WO2023051848A1

WO2023051848A1 - Anti-tumor recombinant collagen, and preparation method and use therefor

Info

Publication number: WO2023051848A1
Application number: PCT/CN2022/135037
Authority: WO
Inventors: 范代娣; 徐茹; 段志广; 宇文伟刚; 严建亚
Original assignee: 陕西巨子生物技术有限公司
Priority date: 2021-09-29
Filing date: 2022-11-29
Publication date: 2023-04-06
Also published as: CN113735966A; CN113735966B

Abstract

The present application provides an anti-tumor recombinant collagen, and a preparation method and use therefor. The amino acid sequence of the recombinant collagen is shown as SEQ ID NO. 1. The present application also provides an anti-oxidation method and a method for inhibiting tumor growth. Compared with natural collagen, the recombinant collagen disclosed by the present application is small in molecular weight and easy to prepare, can be prepared and expressed in high-equal eukaryotic cells such as yeast, has good biological activity of natural collagen, and can effectively support cell adhesion, improve antioxidant damage resistance of the skin, and remarkably inhibit tumor cell proliferation; the recombinant collagen can be used in the fields of food, health care products, biomedicine, etc. The preparation method is safe, easy to amplify, low in production costs and has wide application prospects.

Description

A kind of anti-tumor recombinant collagen and its preparation method and application

technical field

The application belongs to the technical field of genetic engineering, and in particular relates to an anti-tumor recombinant collagen and its preparation method and application.

Background technique

Collagen is one of the most important and abundant proteins in mammals, a structural protein found in the skin, connective tissue and bones of the human body, among other tissues. The content of collagen in the human body is about 30% of the total protein. The structure of collagen is a triple helix consisting of a repeating triplet amino acid sequence named GLY-X-Y, where X and Y can be any amino acid. Recombinant collagen has the advantages of good water solubility, no risk of virus infection, low rejection, biocompatibility, etc., and has been widely used in biomedicine, tissue engineering, food, cosmetics and other fields.

Collagen can give the skin the necessary nutrients, improve the living environment of skin cells and promote the metabolism of tissues. At present, studies have shown that collagen oligopeptides are functional factors that improve skin moisture content, maintain the integrity of skin fiber structure, and repair damaged cells by supplementing the nutrients needed for collagen synthesis. At the same time, it has also been reported that human collagen XV-α1 has the effect of improving sleep, and human collagen IV-α3 has the effect of inducing apoptosis. Therefore, in specific fields, collagen can be precisely constructed through specific amino acid substitutions and amino acid sequence modifications, which can play a more effective role in disease control and treatment, and will also be a safer and more efficient choice.

Contents of the invention

The purpose of this application is to provide an anti-tumor recombinant collagen, which can effectively support cell adhesion, improve the skin's ability to resist oxidative damage, and significantly inhibit tumor cell proliferation. It can be widely used in food, health products, biomedicine and other fields.

The concrete technical scheme of this application is as follows:

1. An anti-tumor recombinant collagen, characterized in that its amino acid sequence is as shown in SEQ ID NO.1.

2. A nucleic acid molecule, characterized in that it encodes the recombinant collagen described in item 1.

3. The nucleic acid molecule according to item 2, characterized in that its sequence is as shown in SEQ ID NO.2.

4. A host cell comprising the nucleic acid molecule described in item 2.

5. The host cell according to item 4, characterized in that the host cell is selected from any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

6. A method for preparing recombinant collagen described in item 1, characterized in that it comprises the following steps:

It is obtained by using the host cell described in Item 4 or 5 for expression, followed by separation and purification.

7. The method according to item 6, wherein the separation and purification methods include salting out, ultrafiltration, affinity chromatography and gel filtration chromatography.

8. The recombinant collagen described in item 1, or the recombinant collagen encoded by the nucleic acid molecule described in item 2 or 3, or the recombinant collagen produced by the host cell described in item 4 or 5 has anti-oxidation, Application in foods, cosmetics, health products and pharmaceutical compositions for inhibiting tumor growth.

9. An anti-tumor pharmaceutical composition, characterized in that it comprises the recombinant collagen described in item 1, or the recombinant collagen encoded by the nucleic acid molecule described in item 2 or 3, or the recombinant collagen described in item 4 or 5 Recombinant collagen produced by host cells.

10. An anti-oxidation method, comprising administering the anti-tumor recombinant collagen described in Item 1 or 8 or the anti-tumor pharmaceutical composition described in Item 9 to a subject.

11. A method for inhibiting tumor growth, comprising administering the anti-tumor recombinant collagen described in item 1 or 8 or the anti-tumor pharmaceutical composition described in item 9 to a subject.

The effect of the invention

1. The recombinant collagen of this application is a brand-new sequence, the length of which is much smaller than that of the natural human collagen gene, and its translated protein has a small molecular weight and is easy to prepare.

2. The recombinant collagen protein prepared in this application is expressed by Pichia pastoris engineering bacteria. The protein has no hidden danger of endotoxin, and the protein carries a histidine tag, which can be purified by specific affinity, and the purification steps are simple.

3. The genetically recombinant collagen prepared by the method of the present application can effectively promote the antioxidant capacity of mammals and inhibit the growth of tumor cells.

Detailed ways

Further illustrate the present application below in conjunction with embodiment, should be understood that embodiment is only for further illustrating and explaining the present application, is not intended to limit the present application.

Unless otherwise defined, technical and scientific terms in this specification have the same meaning as commonly understood by a person skilled in the art. Although methods and materials similar or identical to those described herein can be employed in experiments or practical applications, the materials and methods are described herein below. In case of conflict, the present specification, including definitions, will control and the materials, methods, and examples are presented for purposes of illustration only and not limitation. The present application will be further described below in conjunction with specific examples, but they are not used to limit the scope of the present application.

As used herein, the terms "polypeptide", "peptide", "protein", "protein" are used interchangeably herein to mean a polymer of amino acid residues. That is, descriptions for polypeptides apply equally to descriptions of peptides and descriptions of proteins, and vice versa. The term applies to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally encoded amino acid. As used herein, the term encompasses amino acid chains of any length.

As used herein, the term "nucleic acid molecule" may include those comprising naturally and/or non-naturally occurring nucleotides and bases, for example including those with backbone modifications, which refer to nucleotide Polymers of nucleotides. Such polymers may contain natural and/or unnatural nucleotides and include, but are not limited to, DNA, RNA and PNA. Nucleotide sequence refers to the linear sequence that makes up a nucleic acid molecule.

In some cases, the nucleic acid molecule comprises cDNA, and in some cases, the nucleic acid molecule can be modified for use in the constructs described herein, such as for codon optimization. In some cases, the sequence may be designed to contain terminal restriction site sequences for the purpose of cloning into a vector.

In some cases, nucleic acid molecules can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) of encoding nucleic acids within or isolated from one or more given cells. obtained by PCR amplification.

As used herein, the term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single- or double-stranded form . Unless specifically limited, the term encompasses nucleic acids that contain known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically limited otherwise, the term also means oligonucleotide analogs, including PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioate, phosphoramidate, etc.). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the explicitly designated sequences.

As used herein, the term "vector" is used to describe a nucleic acid molecule that can be engineered to contain a cloned polynucleotide or polynucleotides that can be amplified in a host cell. Vectors include, but are not limited to: single-stranded, double-stranded or partially double-stranded nucleic acid molecules; nucleic acid molecules comprising one or more free ends, without free ends (e.g. circular); nucleic acid molecules comprising DNA, RNA or both; and other polynucleotide species known in the art. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, for example by standard molecular cloning techniques. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) integrate into the genome of the host cell upon introduction into the host cell, thereby replicating along with the host genome. Furthermore, certain vectors are capable of directing the expression of those genes to which they are operably linked. Such vectors are referred to herein as "expression vectors." A recombinant expression vector may comprise a nucleic acid of the present application in a form suitable for expressing nucleic acid in a host cell, which means that the recombinant expression vector includes one or more regulatory elements, which may be based on the The host cell to which the sequence is operably linked is selected.

As used herein, the term "expression" includes any step involved in the production of a variant, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

As used herein, the term "expression vector" means a linear or circular DNA molecule comprising a polynucleotide encoding a variant operably linked to other nucleotides that provide for its expression.

As used herein, the term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, etc., of a nucleic acid construct or expression vector comprising a polynucleotide of the present application. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell mutated due to the replication process. The host cell can be any cell useful in the production of recombinant human-like collagen of the present application.

As used herein, the term "recombinant collagen" refers to collagen produced by recombinant techniques, wherein usually DNA or RNA encoding the expressed protein is inserted into a suitable expression vector and then used to transform host cells to produce the protein. In some exemplary embodiments, DNA or RNA encoding the expressed protein is inserted into the host chromosome by homologous recombination or other means known in the art, and thus used to transform the host cell to produce the protein.

This application provides an anti-tumor recombinant collagen, the amino acid sequence of which is shown in SEQ ID NO.1:

The present application also provides a nucleic acid molecule encoding the above-mentioned anti-tumor recombinant collagen.

In a specific embodiment, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.2:

The present application provides an expression vector comprising the above-mentioned nucleic acid molecule.

For example, one or more nucleic acids encoding the above-mentioned recombinant collagens are cloned into one or more suitable expression vectors, the expression vectors can be any suitable recombinant expression vectors, and can be used for transformation or transfection of any suitable Host. Suitable vectors include those designed for propagation and amplification or for expression or both, such as plasmids and viruses.

The vector may contain regulatory sequences (such as transcriptional and translational initiation and termination codons) specific for the type of host (e.g., bacteria, fungi, plant or animal) into which the vector is to be introduced, as appropriate and considering that the vector is DNA-based Still based on RNA.

In a specific embodiment, the expression vector is pPIC9k.

The present application provides a host cell comprising the above-mentioned nucleic acid molecule.

To produce recombinant collagen, nucleic acid encoding the recombinant collagen can be isolated and inserted into one or more vectors for further cloning and/or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional techniques (eg, by using oligonucleotide probes that are capable of binding specifically to genes encoding recombinant collagen).

The host cell refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include transformants and transformed cells, including the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations.

Methods for introducing vectors into host cells are known, for example, introducing vectors into host cells using electroporation.

In a specific embodiment, the host cell is selected from any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

In a specific embodiment, the host cell is Pichia pastoris.

The present application provides a method for preparing any one of the aforementioned recombinant collagens, which comprises the following steps:

It is obtained by using the above-mentioned host cells for expression, followed by separation and purification.

Expressing the host cells refers to culturing the host cells, and the culture medium and culture conditions are well known to those skilled in the art.

In a specific embodiment, the host cell is Pichia pastoris. After obtaining the Pichia pastoris genetically engineered bacteria, the specific culture conditions are as follows: inoculate the Pichia pastoris genetically engineered bacteria in YPD medium, at 30°C, Cultivate under the condition of 220rpm for 22-24h, until OD ₆₀₀ =18-20 as the seed liquid in the upper tank. After the seed liquid is expanded and cultivated, it is inserted into the NBS 415-19.2L fermenter with an initial volume of 12L according to the inoculation amount of 10%. 28-30°C, pH=5.0-6.0, dissolved oxygen controlled at 20%-30%, when the glycerin is exhausted, start the glycerin feeding culture, and when the wet weight of the bacteria reaches above 180g/L, start the induction culture .

The present application does not make any limitation on the expression method, which can be confirmed as needed, for example, the expression is induced expression, and for the induced expression, the inducer is methanol.

In a specific embodiment, the induction culture is fed with methanol, the temperature of the induction stage is 28° C., the pH is 5.0, and the induction culture is placed in a tank after 48 hours.

The application does not make any limitation on the method of separation and purification, which can be determined according to the method, for example, salting out method, ultrafiltration method, affinity chromatography and gel filtration chromatography can be used.

The present application also provides the above-mentioned recombinant collagen, or the recombinant collagen encoded by the above-mentioned nucleic acid molecule, or the recombinant collagen expressed by the above-mentioned expression vector, or the recombinant collagen produced by the above-mentioned host cell in the preparation of anti-oxidant, tumor-inhibiting Applications in food, cosmetics, health products and pharmaceutical compositions.

The present application also provides an anti-tumor pharmaceutical composition, which includes the above-mentioned recombinant collagen, or the recombinant collagen encoded by the above-mentioned nucleic acid molecule, or the recombinant collagen expressed by the above-mentioned expression vector, or the recombinant collagen produced by the above-mentioned host cell protein.

The present application also provides an anti-oxidation method, which comprises administering any one of the aforementioned anti-tumor recombinant collagen or the aforementioned anti-tumor pharmaceutical composition to a subject.

The present application also provides a method for inhibiting tumor growth, which comprises administering any one of the aforementioned anti-tumor recombinant collagen or the aforementioned anti-tumor pharmaceutical composition to a subject.

In a specific embodiment, the tumor is selected from one of breast cancer, ovarian cancer, gastric cancer, lung cancer, liver cancer, colon cancer, pancreatic cancer, and skin melanoma.

In a specific embodiment, the subject is a mammal, preferably a primate, more preferably a human.

Example

Example 1

Construction and expression of expression engineering bacteria pPIC9K-colA-6His/Pichia pastoris GS115

Chemically synthesize the recombinant collagen gene of the present application (the nucleotide sequence is shown in SEQ ID NO.2). During synthesis, Eco R I and Not I recognition sites and histidine tags were added to the 5' and 3' ends, respectively, and cloned into the expression vector pPIC9K after linearization with the restriction endonuclease Sal I, using Pichia pastoris GS115 In order to express the host bacteria, the obtained pPIC9K-colA-6His clone plasmid was linearized by electroporation and then transformed into GS115. High-copy positive clones were selected by the G418 gradient method, and cultured at 30°C for 72 hours to obtain Pichia genetically engineered bacteria.

Inoculate the Pichia pastoris genetically engineered bacteria obtained above into YPD medium, and when it is cultivated to OD ₆₀₀ =19.2, insert 10% of the inoculum into a NBS 415-19.2L fermenter with an initial volume of 12L, and the culture temperature is 30°C. pH = 5.5, dissolved oxygen is controlled at 20% to 30%. After the glycerin is exhausted, start the glycerin fed culture. When the wet weight of the bacteria reaches above 190g/L, start to feed methanol for induction culture. The temperature of the induction stage is 28°C, pH 5.0, induction for 48h, put in tank, centrifuge to collect supernatant.

Example 2

Purification of recombinant collagen

(1) When the supernatant collected by centrifugation is ultrafiltered to 50% of the initial volume, 3 to 5 times the volume of pure water is added, and then concentrated to 5% of the initial volume by ultrafiltration;

(2) Add 60% saturated ammonium sulfate to the concentrated supernatant, stir at room temperature for 30min, centrifuge at 9000rpm for 10min, collect the precipitate, dissolve the obtained precipitate in 500mL of 0.05M PBS with a pH of 7.0, and pass through a 0.45μm filter membrane filter;

(3) Load 20 mL of the filtered crude protein solution onto the Ni-NAT column at a flow rate of 1 mL/min, and wash 30 mL with 50 mM PBS buffer at pH 7.4;

(3) Elute the target protein with 30mL, 100mM imidazole buffer (50mM PBS with pH 7.4+0.5M NacL) at a flow rate of 2.0mL/min;

(4) Collect the eluted target protein, desalt it through a Sephadex G25 column (GE Healthcare, XK26/20; column volume 50mL), then concentrate it by ultrafiltration to 20-30% of the initial volume, and then place it in a -20°C refrigerator Pre-freeze for 4 hours, then transfer to a vacuum freeze dryer for freeze-drying, and collect the freeze-dried protein after 48 hours.

Example 3

Antioxidant test

Determination of the ability of recombinant collagen to scavenge superoxide anion free radicals The activity of scavenging superoxide anion free radicals of samples was determined by pyrogallol autoxidation method: add 9mL Tris-HCl buffer solution (50mmol/L pH 8.2) and 20μL For the pyrogallol solution, the change in absorbance was monitored at 325 nm and 25° C., and the amount of pyrogallol added was adjusted so that the rate of autoxidation was about 0.07 OD/min. In the experimental group, different amounts of recombinant collagen were added to the above system, and the absorbance value was measured at 325 nm and 25° C. in the same way.

The inhibition rate of recombinant collagen on PR autoxidation was calculated according to the following formula:

Inhibition rate I=(ΔA0-ΔA)/ΔA0×100%

Among them, ΔA0 is the rate of pyrogallol autoxidation, and ΔA is the rate of pyrogallol autoxidation inhibited by recombinant collagen. The definition of activity unit: under the conditions that the reaction temperature is 25°C, the final concentration of pyrogallol is 0.1mM, and the pH value of Tris-HCl buffer solution is 8.2, the inhibitory dose that makes the autooxidation rate of every milliliter of reaction solution inhibited by 50% is 1 vitality unit (U). The results are shown in Table 1.

Table 1 Inhibition rate of different concentrations of recombinant collagen on the autoxidation of pyrogallol

It can be seen from Table 1 that the recombinant collagen prepared by the method of the present application has very significant antioxidant activity, and presents a certain concentration-dependent relationship. The reason may be that the side chain charged groups in the recombinant collagen can strongly increase the polarity of pyrogallol solution, and pyrogallol is more likely to decompose and autoxidize under polar conditions, thereby generating more O ₂ , and finally Exhibits the property of promoting the autoxidation of pyrogallol.

Example 4

Inhibition of tumor cells by human breast cancer cells

Put the 96-well culture plate inoculated with 1.5×10 ⁶ human breast cancer cells in each well into a CO ₂ incubator for 24 hours (saturated humidity, 37°C, 5% CO ₂ ), the blank group was normal cell culture medium, and the experimental group was For recombinant collagen, 3 parallel wells were set for each concentration, and the culture time was 24h, 48h, 72h. After about 4 hours after the end of the culture, 20 μL of MTT solution was added to each well and the culture was continued. The cell inhibition rate was measured by MTT at the end of the culture. The results were statistically analyzed using SPSS 16.0 statistical software, and the experimental data were expressed as mean ± standard deviation. The experimental results are shown in Table 2.

Inhibition rate (%)=(OD value of control group-OD value of experimental group)/OD value of control group

Table 2 Inhibitory rate of different concentrations of recombinant collagen on human breast cancer cells

As can be seen from the results in Table 2, recombinant collagen can inhibit the proliferation of human breast cancer cells when the concentration is 20mg/L, 40mg/L, 60mg/L, 80mg/L, and 100mg/L. L, the inhibition rate of human breast cancer cells reached the maximum.

Claims

An anti-tumor recombinant collagen, characterized in that its amino acid sequence is shown in SEQ ID NO.1.
A nucleic acid molecule, characterized in that it encodes the recombinant collagen of claim 1.
The nucleic acid molecule according to claim 2, characterized in that its sequence is as shown in SEQ ID NO.2.
A host cell, characterized in that it comprises the nucleic acid molecule of claim 2.
The host cell according to claim 4, wherein the host cell is selected from any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.
A method for preparing recombinant collagen according to claim 1, characterized in that it comprises the steps of:

It is obtained by using the host cell described in claim 4 or 5 for expression, followed by separation and purification.
The method according to claim 6, characterized in that the separation and purification methods include salting out, ultrafiltration, affinity chromatography and gel filtration chromatography.
The recombinant collagen described in claim 1, or the recombinant collagen encoded by the nucleic acid molecule described in claim 2 or 3, or the recombinant collagen produced by the host cell described in claim 4 or 5 has antioxidant , Food, cosmetics, health products and pharmaceutical compositions for inhibiting tumor growth.
An anti-tumor pharmaceutical composition, characterized in that it comprises the recombinant collagen according to claim 1, or the recombinant collagen encoded by the nucleic acid molecule according to claim 2 or 3, or the recombinant collagen according to claim 4 or 5 Recombinant collagen produced by the host cells described above.
An anti-oxidation method, characterized by comprising administering the anti-tumor recombinant collagen of claim 1 or 8 or the anti-tumor pharmaceutical composition of claim 9 to a subject.
A method for inhibiting tumor growth, characterized by comprising administering the anti-tumor recombinant collagen of claim 1 or 8 or the anti-tumor pharmaceutical composition of claim 9 to a subject.