Recombinant hydroxylated collagen for inhibiting the growth of breast cancer, its preparation method and application

Abstract

The present invention belongs to the technical field of genetic engineering, and particularly relates to a recombinant hydroxylated collagen for inhibiting the growth of breast cancer, its preparation method and application. A recombinant collagen for inhibiting the growth of breast cancer has an amino acid sequence as shown in SEQ ID NO.1. Compared with natural type III collagen, the recombinant collagen described in the present invention has an improved effect of inhibiting cancer cell proliferation by more than 41%. Through the modular method in the recombinant hydroxylated collagen of the present invention, specific full-length variants capable of targeted binding to DDR can be manufactured, and due to their comparable length to natural collagen, they have a broader range of uses.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to a recombinant hydroxylated collagen for inhibiting the growth of breast cancer, and a preparation method and application thereof. Background Art

[0002] Breast cancer, mammary cancer, is a malignant tumor that occurs in the mammary gland epithelial tissue, and is one of the most common malignant tumors in women. Its incidence is related to various factors, including genetics, reproductive factors, sex hormones, nutrition and diet, environmental factors, etc. Currently, common treatment methods include surgical treatment, radiotherapy, chemotherapy, endocrine therapy, and targeted therapy.

[0003] Collagen is one of the most important and abundant proteins in mammals, and is a structural protein found in the skin, connective tissue, and bones of the human body, as well as other tissues. The content of collagen in the human body is about 30% of the total protein. Collagen is a structural protein and the main component of the extracellular matrix. Type III collagen is composed of three peptide chains that are coiled to the right to form a triple helix. Primary structure analysis shows that a long section of its polypeptide chain sequence is repeated by the Gly-X-Y amino acid sequence. Among them, X is usually proline, and Y is usually hydroxyproline and hydroxylysine, and the latter two amino acids are rarely found in other proteins.

[0004] DDR is discoid in domain receptors, DDRs, which belong to the superfamily of receptor tyrosine kinases, RTKs, and contain two members, DDR1 and DDR2. Related research has confirmed that DDR1 is not only related to the normal growth and development of the body, but also an important factor promoting the occurrence and development of tumors. The main binding site of DDR1 is the GVMGFO sequence from natural type III collagen. Due to the GVMGFO sequence in natural type III collagen, its anti-tumor ability is weak. Summary of the Invention

[0005] The purpose of the present invention is to provide a recombinant hydroxylated collagen for inhibiting the growth of breast cancer, and a preparation method and application thereof, which solves the problem of the weak anti-tumor ability of natural collagen.

[0006] The technical solution adopted by the present invention is a recombinant collagen for inhibiting the growth of breast cancer, the amino acid sequence of which is shown in SEQ ID NO.1, and the nucleotide sequence of which is shown in SEQ ID NO.2.

[0007] The recombinant collagen is used for preparing an anti-breast cancer preparation.

[0008] A sequence with hydroxylation function and enhancing the inhibition of breast cancer growth by the recombinant collagen, the amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence is shown in SEQ ID NO.5.

[0009] An expression vector comprising the nucleotide sequence shown in SEQ ID NO.2.

[0010] An expression vector comprising the nucleotide sequence shown in SEQ ID NO.5.

[0011] A host cell comprising the expression vector described in any one of the above.

[0012] The host cell is any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli and Bacillus subtilis.

[0013] A method for preparing recombinant collagen using the host cell, culturing the host cell in a medium, inducing the expression of recombinant collagen and then purifying it to obtain recombinant collagen.

[0014] The purification method is selected from any one of salting-out method, ultrafiltration method, affinity chromatography method and gel filtration chromatography method.

[0015] The beneficial effects of the present invention are as follows: The present invention provides a recombinant collagen that inhibits the growth of breast cancer, and the amino acid sequence is shown in SEQ ID NO.1. The inventor replaced 8 different sites of the full-length natural type III collagen, increased the number of GVMGFO sites without changing the total length of the natural full-length amino acids, changed the original binding sites to 9, and retained the telopeptide to make it have the potential to form a triple helix structure. The GVMGFO in natural collagen is hydroxyproline, while Pichia pastoris does not contain a hydroxylation gene, so only GVMGFP containing proline can be obtained. Since hydroxyproline plays an important role in maintaining the triple helix structure and biological properties of collagen during synthesis, mammalian cells with prolyl 4-hydroxylase can produce hydroxylated collagen through post-translational modification. Therefore, the obtained recombinant collagen is hydroxylated to prepare recombinant hydroxylated collagen with higher inhibitory effect on cancer cell proliferation and more stable properties.

[0016] Compared with natural type III collagen, the inhibitory effect of the recombinant hydroxylated collagen of the present invention on cancer cell proliferation is increased by more than 41%. Through the modular method in the recombinant hydroxylated collagen of the present invention, specific full-length variants capable of targeted binding to DDR can be manufactured, and due to its length being comparable to that of natural collagen, it has a wider range of uses. Currently, chemotherapy is mostly used for cancer treatment, which can cause harm to normal cells while killing cancer cells. However, using recombinant collagen as a therapeutic component in breast cancer will greatly reduce the adverse reactions to the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a result diagram of colony PCR.

[0018] Figure 2 It is a purification diagram of recombinant hydroxylated collagen.

[0019] Figure 3 It is a result diagram of AO / EB staining. From left to right, they are negative control, natural type III collagen, unhydroxylated recombinant type III collagen, and hydroxylated recombinant type III collagen. DETAILED DESCRIPTION OF THE INVENTION

[0020] In order to enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and drawings.

[0021] In the description of the present invention, unless otherwise specified, the reagents used are commercially available, and the methods used are conventional techniques in the art.

[0022] The inventive concept is as follows: The inventor replaced 8 different sites of the full-length natural type III collagen, increased the number of GVMGFO sites without changing the total length of the natural full-length amino acids, changed the original binding sites to 9, and retained the telopeptides to enable it to have the potential to form a triple helix structure. The GVMGFO in natural collagen contains hydroxyproline, while Pichia pastoris does not contain a hydroxylation gene, so only GVMGFP containing proline can be obtained. Since hydroxyproline plays an important role in maintaining the triple helix structure and biological properties of collagen during synthesis, mammalian cells with prolyl 4-hydroxylase can produce hydroxylated collagen through post-translational modification. Therefore, by hydroxylation modification of the obtained recombinant collagen, recombinant hydroxylated collagen with a higher inhibitory effect on cancer cell proliferation and more stable properties can be prepared.

[0023] The object of the present invention is to provide a recombinant hydroxylated collagen that inhibits the proliferation of breast cancer, which can effectively inhibit the growth of breast cancer cells, can be used as a targeted drug for cancer treatment, and has the advantages of low immunity and high efficiency.

[0024] The present invention provides a type III recombinant collagen for inhibiting the proliferation of breast cancer cells, and its amino acid sequence is shown in SEQ ID NO.1.

[0025] SEQ ID NO.1:

[0026]

[0027] The present invention provides a type III recombinant collagen for inhibiting the proliferation of breast cancer cells. Codon optimization is performed for host cell expression, and a signal peptide cleavage site, an EcoR I restriction site and a Not I restriction site are added at both ends during the design process to facilitate subsequent gene manipulation. After the above optimization, its nucleotide sequence is as shown in SEQ ID NO.2.

[0028] SEQ ID NO.2:

[0029]

[0030] The present invention provides an expression vector, which contains the above nucleic acid molecule. The vector may contain regulatory sequences, such as transcriptional and translational start and stop codons, which are specific to the type of host to which the vector is to be introduced, e.g., bacteria, fungi, plants or animals, taking into account as appropriate whether the vector is DNA-based or RNA-based. In a specific embodiment, the expression vector is pPIC9k, and its nucleotide sequence is as shown in SEQ ID NO.3.

[0031] SEQ ID NO.3:

[0032]

[0033] The present invention provides a sequence that enhances the stability of collagen and converts proline into hydroxyproline, thereby enhancing the inhibition of the proliferation of breast cancer cells. Its amino acid sequence is as shown in SEQ ID NO.4.

[0034] SEQ ID NO.4:

[0035] MTNKFISYNKMETREYLLTILFVIACFMVLNLERREGFETSDRPGVCDGKYYEKIDGFLSDIECDVLINAAIKKGLIKSEVGGATENDPIKLDPKSRNSEQTWFMPGEHEVIDKIQKKTREFLNSKKHCIDKYNFEDVQVARYKPGQYYYHHYDGDDCDDACPKDQRLATLMVYLKAPEEGGGGETDFPTLKTKIKPKKGTSIFFWVADPVTRKLYKETLHAGLPVKSGEKIIANQWIRAVK.

[0036] The present invention provides a sequence with hydroxylation function, which enhances the stability of collagen and inhibits the proliferation of breast cancer cells. Codon optimization is performed for host cell expression, and a signal peptide cleavage site, as well as EcoR I and Kpn I restriction enzyme sites, are added at both ends during the design process to facilitate subsequent gene operations. After the above optimization, its nucleotide sequence is as shown in SEQ ID NO.5.

[0037] SEQ ID NO.5:

[0038] ATGACCAACAAGTTCATCTCTTACAACAAGATGGAAACTCGTGAATACTTGCTGACCATTCTGTTCGTTATCGCTTGTTTCATGGTCTTGAACCTGGAAAGAAGAGAAGGTTTCGAGACTTCTGATAGACCAGGTGTTTGTGACGGTAAGTACTACGAGAAGATCGATGGATTTCTGTCTGACATCGAATGTGATGTGCTGATTAACGCTGCTATCAAGAAGGGTCTGATCAAGTCCGAAGTTGGTGGTGCTACTGAGAACGATCCAATCAAACTTGATCCAAAGTCTCGTAACTCCGAACAAACCTGGTTCATGCCAGGTGAACATGAGGTTATCGACAAGATCCAGAAGAAAACACGAGAGTTTCTGAACTCCAAGAAGCATTGCATCGACAAGTACAACTTCGAAGATGTTCAAGTTGCTAGGTACAAACCAGGTCAGTACTACTACCATCACTACGATGGTGATGATTGCGATGATGCTTGTCCAAAGGATCAGAGACTGGCTACTCTGATGGTTTACCTGAAGGCACCTGAAGAAGGTGGTGGAGGTGAAACCGATTTCCCTACTCTTAAGACCAAGATCAAGCCAAAGAAGGGTACTTCTATCTTCTTCTGGGTTGCTGATCCAGTCACCAGAAAGTTGTACAAAGAGACTTTGCATGCTGGTTTGCCAGTCAAGTCTGGTGAGAAGATTATCGCCAATCAGTGGATCAGAGCTGTTAAG。

[0039] The present invention provides an expression vector, which contains the above nucleic acid molecule. The expression vector may contain regulatory sequences, such as transcription and translation start and stop codons, which are specific to the type of host to be introduced into the vector, such as bacteria, fungi, plants or animals, and consider as appropriate whether the vector is DNA-based or RNA-based. In a specific embodiment, the expression vector is pPICZA, and its nucleotide sequence is shown in SEQ ID NO.6.

[0040] SEQ ID NO.6:

[0041]

[0042] The present invention provides a host cell, which contains the above nucleic acid molecule. 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 primary transformed cells and their progeny, regardless of the number of passages. The progeny may not be exactly the same as the parental cells in terms of nucleic acid content, but may contain mutations.

[0043] In a specific embodiment, the host cell is selected from any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

[0044] In a specific embodiment, the host cell is Pichia pastoris GS115.

[0045] The present invention provides a method for preparing any one of the foregoing recombinant collagens, which comprises the following steps:

[0046] Performing expression using the above host cell, and then performing separation and purification to obtain it.

[0047] The expression of the host cell refers to culturing the host cell, and the culture medium and culture conditions are well-known to those skilled in the art.

[0048] In a specific embodiment, the host cell is Pichia pastoris. After obtaining the Pichia pastoris genetic engineering bacterium, the specific culture conditions are as follows: Inoculate the Pichia pastoris genetic engineering bacterium into YPD medium, and culture it at 30 °C and 220 rpm for 22 h to 24 h until the OD600 is 18 to 20 as the seed liquid for the fermenter. After expanding the culture of the seed liquid, inoculate it into an NBS 415 fermenter with an initial volume of 5 L at an inoculation amount of 10% by volume. The culture temperature is 28 °C to 30 °C, the pH is 5.0 to 6.0, and the dissolved oxygen is controlled at 20% to 30%. When the glycerol is exhausted, start glycerol feeding culture. When the wet weight of the cells reaches more than 180 g / L, start induction culture.

[0049] Regarding the expression mode, the present invention does not make any restrictions, and it can be confirmed according to needs. For example, it can be expressed as induced expression. For induced expression, the inducer is methanol.

[0050] In a specific embodiment, methanol is added dropwise for induction culture. The temperature during the induction stage is 28 °C, the pH is 5.0, and the fermentation is terminated after 48 h of induction.

[0051] Regarding the method of separation and purification, the present application does not make any restrictions, and it can be determined according to circumstances. For example, salting-out method, ultrafiltration method, affinity chromatography method, and gel filtration chromatography method can be used.

[0052] The present invention also provides the above-mentioned recombinant hydroxylated collagen, or the recombinant collagen encoded by the above-mentioned nucleic acid molecule, or the recombinant hydroxylated collagen expressed by the above-mentioned expression vector, or the recombinant hydroxylated collagen produced by the above-mentioned host cell, which has the ability to inhibit the proliferation of breast cancer cells and can be used as a targeted drug in cancer targeted treatment, and has the advantages of low immunity and high efficiency.

[0053] Example 1: Expression of recombinant hydroxylated collagen

[0054] The recombinant collagen gene of the present invention, i.e., the target gene, was chemically synthesized, and the nucleotide sequence was shown in SEQ ID NO. 2. During the synthesis, EcoR I and Not I recognition sites and signal peptide recognition sites were added at the 5' end and 3' end, respectively, and cloned into the expression vector pPIC9K after linearization with restriction endonuclease Sac I to obtain the pPIC9K-RCOL (III) cloning plasmid. Pichia pastoris GS115 was used as the expression host bacteria, and the obtained pPIC9K-RCOL (III) cloning plasmid was linearized by electroporation and transformed into GS115 to obtain GS115-pPIC9K-RCOL (III), and high-copy positive clones were selected by the G418 gradient method.

[0055] The hydroxylation gene of the present invention was chemically synthesized, and the nucleotide sequence was shown in SEQ ID NO.5. During the synthesis, EcoR I and Kpn I recognition sites and signal peptide recognition sites were added at the 5' end and 3' end, respectively, and cloned into the expression vector pPICZA after linearization with restriction endonuclease Sac I to obtain vector pPICZA-PH4. The above-mentioned Pichia pastoris GS115-pPIC9K-RCOL (III) was used as the expression host bacteria, and the obtained pPICZA-PH4 plasmid was linearized and transformed into GS115-pPIC9K-RCOL (III) by electroporation to obtain GS115-pPIC9K-RCOL (III)-pPICZA-PH4 at a higher concentration of zeo + to filter.

[0056] Screening of positive transformants: + Select some transformants from the high-copy positive clones with a marker, pick a small amount of the selected transformants, smear them on the bottom of the PCR tube, then add 50μL of enzyme-free water and mix them by blowing; first heat them in a microwave oven at medium-high temperature for 10 minutes, then immediately put them in a -80℃ refrigerator and freeze them for 10 minutes, repeat 5 times; centrifuge at 12000rpm for 1 minute, take 3μL of the supernatant as a template, and use universal primers, 5'AOX and 3'AOX for PCR reaction. The agarose gel electrophoresis of GS115-pPIC9K-RCOL(III)-pPICZA-PH4 is shown in the figure below. Figure 1As shown. M: marker; Lanes 1 - 3: Transformants 1 - 3.

[0057] The obtained Pichia pastoris genetic engineering bacteria GS115-pPIC9K-RCOL(III) and GS115-pPIC9K-RCOL(III)-pPICZA-PH4 were respectively inoculated into YPD medium and cultured until the OD 600 reached 19.88. Then, they were inoculated into a 5L NBS 415 fermenter with an initial volume at a volume inoculation rate of 10%. The culture temperature was 30°C, the pH was 5.5, and the dissolved oxygen was controlled at 20%. When the glycerol was exhausted, glycerol feeding culture was started. When the wet cell weight reached more than 190 g / L, methanol was added, and induction culture was carried out at a methanol flow rate of 80 mL / h. The temperature during the induction stage was 28°C, the pH was 5.0, and the fermentation was terminated after 48 h of induction. The supernatant was collected by centrifugation.

[0058] Example 2: Purification of Recombinant Collagen and Recombinant Hydroxylated Collagen

[0059] 1. When the supernatant collected by centrifuging the fermentation broth of the two proteins obtained in the above steps was ultrafiltered to 50% of the initial volume, 5 times the volume of pure water was added, and then ultrafiltered and concentrated to 5% of the initial volume.

[0060] 2. Saturated ammonium sulfate accounting for 60% of the total volume of the supernatant was added to the concentrated supernatant, stirred at room temperature for 30 min, centrifuged at 9000 rpm for 10 min, and the precipitate was collected. The obtained precipitate was dissolved in 500 mL of 0.05 M PBS with a pH of 7.0 and then filtered through a 0.22 μm filter membrane.

[0061] 3. A balance buffer was prepared according to the isoelectric point of the protein: namely, 20 mmol / L sodium phosphate buffer with a pH of 6.0, denoted as Solution A; an eluent with a pH of 6.0 was prepared by mixing 20 mmol / L sodium phosphate and 1.0 mol / L NaCl in terms of molar mass, denoted as Solution B. The PBS protein solution obtained in the previous step was diluted with Solution A at a ratio of 10:1 to prepare a sample loading solution, which was filtered and then loaded onto a 25 mL CM-Sepharose cation exchange chromatography column. The column was equilibrated with the balance buffer before loading. After loading, the column was first rinsed with Solution A for 2 column volumes, and then gradient elution was carried out with 70% Solution A and 30% Solution B at a flow rate of 2 mL / min. Each elution fraction was collected and detected by SDS-PAGE. The results are as Figure 2 , M: marker; Lanes 1 - 2: Recovery solutions at two elution time points of unhydroxylated recombinant collagen during the elution stage; Lanes 3 - 4: Recovery solutions at two elution time points of recombinant hydroxylated collagen during the elution stage.

[0062] 4. According to the molecular weight distribution range of the protein obtained after ion exchange chromatography, select a Sephadex 200 gel column to further purify the target protein. AKTA operation process: First, rinse with the equilibration buffer, that is, 0.01 mol / L PBS and 0.05 mol / L NaCl, until the baseline is stable. Then, load the two protein components eluted from the ion exchange column in the previous step onto a gel filtration chromatography column filled with Superdex 200, elute with the eluent, set the flow rate at 10 mL / min, and set the UV detection wavelength at 215 nm. Finally, collect the eluted target protein component after detection by SDS-PAGE electrophoresis.

[0063] 5. Ultrafiltration for desalting; desalting with a G25 desalting column, that is, using 25 mL of G25 packing material. The operation process is similar to the gel filtration chromatography step. Each time, load 6.5 mL, collect about 8 mL, and desalting can be completed 10 min after loading.

[0064] 6. Concentrate by ultrafiltration to 30% of the initial volume, then place it in a -20 °C refrigerator for pre-freezing for 4 h, and then transfer it to a vacuum freeze dryer for freeze-drying. After 72 h, collect the freeze-dried protein, and store the freeze-dried protein sample in a 4 °C refrigerator for later use.

[0065] Example 3: Experiment on inhibiting the proliferation of breast cancer cells by recombinant hydroxylated collagen

[0066] Establish an in vitro cell proliferation experiment model to evaluate the cell activity induced by recombinant hydroxylated collagen. Briefly, inoculate MCF-7 cells in the logarithmic growth phase on a 96-well tissue culture plate so that the cell density in each well is 5×10 3 . And culture for 1 d in a 37 °C, 5% (v / v) CO2 biochemical incubator. Discard the old culture medium, dissolve native type III collagen, recombinant type III collagen, and recombinant hydroxylated type III collagen in DMEM medium at a concentration of 0.2 mg / ml in sequence, filter and sterilize, and add 100 μL to each well in the above 96-well plate. In the negative control group, add 100 μL of fresh complete medium. After incubation for 24 h, use the cck8 method to verify cell proliferation and measure the absorbance at 450 nm. The statistical results of breast cancer cell proliferation are shown in Table 1.

[0067] At the same time, stain the above wells with AO / EB and observe their growth after incubation in the collagen medium under a fluorescence microscope. The AO / EB staining diagram is shown in Figure 3 .

[0068] The results are as follows. Set the group without adding purified recombinant collagen as the negative control, which is 100%. The results are shown in Table 1.

[0069] Table 1 Statistical results of breast cancer cell proliferation

[0070] Negative control Native type III collagen Recombinant human type III collagen Recombinant hydroxylated type III collagen Proliferation rate 100％ 84％ 56％ 43％

[0071] The results show that the type III recombinant hydroxylated collagen of the present invention can significantly inhibit the growth of breast cancer cells, which is consistent with the results of light microscopy observations.

[0072] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.

[0073] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A recombinant collagen for inhibiting the growth of breast cancer, characterized in that, The amino acid sequence is as shown in SEQ ID NO.

1.

2. A gene encoding the recombinant collagen as claimed in claim 1, characterized in that, The nucleotide sequence is as shown in SEQ ID NO.

2.

3. The application of the recombinant collagen according to claim 1, wherein, The recombinant collagen is used for preparing an anti-breast cancer preparation.

4. An expression vector, characterized in that, It contains the nucleotide sequence as described in claim 2.

5. A host cell, characterized in that, It contains the expression vector as described in claim 4.

6. The host cell according to claim 5, characterized in that, The host cell is any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli and Bacillus subtilis.

7. A method for preparing recombinant collagen using the host cell according to claim 5, characterized in that, A recombinant vector is constructed using a hydroxylation gene, the recombinant vector is transformed into the host cell, cultured, and after inducing the expression of recombinant collagen, it is purified to obtain recombinant collagen; The nucleotide sequence of the hydroxylation gene is as shown in SEQ ID NO.

5.

8. The method for preparing recombinant collagen by using the host cell according to claim 7, characterized in that, The purification method is selected from any one of salting out, ultrafiltration, affinity chromatography and gel filtration chromatography.

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