CN1506375A - Bacterial protein azurin with broad-spectrum anti-tumor effect, its use and pharmaceutical composition containing it - Google Patents

Abstract

The invention relates to a bacterial protein azurin with broad-spectrum anti-tumor effect, its coding nucleic acid sequence, and the use of the protein in preparing anti-tumor drugs. The invention also discloses the amino acid sequence of the protein and the nucleic acid sequence encoding the protein. The invention also discloses a pharmaceutical composition containing therapeutically effective dose of azurin. The present invention also relates to methods for preparing said protein and methods for detecting said protein in a sample.

Description

Bacterial protein azurin with broad-spectrum anti-tumor effect, use thereof, and pharmaceutical composition containing it

field of invention

The invention relates to a bacterial protein azurin with broad-spectrum anti-tumor effect, its coding nucleic acid sequence, and the use of the protein in preparing anti-tumor drugs. The invention also discloses the amino acid sequence of the protein and the nucleic acid sequence encoding the protein. The invention also discloses a pharmaceutical composition containing therapeutically effective dose of azurin. The present invention also relates to methods for preparing said protein and methods for detecting said protein in a sample.

Background of the invention

Tumor is a common malignant tumor that seriously endangers human health. At present, there is no particularly effective prevention and treatment strategy (Xu RZ, Gao QK, Wang SJ, et al. Natl Med JChina 78, 504-507, 1998). Therefore, tumor remains a lethal disease for most patients. One of the main reasons is that there is currently no particularly effective drug to completely prevent and cure tumors.

As early as the beginning of the 20th century, it was reported that a very small number of tumor patients had spontaneous tumor regression due to severe bacterial infection. In the past ten years, the inventors have successively observed that many cases of leukemia patients have spontaneous remission of leukemia after severe bacterial infection. The previous explanation for this phenomenon was that the tumor regression was caused by the production of tumor necrosis factor by the body after bacterial infection, and some people believed that it was caused by the high fever of the body after bacterial infection. However, more subsequent studies have shown that tumor necrosis factor and high fever are not the main factors.

In recent years, the inventors have begun to isolate and culture infected bacteria from patients, and have conducted detailed studies on the anti-tumor effects of bacterial metabolites. Through the analysis of more than 100 strains of different bacterial metabolites, it was found that specific bacterial metabolites can significantly induce rapid apoptosis of various human leukemia cells and other tumor cells. On the basis of the separation, purification and identification of the effective components, the inventors unexpectedly found that a protein with a molecular weight of about 16KD, azurin (AZURIN), is the main component for inducing tumor cell apoptosis. Further research results showed that this small molecule protein could not only induce apoptosis of various tumor cells rapidly and efficiently in vitro, but also effectively kill tumor cells in a mouse tumor model. What's more worth mentioning is that this small molecular protein has no obvious side effects and is very stable.

Summary of the invention

One aspect of the present invention provides a bacterial protein azurin with broad-spectrum anti-tumor effects, characterized in that the protein has an amino acid sequence as shown in SEQ ID NO: 2, or has equivalent biological activity Fragments or conservative variants or derivatives thereof.

Another aspect of the present invention provides a nucleic acid sequence encoding the azurin of the present invention, characterized in that it comprises the nucleotide sequence shown in SEQ ID NO: 1, or its degenerate variants.

Another aspect of the present invention provides a method for preparing the azurin of the present invention.

Another aspect of the present invention provides the use of the azurin of the present invention for the preparation of anti-tumor drugs.

Another aspect of the present invention provides an anti-tumor pharmaceutical composition, which contains a therapeutically effective amount of the azurin of the present invention, and optional pharmaceutically acceptable adjuvants.

The present invention also discloses a method for detecting whether the azurin molecule of the present invention exists in a sample, comprising: using the monoclonal antibody or polyclonal antibody prepared by the protein polypeptide sequence of SEQ ID NO.2; using the obtained antibody according to A technique selected from: enzyme immunoassay, immunohistochemical technique, and western blotting to detect the presence or absence of azurin molecules in the sample.

Detailed description of the invention

The present invention relates to a bacterial protein azurin with broad-spectrum anti-tumor effect, characterized in that the protein has an amino acid sequence as shown in SEQ ID NO: 2, or a fragment thereof having equivalent biological activity or Conservative variants or derivatives.

Specifically, the azurin protein of the present invention is a small molecular protein composed of 148 amino acid residues and a molecular weight of about 16KD. Among them, the 1-20 amino acid residues from the N-terminal are the signal peptide of the protein polypeptide molecule, and the signal peptide plays a key role in guiding the protein polypeptide molecule to shuttle freely in the cytoplasm and nucleus of mammalian cells. The 21st-148th amino acid residues are the core sequence of the protein polypeptide molecule.

The term "fragment with equivalent biological activity" or its "conservative variant" as used herein means that the fragment is different from the amino acid sequence described in SEQ ID NO: 2, but retains the basic characteristics of the amino acid , such as fundamental biological, structural, regulatory, or biochemical properties. Usually, the differences at the amino acid level are limited so that the sequence of the reference polypeptide and its conservative variants are generally very similar, and identical in many regions. Conservative variants differ from the amino acid sequence of a reference polypeptide by conservative substitutions, additions, and deletions of one or more amino acids that are identical or similar in nature. Substituted or inserted amino acid residues may or may not be encoded by the genetic code. A variant of a polynucleotide or polypeptide may occur naturally (eg, an allelic variant), or it may be a non-naturally occurring variant. Non-naturally occurring variants of polynucleotides and polypeptides can be produced by mutagenesis techniques or direct synthesis.

Combining the disclosure of the present invention and the conservative amino acid substitutions well known in the art, those of ordinary skill in the art know that the azurin described in the present invention is not limited to the amino acid sequence described in SEQ ID NO: 2, and also includes The equivalent biologically active fragment obtained after the substitution has substantially the same biological function, or a conservative variant thereof.

In one embodiment of the present invention, the azurin has the amino acid sequence shown in SEQ ID NO: 2, which contains the encoded protein and signal peptide of the azurin. In yet another embodiment of the present invention, the azurin has an amino acid sequence as shown in SEQ ID NO:3.

In the present invention, the term "azurin gene" refers to a gene containing a functional regulatory sequence and encoding a biologically functional protein azurin in bacteria such as Pseudomonas aeruginosa. The term also includes all variants or derivatives with biological functions having more than 85% homology with the nucleotide sequence described in SEQ ID NO.1.

Specifically, the present invention provides the amino acid sequence SEQ ID NO.2 of the bacterial protein encoded by the azurin gene, and its core sequence SEQ ID NO.3. SEQID NO.2 is a protein composed of 148 amino acids encoded by the azurin gene, with a molecular weight of about 16KDa, of which 1-20 amino acid residues from the N-terminus are the signal peptide of the protein molecule, which guides the Protein molecules play a key role in free shuttling within the cytoplasm and nucleus of mammalian cells. And SEQ ID NO.3 is the core sequence of the protein, consisting of 127 amino acid residues.

In the present invention, the term "the amino acid sequence of the bacterial protein polypeptide encoded by the azurin gene" not only refers to the amino acid sequence of the protein as described in SEQ ID NO.2, but the term also includes the amino acid sequence described in SEQ ID NO.2. All proteins with a sequence homology of more than 85% and their conservative variants or derivatives with biological functions. The term "the core sequence of the protein" not only refers to the amino acid sequence of the protein as described in SEQ ID NO.3, but also includes all the amino acid sequences that have more than 85% homology with the sequence described in SEQ ID NO.3 and have biological functions. proteins and conservative variants or derivatives.

Another aspect of the present invention relates to a nucleic acid sequence encoding the azurin protein of the present invention or its fragments with equivalent biological activity or conservative variants or derivatives thereof, characterized in that it comprises such as SEQ ID NO : The nucleotide sequence shown in 1, its degenerate variants, or the corresponding nucleotide sequence variants.

Specifically, the present invention provides the gene encoding the above-mentioned azurin, as shown in the nucleotide sequence SEQ ID NO.1. The gene has a total of 1284 nucleotide residues, and its gene structure consists of the 5' end non-coding region (UTR), gene coding region and 3' end non-coding region from the 5' end to the 3' end of the virus gene. . Its 5' end UTR has 488 nucleotides; the gene coding region has 447 nucleotides; the 3' end UTR has 349 nucleotides; the specific arrangement is: from nucleotides 1 to 488 The first nucleotide is the 5' non-coding region, the 489-935th nucleotide is the gene coding region, and the 936-1284th nucleotide is the 3'-terminal non-coding region.

The degeneracy "variant" in the present invention means that due to the degeneracy of the genetic code, the nucleotide sequence encoding the azurin protein may have a variety of base compositions, and its polynucleotide may be different from the reference polynuclear Nucleotides, but retain the basic encoding the protein. A degenerate variant is a change in the nucleotide sequence that does not alter the amino acid sequence of the protein. The corresponding nucleotide sequence variant refers to the nucleic acid sequence encoding the azurin equivalent biologically active fragment or its conservative variant or derivative.

Another aspect of the present invention discloses a method for preparing the azurin of the present invention, which includes, but is not limited to:

1) Combined application of segmented salting-out and SDS-polyacrylamide gel electrophoresis methods to directly separate and purify natural azurin molecules from bacterial cultures;

2) Design PCR primers according to the nucleic acid sequence provided by SEQ ID NO.1, use PCR technology to amplify the gene encoding azurin molecule from bacterial DNA, and then connect it to a suitable expression vector to recombinantly produce azurin molecule; or

3) According to the amino acid sequence provided by SEQ ID NO.2, it is prepared by artificial synthesis.

Specifically, in one embodiment of the present invention, there is provided a method for rapidly separating and purifying natural azurin molecules from the bacterial culture of Pseudomonas aeruginosa, characterized in that the combined application of segmental salting-out and SDS- Isolation and purification of native azurin molecules directly from bacterial cultures of Pseudomonas aeruginosa by polyacrylamide gel electrophoresis.

In the present invention, the term "quick separation and purification of natural azurin molecules" refers to the combined application of saturated ammonium sulfate salting-out technology and SDS-polyacrylamide gel electrophoresis and electroelution methods to obtain high-purity natural azurin molecules directly from bacterial cultures. Azurin molecule. In this term, SDS-polyacrylamide gel electrophoresis can be either a mini-SDS-polyacrylamide gel or a midi-scale or large-scale SDS-polyacrylamide gel.

In yet another embodiment of the present invention, a kind of utilizing SEQ ID NO.1 sequence design primer is provided, application PCR or RT-PCR technology can directly isolate and clone this azurin gene and corresponding from Pseudomonas aeruginosa Regulatory element sequence method. The operation steps are as follows:

(1) Design corresponding PCR or RT-PCR primers.

(2) Genomic DNA or RNA was extracted from Pseudomonas aeruginosa.

(3) PCR amplification of the azurin gene.

(4) The DNA bands amplified by PCR were separated by agarose gel electrophoresis, and the PCR products were purified by SpinColumn.

(5) Clone the PCR product with T-A cloning kit.

Sequencing analysis was performed using a fully automatic DNA sequencer.

In another aspect of the present invention, there is also provided a method for producing the protein polypeptide encoded by the azurin gene by gene recombination technology, the steps are as follows:

(1) operably linking the nucleotide sequence encoding the azurin gene to the expression control sequence to form an azurin expression vector;

(2) transferring the expression vector in step (1) into the host cell to form a recombinant cell of the azurin molecule encoded by the azurin gene;

(3) cultivating the recombinant cells in step (2) under conditions suitable for expressing the azurin;

(4) isolate the viral protein with biological activity.

In yet another aspect of the present invention, the present invention discloses that azurin has broad-spectrum anti-tumor effects, which can be used to prepare anti-tumor drugs, wherein the tumors include but not limited to: myeloid leukemia, lymphoid leukemia, colorectal cancer , lung cancer, liver cancer, gastric cancer.

In the present invention, the term "broad-spectrum anti-tumor effect" means that the azurin described in the present invention has a killing effect on various human tumor cells, and these tumor cells include human myeloid leukemia cells, lymphoid leukemia cells, colorectal cancer cells, lung cancer cells, liver cancer cells, lung cancer cells, gastric cancer cells and other tumor cells.

Based on the analysis of the effect of specific bacterial metabolites on tumor cells, the present inventors discovered for the first time that azurin molecules have highly selective and broad-spectrum anti-tumor effects. Studies on in vitro cell culture systems have shown that the protein can induce rapid apoptosis in various human tumor cells, including human myeloid leukemia cells, lymphoid leukemia cells, colorectal cancer cells, lung cancer cells, liver cancer cells, and lung cancer cells , Gastric cancer cells and other tumor cells.

In addition, the azurin described in the present invention can effectively kill the tumor cells in the mouse tumor model, and has no obvious toxic and side effects on the mice themselves.

Another aspect of the present invention relates to a pharmaceutical composition for tumor treatment, which contains a therapeutically effective amount of the azurin of the present invention, and optional pharmaceutically acceptable adjuvants.

The "therapeutically effective amount" in the present invention refers to the amount that can produce positive effects on the tumor to be treated. Specifically, depending on the type of tumor to be treated, the patient's onset stage, general condition (including age, body weight), and the method of administration, route of administration and other factors, clinicians can judge and adopt a specific "effective treatment" based on their experience. amount.” Generally speaking, the therapeutically effective dose is within the range of 0.01 mg-10 mg protein/kg body weight.

The adjuvants in the composition of the present invention are known to those skilled in the art of formulations, including but not limited to excipients, disintegrants, binders, colorants, etc., can be used as long as the adjuvants do not affect the composition of the present invention. The role of azurin.

In yet another embodiment of the present invention, the pharmaceutical composition for tumor treatment may additionally contain other known anti-tumor drugs, so as to produce a synergistic effect with the azurin of the present invention to treat tumors.

Another aspect of the present invention relates to a method for detecting whether there is azurin molecule in a biological sample, comprising: using a monoclonal antibody or a polyclonal antibody prepared from the protein polypeptide sequence of SEQ ID NO.2; using the obtained antibody according to the selected From: Enzyme immunoassay, immunohistochemical technique, western blotting technique to detect the presence or absence of azurin molecule in the sample.

In particular, the present invention also provides various antibodies against the azurin, including polyclonal antibodies and monoclonal antibodies.

In the present invention, a series of known methods can be used to prepare various specific antibodies against the above-mentioned protein encoded by the azurin gene. For example, the purified above-mentioned azurin gene expression products or their antigen fragments are injected into animals (including, but not limited to, rabbits, sheep, horses, mice, etc.) to produce polyclonal antibodies. Similarly, multiple specific monoclonal antibodies against the azurin can also be prepared by using hybridoma technology well known in the art. The antibodies of the present invention include various antibodies that can inhibit the biological function of the azurin, and can also be antibodies that do not affect its function. Each antibody can be produced by immunizing the above-mentioned azurin fragments or functional domains, and the above-mentioned azurin and its fragments can be produced by recombinant methods or polypeptide synthesizers.

The above-mentioned multiple specific antibodies of the present invention can be used to identify and express the protein encoded by the azurin gene. For example, various specific antibodies of the present invention can be used in combination with techniques such as immunofluorescence test, immunocytochemistry, and flow cytometry commonly used in the art to detect and analyze cells expressing the corresponding azurin. It is also possible to use the antibody of the present invention in conjunction with Western Blot to quantitatively analyze the expression of azurin in cells.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods that do not indicate specific conditions in the following examples, generally follow conventional conditions, such as the experimental conditions described in "Molecular Cloning: Experimental Manual" edited by Sambrook et al., or according to the experimental conditions provided by the reagent or kit manufacturer .

Example

The separation and purification of the azurin of embodiment 1 natural bacterial source

Adopt saturated ammonium sulfate salting-out technology known in the art and SDS-polyacrylamide gel electrophoresis technology (Wang Jiazheng etc., editor-in-chief, "Protein Technology Handbook", Science Press), from the culture of Pseudomonas aeruginosa Isolation and purification of the natural bacterial protein azurin.

a) Inoculate Pseudomonas aeruginosa into 200ml LB culture medium according to the conventional method, and shake and culture at 37°C overnight (about 16 hours).

b) Centrifuge at 10,000 rpm for 15 minutes, take the supernatant, add solid ammonium sulfate to 40% saturation, and ice-bath for 1 hour.

c) Centrifuge at 10,000 rpm for 15 minutes, take the supernatant, add solid ammonium sulfate to 80% saturation, and ice-bath for 1 hour.

d) Centrifuge at 10,000 rpm for 15 minutes, and discard the supernatant. Dissolve the pellet in 1ml deionized water, then add 2 protein electrophoresis loading buffer.

e) Separation of proteins with 12% SDS-polyacrylamide gel and preparative protein electrophoresis.

f) After the electrophoresis, the 16KD protein band (that is, the band corresponding to the azurin of the present invention) was cut out with a scalpel blade, and then the bacterial protein azurin in the gel was eluted by electroelution.

g) Finally, the distilled water is dialyzed overnight with a dialysis bag, and the protein content is quantified by an ultraviolet spectrophotometer.

Example 2 Using the PCR method to prepare the polynucleotide sequence encoding the azurin protein of the present invention

The PCR technology known in the application field is used to isolate and clone the full-length sequence encoding the azurin protein, and the specific operation steps are as follows:

Design PCR primers:

Upstream primer: 5′-GCC CAA GCT TAC CTA GGA GGC TGCTCC ATG CTA-3′ (SEQ ID NO: 4),

Downstream primer: 5′-TGA GCC CCT GCA GGC GCC CAT GAAAAA GCC CGG C-3′ (SEQ ID NO: 5).

Genomic DNA was extracted from Pseudomonas aeruginosa by conventional methods and purified. Take 1 μg of purified genomic DNA as a template and add it to 50 μl PCR reaction system. Add the above primers, dNTP, Taq enzyme, MgCl2, and PCR buffer in sequence.

PCR cycle parameters: 94°C 2'; 94°C 20"/62°C 30"/72°C 10'×35 cycles; 72°C 7'; 4°C.

1% agarose gel electrophoresis was used to separate the DNA band with a size of about 0.5kb, and the PCR product was purified with SpinColumn.

PCR products were cloned using T-A cloning kit.

Automatic DNA sequencer sequencing analysis.

Example 3 Preparation of broad-spectrum anti-tumor bacterial protein azurin by recombinant method

The full-length nucleotide sequence encoding azurin obtained in Example 2 above was constructed into a commercially available expression vector to express and purify the recombinant protein.

To facilitate purification, azurin was prokaryotically expressed in Escherichia coli in the form of 6XHIS fusion protein or GST fusion protein.

Construction of prokaryotic expression vector

According to the sequence provided by SEQ ID NO.1, design PCR primers to amplify the nucleic acid sequence encoding the complete bacterial protein azurin, and clone the amplified DNA fragment into pQE30 or pGEX-T vector after PCR amplification. Then, the calcium chloride method is used to transfer into suitable Escherichia coli, and the recombinants containing the bacterial protein azurin are screened and identified.

Isolation and Purification of Recombinant Fusion Protein Expressing Azurin

Get the recombinant engineered bacteria containing the bacterial protein azurin, put it into a test tube containing 7ml of LB culture solution, and cultivate it overnight at 37°C with shaking. Then, the engineered bacteria were inoculated into fresh LB medium at a concentration ratio of 1:100, and continued shaking culture at 37°C for 3 hours, then added IPTG (final concentration 1mmol/L), and continued shaking culture at 37°C for 3 hours. Take the engineered bacteria liquid induced by IPTG, centrifuge, discard the supernatant, suspend the bacterial pellet with PBS, and then break the bacteria by ultrasonic pulverization. The recombinant bacterial protein azurin was then purified by column centrifugation.

Embodiment 4 Anti-human myeloid leukemia cell effect of azurin described in the present invention

(1) Aseptically inoculate human myeloid leukemia cells HL-60 and K562 in a 96-well cell culture plate, and inoculate 0.1 ml of complete cell culture medium 1640 containing 1×10 ⁵ cells in each well.

(2) Add 0.1 ml of complete cell culture medium 1640 containing different concentrations of azurin prepared as above to each well, mix with the cells, and put the cells in a 37° C. cell incubator (containing 5% CO 2 ) for 48 hours.

(3) The cell culture was terminated, and the viability of the cultured cells was measured by the MTT method.

(4) It was found that: 10-20ug/ml bacterial protein azurin inhibited the growth of the human myeloid leukemia cells HL-60 and K562 above 80%, but had no obvious inhibitory effect on the growth of corresponding normal cells.

(5) The results of flow cytometry analysis showed that: the bacterial protein azurin can significantly induce the apoptosis of the above-mentioned human myeloid leukemia cells HL-60 and K562, after 48 hours of 10-20ug/ml bacterial protein azurin The apoptosis rates of human myeloid leukemia cells HL-60 and K562 were greater than 70%.

Embodiment 5 Anti-human lymphoid leukemia cell effect of azurin described in the present invention

(1) Human lymphoid leukemia cells Jurkat were aseptically inoculated in a 96-well cell culture plate, and 0.1 ml of complete cell culture medium 1640 containing 1×10 ⁵ cells was inoculated in each well.

(2) Add 0.1 ml of complete cell culture medium 1640 containing different concentrations of azurin prepared as above to each well, mix with the cells, and put the cells in a 37° C. cell incubator (containing 5% CO 2 ) for 48 hours.

(3) The cell culture was terminated, and the viability of the cultured cells was measured by the MTT method.

(4) The results showed that: 10ug/ml bacterial protein azurin inhibited the growth of the human lymphoid leukemia cells Jurkat by more than 90%, but had no obvious inhibitory effect on the growth of corresponding normal cells.

(5) The results of flow cytometry analysis showed that after 48 hours of treatment with 10 ug/ml bacterial protein azurin, the apoptosis rate of Jurkat above-mentioned human lymphoid leukemia cells was greater than 85%.

Embodiment 6 The anti-human lung cancer cell effect of astragalin according to the present invention

(1) Aseptically inoculate human lung cancer cells in a 96-well cell culture plate, and inoculate 0.1 ml of complete cell culture medium DMEM containing 1×10 ⁵ cells in each well.

(2) Add 0.1 ml of complete cell culture medium DMEM containing different concentrations of azurin prepared as above to each well, mix with the cells, put the cells in a 37° C. cell incubator (containing 5% CO 2 ) and cultivate for 48 hours.

(3) The cell culture was terminated, and the viability of the cultured cells was measured by the MTT method.

(4) The results showed that: 20ug/ml bacterial protein azurin had obvious inhibitory effect on the growth of the above-mentioned human lung cancer cells, and the inhibition rate was greater than 65%, but had no obvious inhibitory effect on the corresponding normal cell growth.

(5) The results of flow cytometry analysis showed that: the bacterial protein azurin can obviously induce the above-mentioned human lung cancer cells to undergo apoptosis, and the apoptosis rate of 20ug/ml bacterial protein azurin can reach 60% after being treated for 48 hours.

Embodiment 7 Anti-human breast cancer cell effect of azurin of the present invention

(1) Aseptically inoculate human breast cancer cells in a 96-well cell culture plate, and inoculate 0.1 ml of complete cell culture medium DMEM containing 1×10 ⁵ cells in each well.

(2) Add 0.1 ml of complete cell culture medium DMEM containing different concentrations of azurin prepared as above to each well, mix with the cells, put the cells in a 37° C. cell incubator (containing 5% CO 2 ) and cultivate for 48 hours.

(3) The cell culture was terminated, and the viability of the cultured cells was measured by the MTT method.

(4) The results showed that: 15ug/ml bacterial protein azurin had obvious inhibitory effect on the growth of the above-mentioned human breast cancer cells, and its growth inhibition rate reached 75%, but had no obvious inhibitory effect on the corresponding normal cell growth.

(5) The results of flow cytometry analysis showed that the bacterial protein azurin can obviously induce the above-mentioned human breast cancer cells to undergo apoptosis, and the apoptosis rate reaches 70%.

Example 8 In vivo anti-human leukemia effect of azurin according to the present invention

a) Take 20 SCID nude mice of about 20 grams, and subcutaneously inoculate 1×10 ⁶ human leukemia cells on the back of each mouse.

b) One week after inoculation, 20 mice were divided into two groups, A and B, with ten mice in each group. Among them, each mouse in group A was intraperitoneally injected with 100 ug of the bacterial protein azurin prepared as above every day. Simultaneously observe tumor growth and mouse growth.

c) Execute the experimental mice after 4 weeks, measure and record the growth of the tumor on the back of the mice and the growth of the mice themselves.

d) As a result, it was found that the growth of tumors on the back of the mice in the group injected with 100 μg of bacterial protein azurin A per day was significantly inhibited, while the growth of tumors in the back of the mice in the group not injected with bacterial protein azurin B was good. The average tumor weight of mice in group A was only about 1/4 of that in group B of the control group.

e) Observation and analysis of pathological tissue slices showed that actively growing tumor cells were rarely seen in the tumor tissue of the experimental group injected with bacterial protein azurin, while the growth of tumor cells in the control group was very active.

f) The mice in the experimental group injected with the bacterial protein azurin had no obvious side effects during the whole experiment.

Sequence Listing

<110>The Second Affiliated Hospital of Zhejiang University School of Medicine

  Yangshengtang Co., Ltd.

<120> Bacterial protein azurin with broad-spectrum anti-tumor effect and its use and pharmaceutical composition containing it

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tgagcccctg caggcgccca tgaaaaagcc cggc 34

Claims (9)

1. A bacterial protein azurin with broad-spectrum anti-tumor effect, characterized in that, the protein has an amino acid sequence as shown in SEQ ID NO: 2, or a fragment with equivalent biological activity or its conservation variants or derivatives. 2. the described azurin of claim 1 is characterized in that, described protein has the aminoacid sequence shown in SEQ ID NO:2. 3. the described azurin of claim 1 is characterized in that, described protein has the aminoacid sequence shown in SEQ ID NO:3. 4. A nucleic acid sequence encoding azurin according to claim 1, characterized in that it comprises the nucleotide sequence shown in SEQ ID NO: 1, or its degenerate variants. 5. A method for preparing azurin as claimed in claim 1, which is selected from 1) Combined application of segmented salting-out and SDS-polyacrylamide gel electrophoresis methods to directly isolate and purify natural azurin molecules from Pseudomonas aeruginosa cultures; 2) Design PCR primers according to the nucleic acid sequence provided by SEQ ID NO.1, amplify the gene encoding the azurin molecule from the bacterial DNA by PCR technology, and then connect it to a suitable expression vector to recombine the azurin molecule; or 3) According to the amino acid sequence provided by SEQ ID NO.2, it is prepared by artificial synthesis. 6. The use of azurin according to claim 1 for the preparation of antitumor drugs, wherein the tumors include myeloid leukemia, lymphoid leukemia, colorectal cancer, lung cancer, liver cancer, and gastric cancer. 7. An antitumor pharmaceutical composition, which contains a therapeutically effective amount of azurin as claimed in claim 1, and optional pharmaceutically acceptable adjuvants. 8. The anti-tumor pharmaceutical composition as claimed in claim 7, which optionally contains one or more other known anti-tumor drugs in a therapeutically effective amount. 9. A method for detecting whether there is an azurin molecule in a sample, comprising: utilizing a monoclonal antibody or a polyclonal antibody prepared from the protein polypeptide sequence of SEQ ID NO.2; the basis for using the obtained antibody is selected from: enzyme immunoassay, The presence or absence of azurin molecules in samples was detected by immunohistochemical techniques and western blotting techniques.