor is a MUC1-positive tumor; more preferably, the tumor is colorectal cancer.

[0046] The present invention also provides the use of the protein MBP gene and / or the protein MUC1-N gene in the preparation of drugs for the prevention and / or treatment of tumors, characterized in that the nucleotide sequence of the MUC1-N gene is as shown in SEQ ID NO.3, and the MBP gene is as shown in SEQ ID NO.6.

[0047] According to the present invention, the tumor includes all tumors expressing MUC1, including colorectal cancer expressing MUC1.

[0048] The present invention also provides the use of the protein MBP gene and / or the protein MUC1-N gene in the preparation of drugs for the prevention and / or treatment of colorectal diseases, characterized in that the nucleotide sequence of the MUC1-N gene is as shown in SEQ ID NO.3, and the nucleotide sequence of the MBP gene is as shown in SEQ ID NO.6.

[0049] The beneficial effects of this invention are:

[0050] 1. Before optimization, the expression level of the MBP-Muc1-N sequence was only 2% of the total protein. The optimized MBP-Muc1-N sequence of this invention accounted for 51% of the total protein expression, an increase of 25.5 times. After affinity column purification, the protein expressed by the unoptimized gene required a 10-fold concentration before loading for observation, yielding only 0.8 mg of purified protein per 100 ml of culture. In contrast, the optimized MBP-Muc1-N sequence yielded 9.6 mg, an increase of 12 times. This significantly improved expression level and yield, greatly reducing production costs.

[0051] 2. The fusion protein of this invention has a significant inhibitory effect on the growth of colon cancer cells and prolongs the lifespan of mice with cancer. The shortest survival time of mice with cancer was extended from 30 days to 50 days, which is a 60% extension of their lifespan. Furthermore, as shown in the specific embodiments, the drug takes effect 6 days after injection, with a maximum inhibition rate of 48.7%. Attached Figure Description

[0052] Figure 1 : Gel images of proteins after expression and purification;

[0053] Figure 2 Results of experiments on the inhibition of colorectal cancer cell proliferation by MBP-Muc1-N;

[0054] Figure 3 Experimental results showing that MBP-Muc1-N prolongs the lifespan of mice with colorectal cancer. Detailed Implementation

[0055] Example 1: Construction and expression of fusion protein

[0056] 1. Genetic optimization

[0057] (1) Optimization of the MUC1-N gene

[0058] Based on the characteristics of the Muc1-N amino acid sequence SEQ ID NO.1, this invention optimizes the codons to obtain SEQ ID NO.3, and after comparison, 28% of the base sequence was optimized and modified;

[0059] ①SEQ ID NO.1

[0060]

[0061] ② Gene sequence before optimization SEQ ID NO.2

[0062]

[0063] ③ Optimized gene sequence SEQ ID NO.3

[0064]

[0065]

[0066] ④ Comparison of genes before and after optimization 1 (homology 72%, modification 28%)

[0067]

[0068] (2) Optimization of MBP gene

[0069] Based on the characteristics of the MBP amino acid sequence SEQ ID NO.4, codon optimization was performed using DNA software to obtain the optimized sequence SEQ ID NO.6. After comparison, the optimized sequence showed a 15% change in base sequence.

[0070] ①Amino acid sequence SEQ ID NO.4

[0071]

[0072] ② Gene sequence before optimization SEQ ID NO.5

[0073]

[0074]

[0075] ③ Optimized gene sequence SEQ ID NO.6

[0076]

[0077] ④ Comparison of genes before and after optimization 1 (homology 85%, modification 15%)

[0078]

[0079] (3) Synthesis of MUC1-N fusion with MBP optimized gene sequence

[0080] The sequential tandem expression of MBP and Muc1-N yielded the fusion protein sequence SEQ ID NO.7, which is as follows:

[0081] KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEI PALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSA GINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGIEGRISGVTSAPDT RPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAH

[0082] The preparation method is as follows: the MBP and Muc1-N fusion protein is synthesized in tandem according to their gene sequences. To this end, oligonucleotide sequences 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, and 1a_30 were synthesized first, followed by sequences 1b_1, 1b_2, 1b_3, and 1b_4. Gene amplification was performed using 1-seq2 and 1-R sequences to obtain the optimized MUC1-N fusion MBP gene sequence.

[0083]

[0084]

[0085] Example 2. Protein purification and sample preparation process

[0086] The 5' PCR primer for the fusion gene was supplemented with an NcoI restriction site, and the 3' PCR primer was supplemented with an EcoI restriction site. The amplified gene was double-digested and inserted into the pET26b(+) *E. coli* expression vector, which was also double-digested. After screening with resistant bacterial culture plates and single-clone selection, the bacteria were cultured in kanamycin-resistant medium and IPTG-induced operon expression was performed. The unoptimized sequence and the optimized sequence MBP-Muc1-N were tested separately. The final whole bacterial culture was pretreated with SDS-containing buffer at 95°C and analyzed by 5%–12% polyacrylamide gel electrophoresis.

[0087] The results are as follows Figure 1 As shown, the expression level of the unoptimized MBP-Muc1-N sequence was only 2% of the total protein, while the optimized MBP-Muc1-N sequence accounted for 51% of the total protein, an increase of 25.5 times. After affinity column purification, the protein expressed by the unoptimized gene was concentrated 10 times before it could be loaded for observation. The yield of the purified protein after concentration was 0.8 mg per 100 ml of culture, while the yield of the optimized MBP-Muc1-N sequence was 9.6 mg, an increase of 12 times.

[0088] Example 3. Activity test of the optimized fusion protein

[0089] 1. Animal experiments with MBP-Muc1-N

[0090] 1. Materials

[0091] Experimental reagents: Recombinant MBP-MUC1-N fusion protein was prepared using the method of this invention; MC38 colon cancer cells were purchased from the National Experimental Cell Resource Center; and physiological saline was purchased from Beijing Tiantan Biological Products Co., Ltd.

[0092] Experimental animals: C57 BL / 6J mice were purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0093] 2. Methods

[0094] MC38 colon cancer cells were divided into groups of 1.0 × 10⁻⁶. 6 One cell per mouse was injected into the right axilla of C57BL / J mice. After 7 days, the tumor diameter reached 0.5 cm, and the first dose of MBP-Muc1-N was injected into the leg muscle. The mice were given the vaccine twice a week on days 4, 6, 10, and 12 following the first injection of MBP-Muc1-N, and the tumor size was measured three times.

[0095] 3. Results

[0096] Table 1. The tumor-inhibiting effect of MBP-Muc1-N

[0097]

[0098] The result is from Figure 2 As can be seen, the tumor shrank significantly after injection of MBP-Muc1-N. Particularly, the tumor shrank remarkably at 6 and 12 days after the first injection of MBP-Muc1-N. Figure 2 As shown in Table 1, the drug took effect 6 days after injection, with a maximum inhibition rate of 48.7%. Figure 3 As can be seen, compared with the control group injected with saline, injection of MBP-Muc1-N prolonged the survival time of MC38 colon cancer mice. The shortest survival time of cancer-affected mice was extended from 30 days to 50 days, which is a 60% extension of the lifespan of cancer-affected mice.

[0099] 4. Conclusion

[0100] This invention, through animal experiments with the modified MBP-Muc1-N, determined that the modified fusion protein has a significant inhibitory effect on the growth of colon cancer cells. It takes effect 6 days after drug injection, with a maximum inhibition rate of 48.7%, and prolongs the survival of cancer-affected mice by 60%.

[0101] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. SEQUENCE LISTING <110> Yuanben (Zhuhai Hengqin) Biotechnology Co., Ltd. <120> A fusion protein for treating and preventing cancer and its pharmaceutical applications <130> 2021 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 140 <212> PRT <213> unknown <400> 1 Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala 1 5 10 15 Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro 20 25 30 Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr 35 40 45 Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser 50 55 60 Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His 65 70 75 80 Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala 85 90 95 Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro 100 105 110 Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr 115 120 125 Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His 130 135 140 <210> 2 <211> 420 <212> DNA <213> Unknown <400> 2 ggtgtcacct cggccccgga caccaggccg gccccgggct ccaccgcccc cccagcccac 60 ggtgtcacct cggccccgga gagcaggccg gccccgggct ccaccgcccc cccagcccac 120 ggtgtcacct cggccccgga gagcaggccg gccccgggct ccaccgcgcc cgcagcccac 180 ggtgtcacct cggccccgga gagcaggccg gccccgggct ccaccgcgcc cgcagcccac 240 ggtgtcacct cggccccgga gagcaggccg gccccgggct ccaccgcccc ccgagcccac 300 ggtgtcacct cggccccgga caccaggccg gccccgggct ccaccgcccc cccagcccac 360 ggtgtcacct cggccccgga caccaggccg gccccgggct ccaccgcccc cccagcccac 420 <210> 3 <211> 420 <212> DNA <213> Synthetic <400> 3 ggtgttactt ctgctcctga tactcgtcct gctcctggtt ctactgcacc gccagcacat 60 ggcgtgacgt ctgcgccaga tacccgtccg gcaccgggtt ccaccgcccc accggcacac 120 ggcgtaacct ccgcgccaga cacccgtcca gcgccaggtt ctaccgctcc gcctgctcat 180 ggtgttacct ctgccccgga cactcgtccg gctccaggtt ctactgcccc gccagctcat 240 ggcgtcactt ccgccccgga tacccgtcct gccccgggct ctactgcgcc tccggctcac 300 ggcgttacct ctgcaccgga tactcgtccg gctccgggct ctaccgcacc acctgctcat 360 ggcgtaacga gcgctcctga tacccgtccg gctccgggtt ccactgcacc tccggcccac 420 <210> 4 <211> 388 <212> PRT <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 taaaagat 360 420 gcgaaaggta agagcgcgct gatgttcaac ctgcaagaac cgtacttcac ctggccgctg 480 540 gtgggcgtgg ataacgctgg cgcgaaagcg ggtctgacct tcctggttga cctgattaaa 600 aacaaacaca tgaatgcaga caccgattac tccatcgcag aagctgcctt taataaggc 660 gaacagcga tgaccatcaa cggccccgtgg 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> Synthetic <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 gtcggcgtg ataacgccgg cgccaaagcg ggcctgacct ttctggtcga cctgatcaaa 600 aacaaaca 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 aattaacgg gcgtccggtc gtcagactgt cgatgaagcg 1080 ctgaaagatg ctcagactaa ctctagctct aacaataaca ataataacaa caacaacaat 1140 ctgggtattg aaggtcgcat ctct 1164 <210> 7 <211> 528 <212> PRT <213> Synthetic <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

1. A fusion protein, the amino acid sequence of which is shown in SEQ ID NO.

7.

2. A polynucleotide encoding the fusion protein of claim 1.

3. The polynucleotide of claim 2, wherein the polynucleotide is composed of the tandem of the MBP gene and the MUC1-N gene, the nucleotide sequence of the MBP gene is shown in SEQ ID NO. 6, and the nucleotide sequence of the MUC1-N gene is shown in SEQ ID NO.

3.

4. A recombinant expression vector for expressing the fusion protein of claim 1, characterized in that, Includes the polynucleotides described in claim 2 or 3.

5. The recombinant expression vector as described in claim 4, wherein the expression vector is the prokaryotic expression vector pET26b(+).

6. A host cell expressing the fusion protein of claim 1, characterized in that, It contains the polynucleotide as described in claim 2 or 3.

7. The method for preparing the fusion protein according to claim 1, comprising the following steps: (1) Amplify the MBP gene and MUC1-N gene; (2) The gene sequences of MBP and MUC1-N genes were tandemly linked to obtain the gene encoding the MBP-MUC1-N fusion protein; The nucleotide sequence of the maltose-binding protein MBP gene is shown in SEQ ID NO.6, and the nucleotide sequence of the MUC1-N gene is shown in SEQ ID NO.3; The amino acid sequence of the fusion protein is shown in SEQ ID NO.

7.

8. The method for preparing the fusion protein according to claim 7 further includes the step of inserting the MBP-MUC1-N fusion protein gene into an Escherichia coli expression vector, wherein the expression vector is a prokaryotic expression vector pET26b(+) and the expression strain is Escherichia coli BL21(DE3).

9. A pharmaceutical composition, characterized in that, It includes the fusion protein of claim 1.

10. Use of the fusion protein of claim 1 in the preparation of a medicament for treating colorectal cancer.

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