A recombinant human type III collagen expressed in tobacco, its coding gene and preparation method

Abstract

The present invention provides a tobacco-expressed recombinant human type III collagen, its coding gene and preparation method, belonging to the field of biotechnology. Specifically, the human type III collagen COL3A1 gene is codon-optimized to obtain the rCOL3A1 gene according to the codon preference of the receptor plant, and a plant binary expression vector containing the rCOL3A1 gene is constructed using this gene. Further, the receptor plant is transformed with Agrobacterium tumefaciens containing the plant binary expression vector containing the rCOL3A1 gene, thereby preparing recombinant human type III collagen. The present invention uses tobacco as a chassis plant for the synthesis of recombinant human type III collagen, provides an excellent cell system for the expression of recombinant human type III collagen, and provides a new solution to the market demand for recombinant full-length human type III collagen.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a recombinant human type III collagen expressed in tobacco, its coding gene and preparation method. Background Art

[0002] Collagen, as the most abundant protein in the human body, plays roles such as repairing the skin barrier, stimulating cell regeneration, and providing structural support, and is widely used in downstream fields such as functional skin care, medical dressings, skin rejuvenation, and biomedical materials. Among them, type III collagen is rich in infant skin, vascular intima, uterine wall, and intestine.

[0003] Generally, animal-derived collagen is extracted from animal tissues and mainly has three types. The first type is extracted from tendon tissues, such as bovine tendon and porcine tendon; the second type is extracted from skin tissues, such as pigskin, cowhide, and fish skin; the third type is extracted from small intestine tissues, such as bovine small intestine and porcine small intestine. However, animal-derived collagen has the following problems: ① virus risk, the risk of transmission of animal-source diseases such as plague and avian influenza, or human infectious diseases such as viral hepatitis and AIDS; ② rejection reaction, even if the source of collagen is safe, xenogeneic collagen may also cause xenogeneic or allogeneic rejection reactions in clinical applications, resulting in aseptic inflammation at the transplantation site and changes in cell number and morphology; ③ poor structural stability, animal-derived collagen has strong rigidity, which will cause molecular chain breakage during the processing process, and the complete structure of collagen will be damaged to a certain extent. Since structure determines its function, problems such as functional uncertainty and instability between batches will occur; ④ cytotoxicity, animal-derived collagen is insoluble in water, and it is difficult to completely remove solvents such as acids, alkalis, and salts used in the processing process, which may produce cytotoxicity.

[0004] The development of genetic engineering and synthetic biology provides new solutions to solve the shortage of type III collagen and potential virus problems.

[0005] Plants have been studied as an expression and production system for pharmaceutical proteins for nearly thirty years. Compared with single-celled microorganisms, multi-cellular plant systems are rich in endomembrane systems and various organelles. The glandular hairs on the plant surface are important sites for metabolite synthesis and storage. This complex spatio-temporal characteristic provides the most suitable environment required for the synthesis of different types of enzymes and metabolites, which is conducive to maintaining protein activity and yield; the complexity of multi-cellular plant systems also provides an excellent model system for synthetic biology research. Plants are rich in a large number of metabolites, which can directly provide precursors for the synthesis of plant active molecules. Therefore, using plants to synthesize important humanized proteins and natural active small molecules has become an inevitable path for scientific and technological development.

[0006] Currently, the plant expression system has been regarded as a promising alternative to animal cells and microbial cultures for the large-scale production of recombinant proteins. For the production methods of using plants as a chassis to produce recombinant proteins, reference can be made to, for example, Chinese patents CN 107827975 and CN 106554971, US patents No. 5750871 and No. 5565347, etc. However, there is no relevant report on the use of plants to express recombinant human type III collagen at present. Summary of the Invention

[0007] In view of the above problems, the present invention provides a recombinant human type III collagen expressed in tobacco, its coding gene and preparation method. This preparation method obtains the full-length human type III collagen, providing a new solution to solve the problems of low activity of the current recombinant humanized type III collagen fragments and the problems existing in the animal-derived type III collagen industry.

[0008] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] A recombinant human type III collagen expressed in tobacco, the amino acid sequence of the recombinant human type III collagen is as shown in SEQ ID NO: 1.

[0010] Further, the amino acid sequence of the recombinant human type III collagen includes:

[0011] a) BiP signal peptide sequence, the amino acid sequence of the BiP signal peptide is as shown in SEQ ID NO: 3;

[0012] b) Recombinant human type III collagen rCOL3A1 and KDEL fusion sequence, the amino acid sequence of the recombinant human type III collagen rCOL3A1 and KDEL fusion sequence is as shown in SEQ ID NO: 4.

[0013] An rCOL3A1 gene encoding the above recombinant human type III collagen, the coding sequence of the rCOL3A1 gene is as shown in SEQ ID NO: 2.

[0014] Further, the rCOL3A1 gene is obtained by codon optimization of the human type III collagen COL3A1 gene according to the codon preference of the receptor plant.

[0015] Further, the receptor plant is tobacco.

[0016] The rCOL3A1 gene is artificially designed on the basis of the human type III collagen COL3A1 gene sequence according to the tobacco codon preference and encodes the same amino acid sequence.

[0017] A plant binary expression vector containing the rCOL3A1 gene, wherein the plant binary expression vector containing the rCOL3A1 gene is obtained by ligating the above rCOL3A1 gene to a plant binary expression vector.

[0018] Further, the plant binary expression vector is the pCambia1300-35S vector.

[0019] An Agrobacterium containing a plant binary expression vector with the rCOL3A1 gene, wherein the Agrobacterium containing the plant binary expression vector with the rCOL3A1 gene is obtained by introducing the above plant binary expression vector containing the rCOL3A1 gene into Agrobacterium.

[0020] A preparation method of recombinant human type III collagen, wherein the preparation method is to use the Agrobacterium containing the above plant binary expression vector with the rCOL3A1 gene to mediate the transformation of a receptor plant (i.e., through the Agrobacterium containing the plant binary expression vector with the rCOL3A1 gene to mediate the transformation of the receptor plant, integrating the rCOL3A1 gene fragment into the genome of the receptor plant), and the obtained genetically transformed positive plant is cultured to prepare the recombinant human type III collagen.

[0021] Further, the preparation method also includes:

[0022] Insertion identification of the rCOL3A1 gene of recombinant human type III collagen in the genetically transformed positive plant;

[0023] And / or protein identification of recombinant human type III collagen in the genetically transformed positive plant;

[0024] The receptor plant is tobacco.

[0025] The beneficial effects of a recombinant human type III collagen expressed in tobacco, its coding gene and the preparation method of the present invention are as follows:

[0026] The present invention utilizes the regionalized expression of plant cells to increase the expression of human type III collagen in tobacco, providing a solution to the problems of limited sources and high prices of human type III collagen at present;

[0027] Tobacco is used as a chassis plant for recombinant protein synthesis. Its molecular biology and genomics have been deeply studied, and there is a mature gene editing and genetic transformation system, which can provide an excellent cell system for the expression of recombinant human type III collagen; the present invention uses tobacco as a chassis plant for the synthesis of recombinant human type III collagen, providing an excellent cell system for the expression of recombinant human type III collagen, and providing a new solution to the market demand for recombinant full-length human type III collagen;

[0028] The present invention can synthesize full-length recombinant human type III collagen in tobacco. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the map of the pC1300-rCOL3A1 plant binary expression vector in Example 2 of the present invention;

[0030] Figure 2 is the entire tobacco genetic transformation flow chart in Example 3 of the present invention; Figure 2 In it, Figure A is the state diagram during the infection process; Figure B is the state diagram during the co-culture process; Figure C is the state diagram during the screening process; Figure D is the state diagram during the rooting process;

[0031] Figure 3 is the identification result diagram of tobacco resistant seedlings in Example 3 of the present invention; Figure 3 In it, M represents the DNA molecular Marker, numbers 1-16 respectively represent the PCR identification results of 16 resistant seedlings, and number 17 represents the PCR identification result of the positive control;

[0032] Figure 4 is the Western Blot result diagram of recombinant human type III collagen in positive transformed tobacco in Example 4 of the present invention; Figure 4 In it, the leftmost strip is the protein Marker, the middle six strips are the Western Blot results of positive transformed tobacco, and the rightmost two strips are the Western Blot results of wild-type tobacco. DETAILED DESCRIPTION OF THE INVENTION

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Many specific details are set forth in the following description in order to fully understand the present invention, but the present invention can also be implemented in other ways different from those described herein. Those skilled in the art can make similar promotions without departing from the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below. The present invention will be further described in detail below with specific embodiments for those skilled in the art to understand.

[0034] In addition, the experimental methods in the specific embodiments disclosed below are all conventional methods unless otherwise specified;

[0035] The materials, reagents, etc. used in the specific embodiments disclosed below can be obtained from commercial channels unless otherwise specified.

[0036] Example 1 Codon Optimization of Human Type III Collagen

[0037] The mature COL3A1 gene in the human body contains 4332 bp (including the stop codon, GC = 57.04%). According to the codon preference in tobacco, and adding the BiP signal peptide coding sequence at its N-terminus and the KDEL coding sequence at its C-terminus, the recombinant human type III collagen expressed in tobacco is obtained, and its amino acid sequence is as shown in SEQ ID NO: 1.

[0038] Among them, the amino acid sequence of the recombinant human type III collagen includes:

[0039] a) The BiP signal peptide sequence, and the amino acid sequence of the BiP signal peptide is as shown in SEQ ID NO: 3;

[0040] b) The recombinant human type III collagen rCOL3A1 and KDEL fusion sequence, and the amino acid sequence of the recombinant human type III collagen rCOL3A1 and KDEL fusion sequence is as shown in SEQ ID NO: 4.

[0041] According to the codon preference in tobacco, the base sequence of the optimized rCOL3A1 gene is as shown in SEQ ID NO: 2, and GC = 59.48%.

[0042] The rCOL3A1 gene is artificially designed (i.e., obtained after codon optimization) on the basis of the human type III collagen COL3A1 gene sequence according to the codon preference in tobacco, and it encodes the same amino acid sequence.

[0043] Example 2 Construction of the rCOL3A1 gene overexpression vector pC1300-rCOL3A1

[0044] Send the rCOL3A1 base sequence to a gene synthesis company, and synthesize the rCOL3A1 gene into the middle of HindIII and BamHI in the pCambia1300-35S vector, named the pC1300-rCOL3A1 plant binary expression vector (i.e., the plant binary expression vector containing the rCOL3A1 gene). Specifically, the pC1300-rCOL3A1 plant binary expression vector is shown in Figure 1 .

[0045] That is, after adding the BiP signal peptide coding sequence and the KDEL coding sequence to the human type III collagen coding sequence with optimized codons, it is constructed into the plant binary expression vector by gene synthesis.

[0046] Example 3 Tobacco genetic transformation of the pC1300-rCOL3A1 plant binary expression vector

[0047] I. Preparation of Agrobacterium competent cells

[0048] 1) Take out the Agrobacterium strain (LBA4404) stored at -80°C. Dip a small amount of bacterial liquid with an inoculation loop sterilized by burning, and streak it on a YEB solid medium plate containing 20 mg / L rifampicin (Rif), and culture it at 28°C for 18 h;

[0049] 2) After the culture is completed, pick a single colony and place it in 5 mL of YEB liquid medium containing 20 mg / L rifampicin (Rif), and culture it at 28°C and 200 rpm for 16 - 24 h (in this example, culture it for 24 h with shaking) to obtain the activated bacterial liquid;

[0050] 3) Inoculate the activated bacterial liquid into 50 mL of YEB liquid medium containing 20 mg / L rifampicin (Rif) according to a volume ratio of 1:100, and culture it at 28°C and 200 rpm until OD 600 is about 0.5;

[0051] 4) Transfer the cultured bacterial liquid to a pre-cooled 50 mL centrifuge tube, centrifuge it at 4°C and 5000 rpm for 10 min, and discard the supernatant;

[0052] 5) Add 10 mL of pre-cooled 0.1 M CaCl₂ aqueous solution, gently suspend the cells, place them on ice for 20 min, and then centrifuge them at 4°C and 5000 rpm for 5 min, and discard the supernatant;

[0053] 6) Add 4 mL of pre-cooled 0.1 M CaCl₂ aqueous solution containing 15% glycerol, and gently suspend;

[0054] 7) Aliquot the Agrobacterium suspension into sterile Eppendorf tubes, 100 μL per tube, and store them in a -80°C refrigerator to obtain Agrobacterium competent cells.

[0055] II. Transformation of Agrobacterium competent cells with plasmid

[0056] 1) Take out the Agrobacterium competent cells from -80°C and place them on ice. After they melt, add 2 μL of the prepared pC1300-rCOL3A1 plant binary expression vector, and pipette and mix well;

[0057] 2) Incubate on ice for 30 min, and place in liquid nitrogen for 5 min;

[0058] 3) Incubate in a 37°C water bath for 5 min;

[0059] 4) Incubate on ice for 5 min, and add 800 μL of LB liquid medium;

[0060] 5) Then culture it at 28°C and 200 rpm for 3 h, and spread it on an LB plate containing 50 μg / mL kanamycin;

[0061] 6) Incubate at 28°C until single colonies are formed to obtain Agrobacterium containing the plant binary expression vector with the rCOL3A1 gene, and store it in a -70°C refrigerator.

[0062] III. Agrobacterium-mediated genetic transformation of tobacco

[0063] 1) Culture of tobacco sterile seedlings

[0064] 11) Put tobacco seeds into a 2 mL centrifuge tube, soak them in 70% ethanol for 1 min, and rinse with sterile water 2 - 3 times (3 times in this example);

[0065] 12) Soak the seeds in a 1% sodium hypochlorite aqueous solution with 1 drop of Tween 20 for about 15 min (shake occasionally during this period), and rinse with sterile water 5 times;

[0066] 13) Inoculate the tobacco seeds on the germination medium (the germination medium uses MS medium or 1 / 2MS medium, with a pH value of 5.8 in both cases. In this example, MS medium with a pH value of 5.8 is used), about 10 seeds per bottle. Place them in a light incubator at 25 ± 1°C, with a light intensity of 2000 lux for 16 h per day, and germinate for about 1 week. Thin out the seedlings according to the number of seedlings on the medium, with no more than 3 seedlings per bottle, and culture for 30 - 40 d to obtain tobacco sterile seedlings for standby;

[0067] 2) Culture of Agrobacterium liquid

[0068] 21) Use an inoculation loop to inoculate the Agrobacterium containing the plant binary expression vector with the rCOL3A1 gene stored in a -70°C refrigerator onto the LB medium containing 1.0% agar, and culture it in a 28°C incubator for 2 d;

[0069] 22) After the incubation in the incubator is completed, pick single colonies into the LB liquid medium, and culture them at 28°C with shaking at 200 rpm overnight until the OD 600 value is approximately 0.5 - 0.6 to obtain the Agrobacterium liquid;

[0070] 3) Infection

[0071] Cut the young leaves of the tobacco sterile seedlings into pieces of 1 cm × 1 cm size, put them into a sterile beaker, add the cultured Agrobacterium liquid, soak for 10 min, shake occasionally during this period, and perform infection (as shown in Figure A in Figure 2 );

[0072] 4) Co-culture

[0073] After the infection is completed, discard the liquid, immediately take out the leaves, blot the surface liquid on the sterilized filter paper, and place them on the co-culture medium, and culture them in the dark at 25°C for 3 d (as shown in Figure B in Figure 2 );

[0074] Among them, the co-culture medium is MS medium, supplemented with 2.25 mg / L of 6-benzylaminopurine (6-BA), 0.3 mg / L of naphthaleneacetic acid (NAA), 30 g / L of sucrose and 7.5 g / L of agar.

[0075] 5) Screening

[0076] After co-culture, the leaves are inoculated onto the screening medium, and cultured under light at 25 °C, with subculture every 2 weeks for about 3 - 4 weeks (as shown in Figure C in Figure 2 );

[0077] Among them, the screening medium is MS medium, supplemented with 2.25 mg / L of 6-benzylaminopurine (6-BA), 0.3 mg / L of naphthaleneacetic acid (NAA), 30 mg / L of hygromycin (Hyg) and 300 mg / L of cephalosporin antibiotics (Cef).

[0078] 6) Rooting

[0079] After co-culture until buds differentiate, the robust resistant buds are cut and inoculated into the rooting medium, and continue to be cultured under light at 25 °C (as shown in Figure D in Figure 2 );

[0080] Among them, the rooting medium is MS medium, supplemented with 30 mg / L of hygromycin (Hyg) and 300 mg / L of cephalosporin antibiotics (Cef).

[0081] 7) Obtaining of resistant seedlings

[0082] After rooting culture until the roots of the resistant seedlings are well-developed and the growth is good, they are acclimatized in the room for one week and then transplanted. Water is thoroughly poured in the first three times, and field management is carried out to obtain resistant seedlings; at the same time, wild-type tobacco is planted as a blank control.

[0083] The whole tobacco genetic transformation process is as shown in Figure 2 ;

[0084] IV. Identification of resistant seedlings

[0085] 1) Extraction of plant genomic DNA (CTAB method)

[0086] 11) Respectively take the plant tissues of 16 resistant seedlings (the plant tissues in this example are leaves), and then place the corresponding plant tissues into 2 mL centrifuge tubes respectively, add 2 sterilized steel beads, freeze with liquid nitrogen, and grind them with a ball mill;

[0087] 12) Respectively add 500 μL of 2×CTAB extraction buffer, water bath at 65 °C for 20 min, and invert and mix several times during this period;

[0088] 13) Add 800 μL of phenol, chloroform, and isopropanol with a volume ratio of 25:24:1 respectively, invert and mix well, centrifuge at 12000 rpm for 10 min, and transfer the upper aqueous phase to a new 2 mL centrifuge tube;

[0089] 14) Add 0.6 volume times of isopropanol to the obtained aqueous phase, and precipitate at -20 °C for more than 30 min;

[0090] 15) After precipitation, centrifuge at 12000 rpm for 10 min, discard the supernatant, and wash twice with 75% ethanol;

[0091] 16) Air-dry the residual alcohol at room temperature, dissolve with 50 μL of distilled water to obtain the corresponding plant genomic DNA, and store at -20 °C.

[0092] 2) Identification of resistant seedlings by PCR

[0093] To save the cost of primer design and the stability of PCR conditions, the present invention uses the marker gene on the vector for identification (i.e., to identify whether the plant genomic DNA contains hygromycin). The pC1300-rCOL3A1 plant binary expression vector is used as a positive control. The specific reaction system for identification by PCR is as follows:

[0094] Table 1 List of reaction systems for identification by PCR

[0095] Component Volume 2×Taq PCR Master Mix 10μL Forward primer (5’-tggcaaactgtgatggacgacac-3’) 0.5μL Reverse primer (5’-ctatttctttgccctcggacgagtgc-3’) 0.5μL DNA template 1μL Sterilized water Up to 20μL

[0096] Reaction procedure for identification by PCR: Pre-denaturation at 94 °C for 3 min; denaturation at 94 °C for 30 sec, annealing at 60 °C for 30 sec, extension at 72 °C for 30 sec, a total of 28 cycles; extension at 72 °C for 5 min.

[0097] In the present invention, a total of 16 resistant seedlings were obtained. After PCR identification, 14 of them were identified as positive, that is, positive transformed tobacco, and the identification results are as Figure 3 shown.

[0098] Example 4 Western blot identification of type III collagen rCOL3A1 protein in genetically transformed positive plant tobacco

[0099] The protein expression levels of 6 positive transformed tobaccos (i.e., genetically transformed positive plant tobacco) were identified by SDS-PAGE and Western blot, and wild-type tobacco was used as a negative control.

[0100] S1. Extraction of total plant protein and SDS-PAGE

[0101] S11. Extraction of total plant protein

[0102] Take 50 - 100 mg of fresh leaves and place them in 2 ml centrifuge tubes. Add steel beads, quickly freeze them in liquid nitrogen, then oscillate for 1 min on a tissue grinder. Then add 500 μL of protein extraction buffer respectively, oscillate for 15 sec, let it stand for 5 min, and then centrifuge at 4°C, 12000 g × 15 min, and take the corresponding supernatant;

[0103] Among them, the protein extraction buffer is 50 mM Tris - HCl with a pH value of 7.5, and cOmplete ULTRA protease inhibitor (Roche) is added;

[0104] S12. Determine the protein concentration using the Bradford method

[0105] Adjust the protein concentration in the supernatant to 1 μg / μL uniformly to obtain the corresponding protein solution;

[0106] Take 21 μL of the protein solution, then add 7 μL of 4X sample loading buffer, mix well, then boil the protein at 95°C for 8 min, and finally load the sample and run SDS - PAGE (protein gel electrophoresis);

[0107] Among them, 4X sample loading buffer is 250 mM Tris–HCl with a pH value of 6.8, and 8% (w / v) sodium dodecyl sulfate (SDS), 0.2% (w / v) bromophenol blue, 40% (v / v) glycerol and 20% (v / v) β - mercaptoethanol are added;

[0108] S2. Western blot

[0109] After SDS - PAGE, use the wet transfer method to transfer the protein on the protein gel to the PVDF membrane. The specific operation method is as follows;

[0110] S21. Prepare 1X transfer buffer; among them, 1X transfer buffer is prepared by mixing 100 mL of 10X transfer buffer, 200 mL of methanol and 700 mL of ddH2O;

[0111] S22. Cut a PVDF membrane a little larger than the protein gel (about 9 cm * 6 cm);

[0112] S23. Prepare ice in advance;

[0113] S24. Soak the PVDF membrane in methanol with forceps;

[0114] S25. Peel the gel, gently scrape off the stacking gel, and avoid scratching the separating gel;

[0115] S26. Transfer the membrane. Place the membrane transfer clip in a tray filled with 1X membrane transfer buffer, with the black side down (negative electrode), and place the special sponge → filter paper → gel → PVDF membrane → filter paper → sponge in sequence; after placing them in sequence, expel the air bubbles, clamp the membrane transfer clip, place it in the membrane transfer tank, pour in 1X membrane transfer buffer, place an ice box in the tank to cool down, and apply a constant current voltage of 300 mA for 1.5 h with a constant current.

[0116] The results of Western Blot are as Figure 4 shown, indicating that in the present invention, the size of recombinant type III collagen rCOL3A1 is approximately 110 Kda, while the predicted size of mature type III collagen is 95 Kda. It is speculated that the tobacco in the present invention has post-translational modification of the amino acids of recombinant type III collagen.

[0117] Other parts not described in detail are all prior arts. Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. Those of ordinary skill in the art can also obtain other embodiments according to this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing recombinant human type III collagen in tobacco, characterized in that, The preparation method is to ligate the coding sequence of the recombinant human type III collagen to a plant binary expression vector, introduce the plant binary expression vector into tobacco, culture the obtained genetically transformed positive plant tobacco, and prepare the recombinant human type III collagen; The plant binary expression vector is the pCambia1300-35S vector; The amino acid sequence of the recombinant human type III collagen is as shown in SEQ ID NO: 1; The amino acid sequence of the recombinant human type III collagen is successively a BiP signal peptide sequence, an rCOL3A1 sequence, and a KDEL sequence from the N-terminus to the C-terminus; The encoding rCOL3A1 The nucleotide sequence of the gene is shown in SEQ ID NO:

2.

2. The preparation method of recombinant human type III collagen in tobacco according to claim 1, characterized in that, The specific process of introducing the plant binary expression vector into tobacco is to use Agrobacterium-mediated transformation of tobacco with a plant binary expression vector containing rCOL3A1 gene.

3. The preparation method of recombinant human type III collagen in tobacco according to claim 2, characterized in that, The said inclusion rCOL3A1 The Agrobacterium tumefaciens of the plant binary expression vector containing the rCOL3A1 gene is obtained by introducing the plant binary expression vector containing the gene into Agrobacterium tumefaciens.

4. The preparation method of recombinant human type III collagen in tobacco according to claim 1, wherein The preparation method further includes: Identification of the insertion of the rCOL3A1 gene in genetically transformed positive plants; and / or protein identification of the recombinant human type III collagen in the genetically transformed positive plant.

Images