WO2004091650A1

WO2004091650A1 - Application of cardiac troponin i in preparing antitumour medicaments

Info

Publication number: WO2004091650A1
Application number: PCT/CN2004/000339
Authority: WO
Inventors: Fengming Liu
Original assignee: Fengming Liu
Priority date: 2003-04-15
Filing date: 2004-04-12
Publication date: 2004-10-28
Also published as: CN1294987C; CN1537633A

Abstract

The invention provides methods of using cardiac troponin I to prepare antitumour medicaments. The invention also provides the effective dose of cardiac troponin I which is from 1µg to 1mg/kg of patient weight/day. Antitumour medicaments of the invention comprise one or more pharmaceutically acceptable carriers. The invention further provides genes encoding cardiac troponin I which express highly in Escherichia coli and Pichia pastoris expression system.

Description

Application of cardiac troponin I in preparing antitumor drugs

The present invention relates to new uses of compounds, and particularly to new uses of cardiac troponin I.

Background technique

Malignant tumors are one of the most serious diseases that endanger human health. At present, the common methods for treating malignant tumors are: surgical treatment, chemotherapy, radiation therapy, biological therapy, and traditional Chinese medicine. Among them, surgical treatment, chemotherapy, and radiation therapy are the most commonly used. Surgical treatment, as the first choice and main program for most cancer treatments, has enabled countless cancer patients to recover, prolong life or improve quality of life. However, in terms of tumor treatment, surgical treatment is only applicable to patients with certain conditions and ranges, and because of tumor recurrence and metastasis, surgical resection cannot always achieve the goal of radical cure. The use of chemotherapy and radiation therapy also plays a very important and indispensable role in the treatment of tumors, and is also constantly developing. However, currently used chemotherapy drug therapy and radiation therapy have an invasion of all active proliferative cells in the body, such as the hematopoietic system, gastrointestinal tract, etc., so patients with chemoradiation often have reduced blood vision, gastrointestinal bleeding, nausea, and vomiting. And other symptoms. In addition, due to the patient's physical and mental endurance, and tumor cell resistance, and other reasons, radiotherapy and chemotherapy cannot kill all tumor cells, leaving the recurrence and metastasis of the tumor and the deterioration of the disease Hidden danger. Biological therapy and traditional Chinese medicine therapy are also reasonable anti-tumor methods, but further research is needed to improve the efficacy. Therefore, the development of antineoplastic drugs with obvious therapeutic effects and safety is an urgent task facing the medical community.

Studies have shown that the unlimited invasive growth and metastasis of malignant solid tumors depend on the continuous generation of new blood vessels, so inhibiting angiogenesis can significantly inhibit tumor growth. Inhibiting angiogenesis and blocking blood supply to tumors is a new strategy that is different from conventional antitumor therapy, also known as "tumor starvation therapy", and has become a hot spot in antitumor therapy research. Under normal physiological conditions, in addition to the ovulation, luteal generation, pregnancy, and other processes of the female reproductive system, there is a short period of neovascularization, and the adult vasculature is in a relatively static state. However, in tumor tissues, in order to adapt to the rapid growth of tumor tissues, angiogenesis is very active, and blood is provided for tumor tissue growth in a timely manner. 'There are two sources of microvessels in tumor tissues: one is vascular endothelial growth factor produced by tumor cells, which induces tumors to generate microvessels; the other is that the host blood vessels remaining in the tumors gradually become tumor blood vessels. Tumorization. Unlike normal blood vessels, the vascular endothelial cells of tumor blood vessels are incomplete, the basement membrane is sparse and leaks easily. After tumor angiogenesis, it will not be further differentiated into arteries and veins, without smooth muscles and nerve endings. It is a passive blood vessel. Its blood flow is completely dependent on the systemic circulation. The new microvessels are the first stop for tumor invasion and metastasis. Tumor cells enter the blood circulation through the tumor microvessel wall and transfer to distant places.

Solid tumor growth requires a large blood supply. Many experimental studies have confirmed that inhibiting neovascularization in animals can effectively cut off the blood supply that provides nutrients for tumor growth, causing the tumor to shrink and even disappear. Application of anti-neovascular drugs TNP-470 and monoclonal antibodies against bFGF and VEGF, mutant VEGF receptor F1K-1 competitively blocks VEGF signaling, α ν β 3 overexpression induces endothelial cell apoptosis, and specific inhibition of Angiostatin Tumor neovascularization can inhibit tumor growth. It is a new and promising antitumor drug, which can make up for the limitations of current clinical tumor treatment methods and replace some treatment methods. Since these drugs inhibit tumor growth by inhibiting the formation of new blood vessels, the growth of human normal tissue cells does not depend on the formation of new blood vessels, so there are almost no toxic side effects on normal tissues. These drugs do not directly affect tumor cells, so they have a broad spectrum of tumor inhibition without drug resistance. They are currently recognized internationally as effective, safe and promising anti-tumor drugs. They are mainly used in situ and metastatic solid tumors. Surgical treatment of tumors and adjuvant tumors can also be used to prevent tumor recurrence and metastasis. Therefore, the development of an anti-tumor drug with an inhibitory effect on angiogenesis has important clinical significance in the prevention and treatment of malignant tumors.

Cardiac troponin I is one of the three subunits of cardiac troponin. It is often used as a marker for the diagnosis of myocardial infarction. It has the sequence of SEQ ID N _2: 1 amino acid residue in the sequence listing and consists of 210 amino acids. Residue composition.

E. coli and Pichia pastoris expression systems are one of the most successful expression systems for the expression of foreign proteins. They have developed rapidly in recent years, and hundreds of proteins have now been expressed in the expression system. Pichia yeast is a methanol nutritional yeast, which grows rapidly, has simple culture conditions, and can be continuously cultured at high density. The genetic operation is similar to that of Saccharomyces cerevisiae, and the technology is quite mature. It is a high-expressing strain commonly used in industrial production.

Invention Disclosure

The object of the present invention is to provide a new use of cardiac troponin I.

Studies by the inventors of the present invention have shown that cardiac troponin I has the effect of inhibiting tumor proliferation. Therefore, the technical solution of the present invention is:

Application of cardiac troponin I in the preparation of drugs for suppressing tumors.

When necessary, one or more pharmaceutically acceptable carriers may be added to the medicine using cardiac troponin I as an active ingredient. The carrier includes a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, and a lubricant that are conventional in the pharmaceutical field. Wait.

In the medicine of the present invention, the effective dose of cardiac troponin I is 1 microgram to 1 milligram per kilogram of body weight per day, and can be made into injections, lyophilized powder injections and other forms. The aforementioned various dosage forms can be prepared according to a conventional method in the field of pharmacology. Another object of the present invention is to provide a cardiac troponin I-encoding gene that is highly expressed in Pichia and E. coli expression systems.

There are two types of cardiac troponin I encoding genes provided by the present invention. The first is a cardiac troponin I encoding gene that is highly expressed in the Pichia expression system and is one of the following nucleotide sequences:

1) SEQ ID Ns in the sequence list: 2;

2) a polynucleotide encoding the protein sequence of SEQ ID No: 1 in the sequence listing;

3) The DNA sequence defined by SEQ ID No _: 2 in the sequence listing has more than 90% homology, and a DNA sequence encoding the same functional protein.

The DNA sequence of Sequence 2 in the Sequence Listing is composed of 633 nucleotides and encodes the protein of Sequence 1 in the Sequence Listing.

Both expression vectors and cell lines containing the above genes fall within the protection scope of the present invention, such as

PPIC9K-AAF vector (physical map shown in Figure 1) and GS115 Pichia yeast cell line.

The second cardiac troponin I encoding gene provided by the present invention is a cardiac troponin I encoding gene that is highly expressed in an E. coli expression system, and is one of the following nucleotide sequences:

1) SEQ ID No _: 3 in the sequence listing;

2) a polynucleotide encoding the protein sequence of SEQ ID No _: 1 in the sequence listing;

3) A DNA sequence defined by SEQ ID No _: 3 in the Sequence Listing with more than 90% homology and a DNA sequence encoding the same functional protein.

The DNA sequence of Sequence 3 in the Sequence Listing is composed of 633 nucleotides and encodes the protein of Sequence 1 in the Sequence Listing.

Both expression vectors and cell lines containing the above genes belong to the protection scope of the present invention, such as the pET-AAF vector (the physical map is shown in Figure 4) and the BL21-DE3 E. coli cell line.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a physical map of the expression plasmid pPIC9K-AAF

Figure 2 shows the digestion electrophoresis map of plasmid PPIC9K-AAF

Figure 3 shows the electrophoresis of Pichia GS115 culture medium after methanol induction.

Figure 4 is a physical map of the expression plasmid pET-AAF Figure 5 is a restriction enzyme electrophoresis map of the expression plasmid pET-MF

Figure 6 shows the electrophoresis profile of BL21-DE3 E. coli culture medium after IPTG induction.

The best way to implement the invention

Example 1.Pichia yeast in vitro expression of cardiac troponin I

(1) Synthesis of cardiac troponin I gene

The full-length sequence of Sequence 2 in the Sequence Listing was artificially synthesized, and a Ndel digestion site was added to its 5 'end and a Notl digestion site was added to its 3' end to obtain the sequence AAF1.

(2) Expression of cardiac troponin I in vitro

a. Load the artificially synthesized full sequence into the pGEM-T-EASY plasmid to construct the plasmid pGEM-AAF1. b. Construction of expression plasmid

The high-efficiency expression plasmid pPIC9K purchased from Invitrogen, USA was used to digest the pGEM-AAFl plasmid and pPIC9K plasmid with Ndel and Notl, respectively. The target fragments were collected by gel electrophoresis, and ligated with T 4 ligase at 16 ° C overnight. Transformation, picking bacteria, and amplification to obtain expression plasmids by conventional methods

For PPIC9K-AAF, the results are shown in Figure 1. The results of enzyme digestion identification are shown in Figure 2, which proves that the structure of the expression plasmid is correct.

c. The plasmid PPIC9K- AAF was linearized with Sacl, and the linearized pPIC9K-AAF was introduced into Pichia pastoris GS115 (purchased from Invitrogen) using Zhejiang Xinzhi Electric Gene Introducer, which was cultured, selected, and screened. In the process, a monoclonal bacterium resistant to G418 mg / ml was obtained.

d. Select the monoclonal bacteria, inoculate them in 5ml medium, incubate at 30 ° C overnight, then transfer to 250ml medium, and continue to culture to 0D ₆ . . = 10-20, collect bacterial cells, and dilute to 0D ₆₀ with carbon-free medium.

= 50, continue to cultivate for 24 hours, add methanol to a final concentration of 1% to induce expression, and add methanol every 24 hours to 96 hours. The supernatant was centrifuged to perform gel electrophoresis analysis and functional determination. 10 ml of the supernatant was taken, electrophoresed by 12% SDS-PAGE, and stained by Coomassie Blue. As shown in Figure 3, the results show that at 25kD, there is induced protein expression, and according to the BSA control, the expression is calculated as

(3) Functional determination of cardiac troponin I of the present invention

Twenty-four Kunming mice were divided into two groups: the methanol-induced supernatant and the methanol-induced supernatant. The mice were subcutaneously inoculated with the S180 ascites cell line. Three days later, the mice were injected subcutaneously with the methanol-induced supernatant and methanol-induced supernatant. 5ml / head, injection every 12 hours, continuous injection for 7 days. The tumor group was observed and excised and weighed. As a result, the tumor weight of the supernatant before methanol induction and the supernatant after methanol induction was 0.82 + 0.22 g and 0.1 11 + 0.05 g, respectively, and the tumor inhibition rate was 86. . 6%. Example 2. E.coli expression of cardiac troponin I in vitro

(1) Synthesis of artificial gene: The synthetic gene sequence 3 has a full length of 1-210 bases, respectively, with a Snabl digestion site at the 5 end and a Notl digestion site at the T end to obtain the sequence AFF2.

(2) Expression of cardiac troponin I in vitro

a. The artificially synthesized full sequence is loaded into the pGEM-T-EASY plasmid to construct the pGEM-AAF2 cloning vector.

b. Construction of expression plasmid

The high-efficiency expression plasmid pET21b purchased from Novagen Corporation was used to cut the PGEM-AAF2 plasmid and pET21b plasmid with Snabl and Notl, respectively. The target fragments were collected by gel electrophoresis and ligated with T 4 ligase 16Ό overnight. After transformation by conventional methods, picking bacteria, and amplification to obtain the expression plasmid pET-AAF, the structure of which is shown in Figure 4. The results of enzyme digestion identification, as shown in Figure 5, prove that the structure of the expression plasmid is correct.

c. The plasmid pET-AAF was introduced into BL21-DE3 E. coli by transformation method, and after culturing, selection, screening and other processes, monoclonal bacteria were obtained.

d. Select the monoclonal bacteria, inoculate them in 5 ml medium, incubate at 37 ° C overnight, then transfer to 250 ml medium, and continue to grow to OD ₆ . . = 0.8, IPTG was added to a final concentration of 1% 37% to induce expression for 4 hours. The pellet was collected by centrifugation, suspended with lysate, sonicated, centrifuged at 10,000 g for 30 minutes, the supernatant was discarded, the pellet was washed twice, and finally dissolved with 8 M urea, and dialyzed against 50 mM phosphate buffered saline overnight. Take 10ul, electrophoresis with 12% SDS-PAGE, and stain with Coomassie Blue. As shown in FIG. 6, the results show that there is induced protein expression at the position of 24KD, and according to the BSA control, the expression amount is calculated to be 85 mg / L.

(3) Functional determination of cardiac troponin I of the present invention

Omg protein / kg ， Inject every 12 hours for 7 consecutive days. The tumor tissue was observed and excised, and weighed. As a result, the weight of the tumor in the phosphate buffer solution and the expression purification group was 1. 02 + 0. 28 grams and

0. 15 + 0. 06 grams, the tumor inhibition rate was 85.3%.

Example 3. In vivo stability test of myocardial troponin I in E.coli expression purification solution:

Twenty-four Kunming mice were divided into phosphate buffer solution and expression purification group, and the phosphate buffer solution and expression purification solution were injected subcutaneously at 2.0 mg / kg protein, 8 hours after injection, blood was collected from the fundus vein, and the serum was separated. Serum cardiac troponin I concentration was measured by conventional enzyme-linked immunosorbent assay. Results in phosphate buffer and The concentration of serum cardiac troponin I in the purified solution group was 0.52 + 0.08ng / ml,

3. 55 + 0. 12ng / ml, indicating that cardiac troponin I has higher stability in vivo.

Industrial applications

The present invention creatively discloses the antitumor activity of cardiac troponin I. Drugs using cardiac troponin I as an active ingredient do not directly affect tumor cells. Inhibiting tumors has a broad spectrum without producing drug resistance, which has far-reaching practical significance.

Claims

Claim

1. Application of cardiac troponin I in the preparation of drugs for inhibiting tumors.

2. The use according to claim 1, characterized in that in the tumor suppressing drug, the effective dose of cardiac troponin I is 1 microgram-1 mg / kg body weight / day.

3. The use according to claim 1 or 2, characterized in that: one or more pharmaceutically acceptable carriers are further added to the preparation of the medicine for inhibiting tumors.

4. Cardiac Troponin I gene, which is highly expressed in the Pichia expression system, is one of the following nucleotide sequences:

1) SEQ ID No _: 2 in the sequence listing;

2) It has more than 90% homology with the DNA sequence defined by SEQ ID No _: 2 in the Sequence Listing, and has a Pichia codon-encoding DNA sequence of SEQ ID No _: 1.

5. An expression vector and a cell line containing the gene according to claim 4.

6. The expression vector and cell line according to claim 5, wherein: the expression vector is PPIC9K-AAF; and the cell line is Pichia GS115.

7. Cardiac troponin I gene, which is highly expressed in the E. coli expression system, is one of the following nucleotide sequences:

1) SEQ ID No _: 3 in the sequence listing;

2) It has more than 90% homology with the DNA sequence defined by SEQ ID No _: 3 in the Sequence Listing, and has the DNA sequence of E. coli preferred codon coding SEQ ID No _: 1.

8. An expression vector and a cell line containing the gene according to claim 7.

9. The expression vector and cell line according to claim 8, wherein: the expression vector is pET-AAF; and the cell line is BL21-DE3 E. coli.