Fusion protein HSA-ⅡB-P53 with the function of promoting tumor cell apoptosis and its application

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a novel fusion protein HSA-ⅡB-P53 having the function of promoting tumor cell apoptosis and its application. The fusion protein provided by the present invention is obtained by fusing the ⅡB functional region of human serum albumin with the P53 protein.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a novel fusion protein HSA-ⅡB-P53 having the function of promoting tumor cell apoptosis and application thereof. Background Art

[0002] The occurrence and invasion of malignant tumors are often accompanied by the loss of key proteins or mutations of key genes. These missing key proteins or key genes usually play a role in ensuring that cells stop growing or die at the right time. Therefore, once key proteins are lost or key genes mutate, the growth of malignant tumor cells will get out of control and form cancer. Based on this, a strategy that can effectively compensate for the loss of key proteins or inhibit key gene mutations is proposed, which helps promote the "return" of tumor cells to normal programmed cell death.

[0003] Existing studies have shown that in the body, the most important role of P53 protein is to regulate cells to stop growing or dying at the right time to prevent cell canceration. When a malignant tumor is formed, P53 protein will be severely deficient in malignant tumor tissue, resulting in the uncontrolled occurrence of malignant tumors. Therefore, there is a method of preparing recombinant P53 protein in the prior art to increase the concentration of P53 in cancer cells to change the situation where the expression of related proteins is too low due to P53 gene mutation. For example, the patent with publication number CN112156189A (20210101) and invention name “A HER2+ breast cancer targeting protein composite nanoparticle and its preparation method and application” discloses a HER2+ breast cancer targeting protein composite nanoparticle, which is a micelle loaded with a complex cross-linked with ER antibody and recombinant P53 protein. However, the composite nanoparticles of the patent exert tumor killing effects mainly relying on the synergistic effect of small molecule compounds and p53 protein; and the composite nanoparticles can only specifically target HER2-positive breast cancer cells, so their targeting has certain limitations. In addition, the strategy of designing targeted antibodies based on markers expressed on the tumor surface has certain safety risks, because some normal cells also express non-tumor-specific antigens at low levels on their surfaces.

[0004] In summary, it is necessary to propose new development methods and strategies to supplement the shortcomings of existing technologies. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a novel fusion protein HSA-ⅡB-P53 having the function of promoting tumor cell apoptosis, and a preparation method and application thereof. The specific technical scheme is as follows.

[0006] A fusion protein with the function of promoting tumor cell apoptosis, the fusion protein is obtained by fusing the IIB functional region of human serum albumin with P53 protein, and the amino acid sequence of the fusion protein is shown in SEQ ID NO.1.

[0007] A recombinant expression vector comprises the above fusion protein having the function of promoting tumor cell apoptosis.

[0008] Furthermore, the types of the vector include plasmid vectors, phage vectors or animal and plant virus vectors, etc.

[0009] A recombinant bacterium expresses the above fusion protein with the function of promoting tumor cell apoptosis.

[0010] Furthermore, the types of bacteria include one or more selected from Escherichia coli, Bacillus subtilis, Pichia pastoris and / or Saccharomyces cerevisiae.

[0011] The application of the above fusion protein with the function of promoting tumor cell apoptosis in the preparation of anti-tumor drugs.

[0012] Furthermore, the dosage form of the drug includes injection.

[0013] Furthermore, the tumor includes lung cancer, kidney cancer and / or breast cancer.

[0014] Furthermore, the drug also includes a stabilizer, for example, sodium octanoate.

[0015] Furthermore, the effective dosage concentration of the fusion protein having the function of promoting tumor cell apoptosis in the drug is not less than 400 μg / L.

[0016] Beneficial technical effects

[0017] The present invention first proposes a novel fusion protein HSA-ⅡB-P53 with the function of promoting tumor cell apoptosis. The fusion protein is synthesized based on natural P53 protein and combined with the ⅡB functional region of human serum albumin (HSA) with blood long circulation effect. The fusion protein HSA-ⅡB-P53 has good affinity for tumor cells, can enter the interior of cells through the action of cell membrane fusion, replenish the P53 protein deficiency in tumor cells or regulate the P53 gene disorder, and play a normal and orderly regulatory function, thereby causing tumor cells to apoptosis. For normal cells, the replenishment of P53 will not cause the death of normal cells, so the fusion protein HSA-ⅡB-P53 has a universal ability to enter cells and is highly safe. The experiment of the present invention confirms that the fusion protein HSA-ⅡB-P53 has a good anti-tumor effect in cell experiments, so it can be further proved that the fusion protein HSA-ⅡB-P53 has a high efficiency in entering cells.

[0018] Furthermore, due to the long circulation time of HSA, after HSA-ⅡB and P53 are fused, the fusion protein HSA-ⅡB-P53 obtained has a longer half-life and a longer circulation time in the blood, which is beneficial to the enrichment of the fusion protein in tumor tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0020] Figure 1 The crystal structure prediction diagram of the fusion protein HSA-ⅡB-P53 prepared by the present invention;

[0021] Figure 2 This is the SDS-PAGE image of the fusion protein HSA-ⅡB-P53 purified by the present invention;

[0022] Figure 3 This is a diagram showing the stability verification results of the fusion protein HSA-ⅡB-P53 preparation prepared by the present invention;

[0023] Figure 4 This is a diagram showing the hydrophilicity analysis results of the fusion protein HSA-ⅡB-P53 prepared in the present invention;

[0024] Figure 5 This is a result verification diagram of the targeted entry of GFP-labeled fusion protein HSA-ⅡB-P53 into cells in one of the examples;

[0025] Figure 6 This is an experimental diagram of the effect of the fusion protein HSA-ⅡB-P53 on the survival rate of L929 cells (mouse epithelial fibroblasts) in one of the examples;

[0026] Figure 7 This is an experimental diagram of the effect of the fusion protein HSA-ⅡB-P53 on the survival rate of HEK293T cells (human embryonic kidney cells) in one of the examples;

[0027] Figure 8 This is an experimental diagram of the effect of the fusion protein HSA-ⅡB-P53 on the survival rate of 4T1 cells (mouse breast cancer cells) in one of the examples;

[0028] Fig. 9This is an experimental diagram of the effect of the fusion protein HSA-ⅡB-P53 on the survival rate of A549 cells (human non-small cell lung cancer cells) in one of the examples. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0030] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0031] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0032] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0033] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0034] In this specification, some embodiments may be disclosed in a format of being within a certain range. It should be understood that such description of "being within a certain range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the range The description of should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within this range, for example, 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0035] Materials and Reagents

[0036] Human embryonic kidney cells 293T were purchased from the Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences, catalog number SCSP-502, cell name: human embryonic kidney cells.

[0037] Escherichia coli BL21 (DE3) competent cells were purchased from Beijing Solebow Technology Co., Ltd. pET-22b (+) plasmid is a commercially available Escherichia coli expression vector purchased from Beijing Qingke Biotechnology Co., Ltd. The vector tags are N-pelB and C-His, and the vector resistance is Ampicillin. Restriction endonucleases NdeI and XhoI were purchased from NEB (Beijing) Co., Ltd. His-tag protein purification resin (nickel column) was purchased from Shanghai Lianmai Bioengineering Co., Ltd., item number LM-616. IPTG, ampicillin, and DMSO (dimethyl sulfoxide) were all purchased from Beijing Solebow Technology Co., Ltd. DMEM medium was purchased from GIBCO. CCK8 reagent was purchased from Chongqing Baoguang Technology Co., Ltd. and used according to the reagent instructions. .

[0038] Culture medium

[0039] Each liter of LB medium contains: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, and the pH is adjusted to 7.0.

[0040] Preparation method: Dissolve 5g yeast extract, 10g tryptone, and 10g sodium chloride in 950mL double distilled water, adjust the pH to 7.0 with sodium hydroxide solution, and dilute to 1L with double distilled water. If preparing solid culture medium, add agar at 1.5g / 100mL. Sterilize at 121℃ with high pressure steam for 30min.

[0041] The experimental reagents not specifically described in the present invention are all conventional reagents in the art, and can be prepared according to conventional methods in the art or purchased from relevant reagent suppliers; the experimental methods not specifically described are all conventional methods in the art, and reference can be made to relevant experimental manuals, such as molecular cloning experimental manuals or instructions of relevant reagent manufacturers.

[0042] Example 1

[0043] This example provides an example of a method for HSA-ⅡB-P53 gene design and protein expression.

[0044] 1. Genetic Design

[0045] A gene was designed, which encodes a protein containing the IIB region of HSA protein and P53 protein, with a total length of 607 amino acids (aa), named as fusion protein HSA-IIB-P53, and its amino acid sequence is shown in SEQ ID NO.1; the nucleotide sequence encoding the protein is shown in SEQ ID NO.2, with a total length of 1821 base pairs (bp). NdeI restriction sites (CATATG) and XhoI restriction sites (CTCGAG) were added to the 5' and 3' ends of the gene, respectively, and the nucleotide sequence of the gene with restriction sites is shown in SEQ ID NO.3, with a total length of 1830bp. The synthesized gene was sequenced and verified, and the gene with the correct sequence was used for subsequent vector construction. The sequences involved in this embodiment are shown in Table 1.

[0046] Table 1

[0047]

[0048]

[0049]

[0050] 2. Vector Construction

[0051] The pET-22b(+) plasmid was used as an expression vector. The pET-22b(+) plasmid and the target gene (SEQ ID No.1) were subjected to double restriction digestion reaction with restriction endonucleases NdeI and XhoI, respectively, and then the target gene was connected to the pET-22b(+) vector by ligation reaction. It is understood that this embodiment can also be implemented by using a phage vector or an animal or plant virus vector.

[0052] 3. Recombinant Bacterial Transformation

[0053] (1) Take out the competent Escherichia coli BL21 (DE3) cells (Beijing Solebow) from the -80°C freezer and place them on ice for 5 min.

[0054] (2) After the glycerol containing the BL21(DE3) competent cells has melted, add the competent cells to the ligation product, pipette and release the pipette tip 3-4 times to mix, and place on ice for 30 min.

[0055] (3) Quickly wipe the tube wall dry with absorbent paper, then heat shock at 42°C for 90 seconds, and immediately place on ice for 2 minutes.

[0056] (4) Add 800 μL of LB liquid culture medium under sterile conditions and culture at 37°C, 150 rpm for 45 min.

[0057] (5) Collect the cells by centrifugation at 8000 rpm for 5 min, discard part of the supernatant, and use the remaining 100 μL of supernatant to resuspend the E. coli. Then, spread it evenly on LB solid culture medium containing 100 μg / mL ampicillin, place it in a 37°C incubator, and invert and culture for 10-16 h.

[0058] In this embodiment, Escherichia coli is preferably used as the recombinant bacteria. It is understandable that this embodiment can also be implemented using Bacillus subtilis, Pseudomonas aeruginosa or Saccharomyces cerevisiae strains.

[0059] (6) Pick a single clone and inoculate it into liquid LB medium containing 100 μg / mL ampicillin. After culturing at 37°C for 10-16 h, identify the positive clone.

[0060] 4. Protein Expression and Purification

[0061] (1) Inoculation: Prepare liquid LB medium and sterilize it. Place the sterilized liquid LB medium in a clean bench and cool it to room temperature. Add ampicillin to the LB medium in the clean bench and mix it to a final concentration of 100 μg / mL. Then, inoculate 200 μL / L of Escherichia coli (positive clone) containing the target gene plasmid into the LB medium. Place the LB medium in a shaker and culture it at a speed of 170 rpm and a temperature of 37° C. for 8-10 h.

[0062] (2) Induction: After culturing on a shaking platform for 8-10 h, take out 2 mL of the bacterial solution and measure its OD600 value using a spectrophotometer. When the OD600 value of the bacterial solution reaches 0.6-0.8, add IPTG with a final concentration of 200 μL / mL to the bacterial solution, and then culture on a shaking platform at 37°C and 170 rpm for 8 h.

[0063] (3) Purification: After adding IPTG and culturing on a shaking platform for 8 h, the bacterial solution was removed and centrifuged at 8000 rpm for 5 min at 4°C. After centrifugation, the supernatant was removed and the precipitate was retained (the precipitate is Escherichia coli);

[0064] (4) Ultrasonic disruption: The E. coli was ultrasonically disrupted and then centrifuged. The precipitate obtained by centrifugation contained the target protein. The precipitate obtained by centrifugation was first washed with washing solution I (50 mmol / L Tris-HCl, 1 mol / L urea, 10 mL / L Triton X-100), then washed with washing solution II (50 mmol / L Tris-HCl, 2 mol / L urea, 5 mL / L Triton X-100), and then dissolved with inclusion body dissolution solution (50 mmol / L Tris-HCl, 8 mol / L urea, 100 mmol / L NaCl);

[0065] (5) Finally, the inclusion body solution was subjected to gradient renaturation as follows: the inclusion body solution was placed in a dialysis bag (Beijing Solebow Technology Co., Ltd., catalog number: YA1071), and the dialysis bag was placed in 6M, 4M, 2M, 1M, and 0.5M urea solutions in sequence for gradient renaturation. The renaturation was performed for 2-4 hours at each urea concentration, and the renaturation was performed at a low temperature of 4°C. The renaturation solution was purified by His-tag protein purification resin (nickel column) to obtain the fusion protein HSA-ⅡB-P53 (the gene sequence of the target protein was designed with a histidine tag to facilitate protein purification). After purification, polyacrylamide gel electrophoresis (SDS-PAGE) was used to identify whether the purified target protein was successfully obtained. The results are as follows. Figure 2 As shown, a target protein of about 67.286 kDa was obtained, which was consistent with the relative molecular mass prediction result.

[0066] Example 2

[0067] This example provides an example of a method for preparing a fusion protein HSA-ⅡB-P53 preparation.

[0068] (1) Weigh 5 mg of the purified fusion protein HSA-ⅡB-P53 novel protein;

[0069] (2) dissolving in 1 mL of normal saline under magnetic stirring to prepare a fusion protein HSA-ⅡB-P53 normal saline solution with a mass concentration of 5 mg / mL;

[0070] (3) adding an appropriate amount of sodium octanoate to the saline solution of the fusion protein HSA-ⅡB-P53 at a ratio of 0.16 mmol sodium octanoate per gram of the fusion protein HSA-ⅡB-P53 to inhibit the growth and reproduction of microorganisms, improve the stability of the fusion protein HSA-ⅡB-P53 and protect its protein activity;

[0071] (4) The fusion protein HSA-ⅡB-P53 physiological saline solution to which sodium octanoate has been added in the above (3) is ultrasonically dispersed for 10 minutes to uniformly disperse the various solutes in the solution, and then sterilized and impurities are removed through a 0.22 μm sterile filter to obtain a fusion protein HSA-ⅡB-P53 preparation. The dosage form of the fusion protein HSA-ⅡB-P53 preparation is preferably an injection.

[0072] Example 3

[0073] This example provides stability verification of the fusion protein HSA-ⅡB-P53 injection.

[0074] Further, the stability of the fusion protein HSA-ⅡB-P53 injection prepared as above was investigated. The fusion protein HSA-ⅡB-P53 injection and the protein injection prepared without adding sodium octanoate (as a control) were respectively placed at 4°C for static treatment. The concentration of the fusion protein HSA-Ⅱ-P53 was determined once every 24 hours by the BCA method to determine the stability of the protein. The results are as follows: Figure 3 shown.

[0075] The results showed that the stability of the fusion protein HSA-ⅡB-P53 injection with the addition of sodium octanoate was greatly improved compared with the control group, and it would hardly degrade within 15 days, proving the prospect of subsequent clinical development and application.

[0076] Example 4

[0077] This example provides verification of the hydrophilicity and hydrophobicity of the fusion protein HSA-ⅡB-P53.

[0078] Furthermore, the hydrophilicity and hydrophobicity of the novel protein HSA-ⅡB-P53 were investigated. Figure 4 The horizontal axis represents the amino acid number of the protein, and the vertical axis represents the hydrophilicity. The larger the vertical axis value, the more hydrophobic it is; if it is a negative value, it represents hydrophilicity.

[0079] The results showed that the average hydrophilicity value of the fusion protein HSA-ⅡB-P53 was -0.679, indicating that the fusion protein HSA-ⅡB-P53 was hydrophilic overall.

[0080] The basic information of the novel HSA-ⅡB-P53 protein is shown in Table 2.

[0081] Table 2

[0082]

[0083] Example 5

[0084] This example provides verification of the efficiency of the fusion protein HSA-IIB-P53 entering cells.

[0085] Experimental method: The fusion protein HSA-ⅡB-P53, natural human serum albumin (HSA) and natural P53 protein were labeled with FITC, and 200 μg of protein was added to each T75 culture flask, and the three FITC-labeled proteins were co-cultured with A549 cells. After 4 hours of co-culture, the A549 cells in the three culture flasks were centrifuged and collected in sequence, and the cell pellets were washed three times with sterilized PBS buffer. The cells were then resuspended with sterilized PBS buffer and finally the average fluorescence intensity of A549 after co-culture of the three proteins was measured by flow cytometry.

[0086] Results Figure 5 The results showed that compared with the natural has control group and the P53 control group, the mean fluorescence intensity of A549 cells co-cultured with the fusion protein HSA-ⅡB-P53 was significantly enhanced. The results showed that the fusion protein HSA-ⅡB-P53 can efficiently enter tumor cells.

[0087] Example 6

[0088] This example provides an evaluation of the toxicity of the fusion protein HSA-ⅡB-P53 to normal cells.

[0089] The toxicity of the fusion protein HSA-ⅡB-P53 to human embryonic kidney cells 293T and mouse epithelial fibroblasts L929 was studied by CCK8 method to evaluate its biosafety.

[0090] (1) In a 96-well plate, 1 × 10 4 Human embryonic kidney 293T cells were cultured in DMEM medium, and 5 experimental groups and 1 control group were set up, with 3 replicate wells in each group.

[0091] (2) After culturing the cells at 37°C for 24 hours, the fusion protein HSA-ⅡB-P53 was added to the wells of the experimental group (experimental wells), so that the final concentrations of the fusion protein HSA-ⅡB-P53 in the five experimental groups were 10 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, and 400 μg / ml, respectively. No fusion protein HSA-ⅡB-P53 was added to the wells of the control group (control wells).

[0092] (3) Continue to culture at 37°C for 24 h, and then use the CCK8 method to detect the number of live cells. The steps are as follows: add 10 μL of CCK8 reagent to each well and incubate at 37°C for 3 h. Then use an enzyme reader to measure the absorbance of each well at 450 nm.

[0093] The cell viability was calculated as follows:

[0094] [Absorbance of experimental wells (culture medium containing cells, CCK8 reagent, protein sample) - absorbance of blank wells (culture medium without cells and protein sample, CCK8 reagent)] / [Absorbance of control wells (culture medium containing cells, CCK8 reagent, no protein sample) - absorbance of blank wells (culture medium without cells and protein sample, CCK8 reagent)]×100%.

[0095] The cell survival rate of each group is the average of the cell survival rates of the three replicate wells in the group. The cell survival rate of the control group is 100%, and the results are shown in Figure 2. Figure 6 and Figure 7As shown in the figure, the cells showed a higher survival rate after adding 10-400μg / mL concentration of fusion protein HSA-ⅡB-P53. When the concentration of fusion protein HSA-ⅡB-P53 reached 400μg / ml, the survival rate of L929 cells and HEK293T cells remained above 90%, proving that the fusion protein HSA-ⅡB-P53 has low toxicity to normal cells and good biocompatibility.

[0096] Example 7

[0097] This example provides an evaluation of the anticancer efficacy of the fusion protein HSA-IIB-P53.

[0098] (1) In a 96-well plate, 1×10 4 A549 (lung cancer human alveolar basal epithelial cells) cells and 4T1 (mouse breast cancer cells) cells were added to DMEM culture medium, and 5 experimental groups and 1 control group were set up respectively, with 3 replicate wells in each group.

[0099] (2) After culturing the cells at 37°C for 24 hours, the fusion protein HSA-ⅡB-P53 was added to the wells of the experimental group (experimental wells), so that the final concentrations of the fusion protein HSA-ⅡB-P53 in the five experimental groups were 10 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, and 400 μg / ml, respectively. No fusion protein HSA-ⅡB-P53 was added to the wells of the control group (control wells).

[0100] (3) Continue to culture at 37°C for 24 h, and then use the CCK8 method to detect the number of live cells. The steps are as follows: add 10 μL of CCK8 reagent to each well and incubate at 37°C for 3 h. Then use an enzyme reader to measure the absorbance of each well at 450 nm.

[0101] The cell viability was calculated as follows:

[0102] [Absorbance of experimental wells (culture medium containing cells, CCK8 reagent, protein sample) - absorbance of blank wells (culture medium without cells and protein sample, CCK8 reagent)] / [Absorbance of control wells (culture medium containing cells, CCK8 reagent, no protein sample) - absorbance of blank wells (culture medium without cells and protein sample, CCK8 reagent)]×100%.

[0103] The results are as follows Figure 8 and Fig. 9 shown. Figure 8 and Fig. 9The results of the toxicity of the new HSA-ⅡB-P53 protein to two types of tumor cells are shown respectively. When the concentration of the new HSA-ⅡB-P53 protein reaches 400μg / ml, the survival rate of 4T1 cells is only about 50%, and the survival rate of A549 cells is less than 50%, indicating that the fusion protein HSA-ⅡB-P53 can not only enter tumor cells smoothly, but also has strong cytotoxicity to tumor cells and can effectively promote tumor cell apoptosis.

[0104] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A fusion protein having the function of promoting apoptosis of tumor cells, characterized in that: The fusion protein is obtained by fusing the IIB functional region of human serum albumin with the P53 protein. The amino acid sequence of the fusion protein is shown in SEQ ID NO.

1.

2. A recombinant expression vector, characterized in that: It comprises a gene encoding the fusion protein having the function of promoting tumor cell apoptosis as claimed in claim 1.

3. The recombinant expression vector according to claim 2, characterized in that The types of the vector include plasmid vectors, phage vectors or animal and plant virus vectors.

4. A recombinant bacterium, characterized in that Expressing the fusion protein having the function of promoting tumor cell apoptosis as claimed in claim 1.

5. The recombinant bacterium according to claim 4, characterized in that The bacteria may be selected from Escherichia coli and / or Bacillus subtilis.

6. Use of the fusion protein having the function of promoting tumor cell apoptosis according to claim 1 in the preparation of anti-tumor drugs, characterized in that: The tumors include lung cancer and / or breast cancer.

7. The use according to claim 6, characterized in that The dosage form of the drug includes injection.

8. The use according to claim 6, characterized in that The drug also includes a stabilizer.

9. The use according to claim 6, characterized in that The effective dosage concentration of the fusion protein having the function of promoting tumor cell apoptosis in the drug is not less than 400 μg / L.

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