Recombinant human III type collagen and expression method thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to recombinant human collagen III and an expression method of the recombinant human collagen III. The amino acid sequence of the recombinant human collagen III is shown as SEQ ID No.1; the amino acid sequence of the recombinant human III type collagen comprises a plant secretion signal peptide Apo, a human III type collagen Gln24-Leu 1466 segment and a 6His tag. Besides, tobacco is used as a carrier, an agrobacterium tumefaciens-mediated instantaneous conversion technology is applied, efficient and stable expression of the recombinant human-derived III-type collagen is achieved, the ethical and safety problems of a traditional collagen source are effectively solved, and a new way is established for production of recombinant protein. The innovative achievement has wide application prospect and important scientific value, and is expected to promote sustainable development and progress in the field of biotechnology.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a recombinant human type III collagen and an expression method thereof. Background Art

[0002] As an important structural protein, collagen is widely present in organisms and plays a vital role in providing support and elasticity to the extracellular matrix. Different types of collagen (type I, II, III, etc.) have their own functional characteristics. Type III collagen plays an important role in the structure of skin, blood vessels and internal organs, and is widely used in the medical, cosmetic and food industries. Especially in wound repair and tissue engineering, type III collagen has become a research hotspot in the field of recombinant protein production due to its excellent biocompatibility and ability to promote tissue regeneration.

[0003] At present, traditional sources of collagen mainly rely on animal tissues, especially connective tissues of pigs and cows. However, this method not only has ethical and safety issues, but also faces the challenge of difficult to control production and quality. In order to solve these problems, scientists have gradually turned to genetic engineering methods, especially the expression of collagen in microorganisms, animal cells and even plants through recombinant technology. The biggest advantage of recombinant collagen production is that it can accurately control the quality and structure of the protein without the risk of viral contamination, and the production cost is low.

[0004] Plant expression system, as an important expression platform, has gradually become a good platform for recombinant protein production due to its high cost-effectiveness, simple operation and strong scale-up production capacity. Tobacco has high transformation efficiency and expression potential, and is capable of complex post-translational modifications, such as glycosylation and hydroxylation, which are essential for the function and stability of proteins such as collagen. With the continuous development of these technologies, plant expression systems are expected to play an increasingly important role in the production of recombinant collagen, especially in the production of high-quality type III collagen, and will have broader application prospects in the future. Summary of the invention

[0005] The purpose of the present invention is to provide a recombinant human type III collagen and an expression method thereof, which utilizes tobacco as an expression system and obtains a high yield of recombinant protein through Agrobacterium-mediated transient transformation, thereby avoiding the ethical issues of traditional animal-derived collagen and having the advantages of low cost and high safety, and is widely applicable to the medical and beauty industries.

[0006] The objective of the present invention is achieved through the following technical solutions: The present invention provides a recombinant human type III collagen protein, the amino acid sequence of the recombinant human type III collagen protein is shown as SEQ ID No.1.

[0007] Furthermore, the amino acid sequence of the recombinant human type III collagen includes a plant secretion signal peptide Apo, a human type III collagen Gln24-Leu1466 segment and a 6´His tag.

[0008] Furthermore, the amino acid sequence of the plant secretion signal peptide Apo is shown as SEQ ID No. 2.

[0009] The present invention also provides a gene encoding the recombinant human type III collagen, and the gene sequence is shown in SEQ ID No. 3.

[0010] Furthermore, the sequence of the gene is obtained after being optimized according to the codon preference of tobacco.

[0011] The present invention also provides a plant binary expression vector comprising the recombinant human type III collagen-related coding gene, wherein the plant binary expression vector is a pBI121 vector.

[0012] The present invention also provides a method for expressing the recombinant human type III collagen, comprising the following steps: (1) Transforming the plant binary expression vector containing the coding gene according to any one of claims 4 to 5 into Agrobacterium, and obtaining positive Agrobacterium by culturing and PCR identification and screening; (2) Cultivate the positive Agrobacterium obtained in step (1) and the Agrobacterium carrying the silencing suppressor P19 overnight, centrifuge and discard the supernatant the next day, suspend with infection buffer, and incubate in the dark for 2-3 hours; (3) Mix the positive Agrobacterium suspension in step (2) and the P19 Agrobacterium suspension and adjust the OD of each bacterial suspension. 600 When the concentration is 0.6-0.8, use the needle of a 1 mL syringe to gently make a wound on the back of the tobacco leaf, and use a syringe without the needle to inject the mixed bacterial solution into the tobacco leaf from the wound; (4) culturing the injected tobacco leaves in step (3) in a darkened state overnight, and then culturing under normal light for 48-72 hours; (5) Take the tobacco leaves after light culture in step (4), grind them with liquid nitrogen, add the extract, and centrifuge them to obtain the supernatant containing the recombinant human type III collagen.

[0013] Furthermore, in step (5), the formula of the extract is: 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EDTA, 1 mM PMSF, 1 / 100 Cocktail (V / V).

[0014] The present invention also provides a short fragment form G1 of the recombinant human type III collagen, and the short fragment form G1 of the recombinant human type III collagen consists of the amino acid sequence shown in SEQ ID No.4.

[0015] The present invention also provides a gene encoding the short fragment form G1 of the recombinant human type III collagen, and the sequence of the gene encoding is shown in SEQ ID No.5. Beneficial Effects

[0016] The core of the present invention is to obtain a type III collagen sequence suitable for expression in tobacco chassis through a codon optimization strategy. At the same time, the ability to customize the expression of recombinant collagen is improved by combining plant secretion signal peptides and tag protein technology. This means that researchers can flexibly design and express recombinant collagen with specific functions and characteristics according to actual needs. This progress will bring new development opportunities for the application of type III collagen in the fields of medical treatment, beauty, etc., and promote the efficient production and promotion of recombinant collagen.

[0017] In addition, the present invention uses tobacco as the expression receptor and Agrobacterium-mediated transient transformation technology to achieve efficient and stable expression of recombinant human type III collagen in plants. Compared with traditional animal-derived collagen, this system not only effectively avoids ethical disputes, but also significantly reduces production costs and improves product safety, laying a solid foundation for the widespread application of collagen.

[0018] At the same time, the present invention verified the expression and localization of the recombinant protein in tobacco leaves through advanced technologies such as laser confocal microscopy and Western blot, fully proving the effectiveness and reliability of the expression system. This verification result provides strong technical support for subsequent in-depth research.

[0019] In summary, this application not only effectively solves the ethical and safety issues of traditional collagen sources, but also opens up a new and efficient way for the production of recombinant proteins by providing a recombinant human type III collagen protein and its encoding gene based on a plant expression system, as well as an expression verification method in tobacco. This innovative achievement has broad application prospects and important scientific value, and is expected to promote the sustainable development and progress of the field of biotechnology. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 Schematic diagram of the construction of plant binary expression plasmid for recombinant human type III collagen.

[0022] Figure 2 Comparison of transiently transformed tobacco leaves. The left picture Mock is a negative control, i.e., a leaf injected with the untransformed plant binary vector bacterial solution; the right picture is a tobacco leaf injected with the positive bacterial solution, transiently expressing the Apo-COL3A1-G1-GFP-6His protein for about 72 hours.

[0023] Figure 3 This is the subcellular localization map of the fusion protein Apo-COL3A1-G1-GFP-6His, which is a recombinant human type III collagen G1 segment targeted to the extracellular space and GFP.

[0024] Figure 4 This is a Western blot result of tobacco transient expression, where the first lane is the total protein of leaves injected with Apo-COL3A1-G1-GFP-6His bacterial solution; the second lane is the negative control, that is, the total protein of leaves injected with untransformed plant binary vector bacterial solution; the third lane is the Lambolide P1025 protein marker.

[0025] Figure 5 This is a Western blot result of transient expression of COL3A1-G1-GFP-6His fusion protein in tobacco with different subcellular targeting sequences added. The four lanes on the left are total leaf proteins, and the four lanes on the right are proteins enriched and purified by GFP magnetic beads. VTP is the vacuole targeting sequence, hSP is the human secretion signal peptide, and Apo is the plant secretion signal peptide targeting the extracellular space. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Unless otherwise specified, the chemical reagents, biochemical reagents and materials used in the present invention can be obtained from commercial sources.

[0032] Example 1 Codon Optimization of Human Type III Collagen Human type III collagen COL3A1 contains 1466 amino acids and is encoded by 4401 base pairs. According to the expression requirements of the plant chassis, this embodiment selects human type III collagen after removing the secretion signal, adds a plant signal sequence to its N-terminus, and adds a tag protein to the C-terminus. Its amino acid sequence is shown in SEQ ID NO.1. The above-mentioned plant signal sequence is a signal peptide Apo targeting extracellular space, which is derived from Arabidopsis endo-1,4-beta-glucanase protein and consists of the amino acid sequence shown in SEQ ID No.2. The codon online optimization tool Gensmart was used to refer to the codon preference of tobacco expression, and the sequence of the above-mentioned recombinant collagen was optimized. On this basis, further artificial optimization was performed, and the nucleotide sequence was finally obtained as shown in SEQ ID No.3. The above-mentioned nucleotide sequence reduces the number of rare codons in tobacco, balances the GC content, and is conducive to the efficient expression of the gene in the recipient plant tobacco.

[0033] The optimized recombinant collagen sequence SEQ ID No. 3 in the present invention can be used as a template. Through molecular cloning, according to specific requirements for the expression efficiency, functional effectiveness, etc. of recombinant type III collagen, its full length or partial fragments are selected and combined with different plant subcellular targeting sequences and tag proteins to construct a plant expression project.

[0034] Among them, the N-terminal propeptide, terminal peptide segment and the triple helical segment Gly103-Gly315 connected to the human COL3A1 protein were selected and connected to GFP and 6´His tag proteins to construct Apo-COL3A1-G1-GFP-6His (G1) fusion protein, whose amino acid sequence is shown in SEQ ID No.4 and the encoding gene sequence is shown in SEQ ID No.5. The first 65 amino acids of the G1 fusion protein are located in the non-collagenous region, helping to maintain the overall stability of the collagen molecule. The GFP tag facilitates real-time monitoring of the expression of the recombinant protein, identification of its subcellular localization, and visualization of the expression abundance. The 6´His tag facilitates subsequent purification. The basic physical and chemical properties of the G1 protein were analyzed using the proteomics database (https: / / web.expasy.org / protparam / ). It was found that the theoretical isoelectric point of the sequence was 6.41, which is relatively close to neutral. It has a total of 500 amino acids, a relative molecular weight of 52360.79 Daltons, an instability index of 33.77, and a grand average of hydropathicity of -0.630. Overall, it is relatively stable and a hydrophilic protein, suitable for application in plant chassis and synthetic biology expression.

[0035] Example 2 Construction of plant binary expression vector The sequences SEQ ID No.3 and SEQ ID No.5 were submitted to Nanjing GenScript for full gene synthesis. The above synthesized gene fragments were used as templates for PCR amplification, and the target gene fragments and the pBI121 expression vector backbone were connected by seamless cloning to obtain the corresponding plasmids. They were named pBI121-35S::Apo-COL3A1-6His and pBI121-35S::Apo-COL3A1-G1-GFP-6His respectively. The plasmid map of pBI121-35S::Apo-COL3A1-6His is shown in Figure 1 .

[0036] Example 3 Tobacco transient expression 1. Obtaining Agrobacterium carrying plant binary vectors Take 1µL of the constructed plasmid and add it to the completely thawed Agrobacterium GV3101 competent cells. Flick gently to mix, and let it stand on ice for 5 min. Then, add the competent cells and plasmid mixture to the electroporation cup in the clean bench. Use the BIO-RAD GenePµLserXcell electroporator, set the program to 1800V, 25µF, 200Ω, put it into the electroporation cup for electroporation, and then put the electroporation cup back on ice for 2 min. Then, add 500µL of antibiotic-free LB liquid culture medium in the clean bench, rinse the bacteria in the electroporation cup, and transfer it to a centrifuge tube. Incubate at 30℃, 220rpm for 2-3 hours, and take 50µL to spread on the LB solid plate with dual resistance to kanamycin and rifampicin. After culturing in the dark at 30℃ for 2 days, perform PCR identification to screen the colonies that have successfully transferred into the plant binary vector.

[0037] 2. Agrobacterium-mediated transient expression in tobacco Pick the positive colonies of Agrobacterium obtained in the previous step, add them to 5mL of LB liquid medium with dual resistance to kanamycin and rifampicin, and culture overnight at 30℃ and 220rpm. Synchronously culture Agrobacterium with silencing suppressor P19. The next day, centrifuge the Agrobacterium and discard the supernatant, suspend it with infection buffer, centrifuge it again, and resuspend it to reduce residual antibiotics. Incubate in the dark at 30℃ for 2-3 hours. Among them, the centrifugation parameters of Agrobacterium are 5000rpm, 5min, and the infection buffer formula is 10mMMgCl2, 10mM MES, 200µM AS.

[0038] Determine the OD of bacterial resuspension 600 Absorbance value, mix the positive Agrobacterium suspension and P19 Agrobacterium suspension to ensure that the OD of each bacterium is 600Select 4-6 week old Nicotiana benthamiana and gently make a wound on the back of the young, healthy and fully expanded leaves with a 1 mL syringe needle. Use a syringe without the needle to inject the mixed bacterial solution into the leaves through the wound and wipe off the excess bacterial solution with a paper towel.

[0039] After culturing the tobacco in the dark overnight, the culture was switched to normal light for a total of 48-72 hours.

[0040] Example 4 Observation of the expression of recombinant protein Apo-COL3A1-G1-GFP-6His by UV light Use portable excitation light source and fluorescent protein observation glasses to observe the expression of recombinant proteins on tobacco leaves. Figure 2 As shown, the left picture Mock is a negative control, that is, a leaf injected with untransformed plant binary vector bacterial solution. The right picture shows a tobacco leaf injected with positive bacterial solution and transiently expressing Apo-COL3A1-G1-GFP-6His protein for about 72 hours. The leaf as a whole presents a red background, and the part with recombinant protein expression shows yellow-green fluorescence, indicating that the recombinant protein has achieved good leaf expression.

[0041] Example 5 Confocal laser scanning observation of subcellular localization of Apo-COL3A1-G1-GFP-6His Use a hole puncher to take tobacco leaf discs and make temporary slides. Use a laser confocal microscope Leica TCS SP8 to observe the lower epidermis of the leaves. The excitation light is selected at 488 nm and the receiving band is 500-550 nm. The shooting parameters are 1024×1024 pixels and 50 Hz. Treat the leaf discs with 2M NaCl solution for 2-3 minutes to induce plasmolysis. The results are shown in Figure 3 As shown by Figure 3 It can be seen that Apo-COL3A1-G1-GFP-6His showed clear and bright extracellular localization, indicating that the synthesis and transport of the recombinant protein were normal and achieved high expression at the subcellular level.

[0042] Example 6 Tobacco protein extraction and Western blot COL3A1-G1-GFP-6His fusion protein with different subcellular targeting sequences was expressed by Agrobacterium-mediated tobacco transient expression system, and Western blot was performed. Figure 4 The first lane is the total protein of leaves injected with the extracellular targeting fusion protein Apo-COL3A1-G1-GFP-6His, the second lane is the negative control, that is, the total protein of leaves injected with the untransformed plant binary vector bacterial solution, and the third lane is the Lambolide P1025 protein marker. Figure 5The proteins targeting different subcellular spaces were compared. The four lanes on the left were the total proteins of the leaves, and the four lanes on the right were the proteins enriched and purified by GFP magnetic beads. VTP is the vacuole targeting sequence, hSP is the human secretion signal peptide, and Apo is the plant secretion signal peptide targeting the extracellular space. Western blot results showed that COL3A1-G1-GFP-6His can be well expressed in a variety of subcellular spaces under the guidance of different signal sequences, reflecting that the sequence has a high application potential.

[0043] The specific implementation method of this embodiment is to cut tobacco leaves, add liquid nitrogen and grind them in a pre-cooled mortar, repeat 3-4 times until the leaf tissue is completely turned into powder, transfer the powder to a 2 mL centrifuge tube with a pre-cooled medicine spoon, weigh the powder, add protein extract at a ratio of 1 mL lysate per gram of tissue powder, vortex mix, incubate on ice for 15 minutes, centrifuge at 12000rpm for 10 minutes, aspirate the supernatant to a 1.5 mL centrifuge tube, centrifuge at 12000rpm for 10 minutes, aspirate the supernatant again to a new 1.5 mL centrifuge tube, and obtain a crude extract of type III collagen expressed in tobacco. Take 40 µL of the crude extract, add 10 µL of 5' loading buffer, boil at 95 °C for 10 minutes to denature the protein, and use it for subsequent Western blot detection. The formula of protein extraction solution is: 50mM Tris-HCl pH8.0, 150mM NaCl, 10% glycerol, 1% Triton X-100, 1mM EDTA, 1mM PMSF, 1 / 100 Cocktail (V / V). The formula of 5´ loading buffer is 250mM Tris-HCl (pH6.8), 10% SDS (W / V), 0.5% bromophenol blue (W / V), 5% β-mercaptoethanol (W / V).

[0044] 10 µL of GFP antibody-coupled nanomagnetic beads were mixed with the above tobacco expression crude extract, incubated at 4 °C for 2-4 hours, and then the supernatant was discarded. After washing the beads 3-4 times with washing buffer, 40 µL washing buffer and 10 µL 5 ´ loading buffer were added, and the proteins were denatured by boiling at 95 °C for 10 min. The nanomagnetic beads were from Kangti Life, and the washing buffer formula was 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% glycerol, and 0.02% NP-40.

[0045] Prepare 10% polyacrylamide gel, load 20 μL of sample, run at 80 V for 30 min, 130 V for 90 min, until the bromophenol blue band migrates to the bottom of the gel, then transfer the protein to the PVDF membrane at 200 mA constant flow transfer. Block with 5% skim milk for 1 h, incubate with COL3A1 polyclonal antibody for 2 h, wash 3 times with PBST buffer for 10 min each, incubate with secondary antibody for 1 h, wash 3 times with PBST buffer for 10 min each, and perform chemiluminescence development. Among them, COL3A1 polyclonal antibody is from Sangon Biotechnology, and secondary antibody is from Lamblide.

[0046] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A recombinant human type III collagen, characterized in that: The amino acid sequence of the recombinant human type III collagen is shown in SEQ ID No.

1.

2. The recombinant human type III collagen according to claim 1, characterized in that: The amino acid sequence of the recombinant human type III collagen includes a plant secretion signal peptide Apo, a human type III collagen Gln24-Leu1466 segment and a 6'His tag.

3. The recombinant human type III collagen according to claim 2, characterized in that: The amino acid sequence of the plant secretion signal peptide Apo is shown in SEQ ID No.

2.

4. A gene encoding recombinant human type III collagen according to any one of claims 1 to 3, characterized in that: The gene sequence is shown in SEQ ID No.

3.

5. The gene encoding recombinant human type III collagen according to claim 4, characterized in that: The sequence of the gene is obtained after being optimized according to the codon preference of tobacco.

6. A plant binary expression vector comprising the recombinant human type III collagen-related coding gene according to any one of claims 4 to 5, characterized in that: The plant binary expression vector is a pBI121 vector.

7. A method for expressing recombinant human type III collagen according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Transforming the plant binary expression vector containing the coding gene according to any one of claims 4 to 5 into Agrobacterium, and obtaining positive Agrobacterium by culturing and PCR identification and screening; (2) Cultivate the positive Agrobacterium obtained in step (1) and the Agrobacterium carrying the silencing suppressor P19 overnight, centrifuge and discard the supernatant the next day, suspend with infection buffer, and incubate in the dark for 2-3 hours; (3) Mix the positive Agrobacterium suspension in step (2) and the P19 Agrobacterium suspension and adjust the OD of each bacterial suspension. 600 When the concentration is 0.6-0.8, use the needle of a 1 mL syringe to gently make a wound on the back of the tobacco leaf, and use a syringe without the needle to inject the mixed bacterial solution into the tobacco leaf from the wound; (4) culturing the injected tobacco leaves in step (3) in a dark place overnight, and then culturing under normal light for 48-72 hours; (5) Take the tobacco leaves after light culture in step (4), grind them with liquid nitrogen, add the extract, and centrifuge them to obtain the supernatant containing the recombinant human type III collagen.

8. The expression method according to claim 7, characterized in that: In step (5), the formula of the extract is: 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EDTA, 1 mM PMSF, 1 / 100 Cocktail (V / V).

9. A short fragment form G1 of recombinant human type III collagen according to any one of claims 1 to 3, characterized in that: The short fragment form G1 of the recombinant human type III collagen consists of the amino acid sequence shown in SEQ ID No.

4.

10. A gene encoding the short fragment form G1 of recombinant human type III collagen as claimed in claim 6, characterized in that: The sequence of the coding gene is shown in SEQ ID No. 5.

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