Gene for coding recombinant human-derived III-type collagen, plant expression vector, plant suspension cell line and method for expressing recombinant human-derived III-type collagen by plant suspension cells

Abstract

The invention provides a gene for coding recombinant human-derived III-type collagen, a plant expression vector, a plant suspension cell line and a method for expressing the recombinant human-derived III-type collagen by plant suspension cells. The gene for coding the recombinant human-derived III-type collagen is obtained through codon optimization, the gene can be used for constructing a plant expression vector, and the mRNA stability of the plant expression vector is further improved by adding upstream and downstream regulation elements for regulating a target gene, so that the expression quantity of the plant-derived III-type human collagen is improved. According to the invention, a plant suspension cell is adopted as an efficient platform technology for recombinant protein production, and the recombinant human III-type collagen is successfully expressed; while the expression quantity is relatively high, the biological safety problem is reduced to the greatest extent, and the safety of the product is improved; and the method also has the advantages of short production period, simplified purification process, convenient production operation, significantly reduced production cost and the like.

Description

Technical Field

[0001] The present application relates to the field of genetic engineering technology, and in particular to a gene encoding recombinant human type III collagen, a plant expression vector, a plant suspension cell line, and a method for expressing recombinant human type III collagen in plant suspension cells. Background Art

[0002] As the main structural component of skin, bones, tendons, ligaments and blood vessels, collagen plays a vital role in maintaining the structural stability and function of these tissues. It is not only biodegradable, but also has low antigenicity, low irritation and low toxicity. In addition, collagen can promote cell proliferation, cell adhesion and coordinated reconstruction of new cells. Due to its characteristics, collagen has a wide range of applications in the fields of medicine, beauty and biomaterials.

[0003] Depending on the source, collagen extraction methods are divided into traditional animal extraction methods and recombinant technology methods. Traditional collagen extraction methods mainly rely on extraction from animal tissues, such as pig skin, cow skin, fish skin, etc. However, this method has many limitations, including limited raw material sources, complex extraction process, and unstable product quality. In addition, animal-derived collagen may also be at risk of viral and bacterial contamination and potential immune rejection. Compared with natural collagen extracted from animal tissues, recombinant collagen has better control capabilities (such as water solubility and emulsification), as well as a lower risk of pathogen transmission. However, due to the low yield and high cost of animal and insect expression systems. At this stage, the expression system of recombinant collagen is mainly based on Escherichia coli and yeast, but most of them do not have a triple helix structure.

[0004] Plant-derived collagen has attracted much attention because it avoids the risks of animal-derived pathogen contamination and high endotoxin content, and shows significant advantages in raw material acquisition, production process control, product quality stability, cost-effectiveness, and environmental friendliness. Therefore, there is greater potential to use plants to produce high-yield recombinant human collagen with a triple helix structure. However, compared with other expression systems, the expression level of plant-derived collagen is relatively low. Therefore, the development of an efficient collagen expression method and its supporting vector is of great practical significance for improving the production efficiency of plant-derived collagen. Summary of the invention

[0005] The present application provides a gene encoding recombinant human type III collagen, a plant expression vector, a plant suspension cell line, and a method for expressing recombinant human type III collagen in plant suspension cells to solve the problems existing in the related technologies. The technical solution is as follows:

[0006] In a first aspect, an embodiment of the present application provides a gene encoding a recombinant human type III collagen, the amino acid sequence of the recombinant human type III collagen being shown in SEQ ID NO: 1; the nucleotide sequence of the gene encoding the recombinant human type III collagen being shown in SEQ ID NO: 2.

[0007] In a second aspect, the present application provides a plant expression vector for expressing recombinant human type III collagen, wherein the plant expression vector for expressing recombinant human type III collagen comprises a recombinant gene fragment;

[0008] The recombinant gene fragment includes the promoter CaMV 35S promoter, the cis-regulatory element COR47-5'UTR, the above-mentioned gene nucleotide sequence encoding the recombinant human type III collagen, and the terminator HSPT878 which are connected in sequence.

[0009] In one embodiment, the plant expression vector expressing recombinant human type III collagen is a pCAMBIA1300 plant expression vector;

[0010] The recombinant gene fragment is constructed and placed upstream of the right border of the T-DNA region of the pCAMBIA1300 plant expression vector.

[0011] In one embodiment, the nucleotide sequence of the plant expression vector expressing recombinant human type III collagen is as shown in SEQ ID NO.3.

[0012] In a third aspect, the present application provides a method for constructing a plant expression vector for expressing recombinant human type III collagen, comprising the following steps:

[0013] The promoter CaMV 35S promoter, cis-regulatory element COR47-5′UTR, multiple cloning site sequence, and terminator HSPT878 were constructed and placed upstream of the right border of the T-DNA region of the pCAMBIA1300 plant expression vector to obtain the plant expression vector pCAMBIA-COR47-HSPT878;

[0014] The above-mentioned gene nucleotide sequence encoding the recombinant human type III collagen was constructed into the multiple cloning site position on the plant expression vector pCAMBIA-COR47-HSPT878 by PmeI / Mlu I enzyme ligation method to obtain the plant expression vector pCAMBIA-COR47-1059-HSPT878 expressing the recombinant human type III collagen.

[0015] In a fourth aspect, an embodiment of the present application provides a high-yield plant suspension cell line expressing recombinant human type III collagen, comprising any of the above-mentioned plant expression vectors expressing recombinant human type III collagen.

[0016] In one embodiment, the plant is tobacco, rice or lettuce suspension cells.

[0017] In a fifth aspect, the present application provides a method for expressing recombinant human type III collagen in plant suspension cells, comprising the following steps:

[0018] Transforming any of the above-mentioned plant expression vectors expressing recombinant human type III collagen into Agrobacterium, infecting plant suspension cells through Agrobacterium-mediated infection, and screening suspension cells with stable expression using a suspension cell screening medium;

[0019] The stably expressed suspension cells are cultured with expression medium, the medium is removed, and the cells are resuspended with extraction buffer; the cell suspension is crushed, and the supernatant is collected by centrifugation; the protein is separated to obtain recombinant human type III collagen.

[0020] In one embodiment, the suspension cell screening medium is K427 basal medium + 30 g / L sucrose + 1 mg / L 6-BA + 0.5 mg / L NAA + hygromycin 50 ug / L + cephalosporin 200 mg / L + carbenicillin 200 mg / L.

[0021] In one embodiment, the suspension cell expression medium is SH basal medium + 30 g / L sucrose + 1 mg / L 6-BA + 0.5 mg / L NAA.

[0022] In one embodiment, the extraction buffer consists of 100 mM phosphate buffer at pH 7, 5 mM EDTA and 1% PVP.

[0023] In one embodiment, the mass volume ratio of the suspended cells to the extraction buffer is 1:2.

[0024] In a sixth aspect, the embodiments of the present application provide the application of the recombinant human type III collagen prepared by the above-mentioned gene encoding recombinant human type III collagen, the plant expression vector expressing recombinant human type III collagen, the high-yield plant suspension cell line expressing recombinant human type III collagen, and the method of expressing recombinant human type III collagen in plant suspension cells in the field of food, cosmetics, health care products or pharmaceutical devices.

[0025] The advantages or beneficial effects of the above technical solution include at least:

[0026] The gene encoding recombinant human type III collagen of the present application can be constructed and placed in a plant expression vector, and the plant expression vector can be further improved in terms of mRNA stability by adding upstream and downstream regulatory elements that regulate the target gene, thereby increasing the expression level of plant-derived type III human collagen.

[0027] This application uses plant suspension cells as an efficient platform technology for the production of recombinant proteins, and successfully expresses recombinant human type III collagen. The constructed recombinant plant expression vector is transformed into Agrobacterium and mediated to infect plant suspension cells. After screening, culturing and expressing by plant suspension cells, the exogenous protein with biological activity is successfully isolated under mild conditions; it has a high expression level. And this method minimizes biosafety issues and improves the safety of the product; plant suspension cells basically do not contain plant toxic substances, and they themselves have less fiber, which is conducive to downstream protein purification. It also has the advantages of short production cycle (6 days), simplified purification process, convenient production operation, and significantly reduced production costs.

[0028] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present application will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the multiple drawings represent the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings only depict some embodiments disclosed in the present application and should not be regarded as limiting the scope of the present application.

[0030] Figure 1 The expression vector pCAMBIA-COR47-MCS-HSPT878 expression cassette;

[0031] Figure 2 is the plasmid map of the expression vector pCAMBIA-COR47-1059-HSPT878;

[0032] Figure 3 is the plasmid map of the expression vector pCAMBIA1300-1059;

[0033] Figure 4 Create a diagram for the tobacco suspension cell system;

[0034] Figure 5 This is the growth curve of tobacco suspension cell system;

[0035] Figure 6WB graph of the expression levels of the expression vector prepared for the present application and the expression vector prepared for the comparative example. DETAILED DESCRIPTION

[0036] In the following, only some exemplary embodiments are briefly described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the present application. Therefore, the drawings and descriptions are considered to be exemplary and non-restrictive in nature.

[0037] The use of plants to produce high-yield recombinant human collagen with a triple-helix structure has greater potential, but the relatively low expression level of plant-derived collagen is the main problem. By introducing upstream and downstream regulatory elements of the target gene, the expression level of the target gene can be significantly improved compared to the traditional overexpression vector driven by the CaMV 35S promoter; and compared to other plant (whole plant) expression systems, the suspension cell expression system reduces the process of plant chloroplasts and secondary metabolites. Therefore, the development of an efficient collagen expression method and its supporting vector is of great practical significance for improving the production efficiency of plant-derived collagen.

[0038] The embodiment of the present application provides a gene encoding recombinant human type III collagen, the amino acid sequence of the recombinant human type III collagen is shown in SEQ ID NO: 1; the nucleotide sequence of the gene encoding the recombinant human type III collagen is shown in SEQ ID NO: 2.

[0039] SEQ ID NO: 1

[0040] GKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCCGGVGAAAIAGIGGEKAGGFAPYYGDEPMDFKINTDEIMTSLKSVNGQIESLISPDGSRKNPARNCRDLKFCHPELKSGEYWVDPNQGCKLDAIKVFCNMETGETCISANPLNVPRKHWWTDSSAEKKHVWFGESMDGGFQFSYGNPELPEDVLDVHLAFLRLLSSRASQNITYHCKNSIAYMDQASGNVKKALKLMGSNEGEFKAEGNSKFTYTVLEDGCTKHTGEWSKTVFEYRTRKAVRLPIVDIAPYDIGGPDQEFGVDVGPVCFL。

[0041] SEQ ID NO:2

[0042]

[0043] The amino acid sequence of human type III collagen (COL3A1) is shown in SEQ ID NO: 1; the corresponding gene sequence encoding human type III collagen is optimized according to the preference of the corresponding plant expression system, that is, after the corresponding codon optimization, it can be better expressed in the corresponding plant expression system. Therefore, in this application, the Pme I restriction site GTTTAAAC, the start codon and the 6*His sequence are added to the front end of the gene sequence encoding human type III collagen, and the termination codon TAG and the Mlu I restriction site ACGCGT are added to the back end of the sequence to obtain an optimized gene encoding recombinant human type III collagen, and its sequence is shown in SEQ ID No: 2.

[0044] The present application embodiment provides a plant expression vector for expressing recombinant human type III collagen, wherein the plant expression vector for expressing recombinant human type III collagen comprises a recombinant gene fragment;

[0045] The recombinant gene fragment includes the promoter CaMV 35S promoter, the cis-regulatory element COR47-5'UTR, the gene nucleotide sequence encoding the recombinant human type III collagen described in claim 1, and the terminator HSPT878, which are connected in sequence.

[0046] The expression level of protein is closely related to gene sequence and expression vector, and suitable gene sequence and expression vector can promote high expression of protein. In order to achieve high expression of transgenic, it is not only necessary to optimize codons and promoters, but also important to optimize the upstream and downstream regulatory elements 5-untranslated region (UTR) and transcription terminator of genes. Therefore, the present application designs an expression cassette, in which the coding sequence of collagen is jointly driven by the transcription terminator (HSPT878) of heat shock protein 18.2 (HSP) gene (HSPT878) of cauliflower mosaic virus (CaMV) 35S promoter and Arabidopsis thaliana (HSPT878) and the 5 untranslated region (5′UTR) of cold regulation (COR47) of Arabidopsis thaliana. Due to the addition of upstream and downstream regulatory elements of target gene, compared with the ordinary overexpression vector driven by CaMV 35S promoter, the mRNA stability is improved, the expression of target gene is significantly enhanced, and the expression amount of plant-derived type III human collagen is significantly enhanced.

[0047] In one embodiment, the plant expression vector expressing recombinant human type III collagen is a pCAMBIA1300 plant expression vector;

[0048] The recombinant gene fragment is constructed and placed upstream of the right border of the T-DNA region of the pCAMBIA1300 plant expression vector.

[0049] In one embodiment, the nucleotide sequence of the plant expression vector expressing recombinant human type III collagen is as shown in SEQ ID NO.3.

[0050] The present application embodiment provides a method for constructing a plant expression vector for expressing recombinant human type III collagen, comprising the following steps:

[0051] The promoter CaMV 35S promoter, cis-regulatory element COR47-5′UTR, multiple cloning site sequence, and terminator HSPT878 were constructed and placed upstream of the right border of the T-DNA region of the pCAMBIA1300 plant expression vector to obtain the plant expression vector pCAMBIA-COR47-HSPT878;

[0052] The gene nucleotide sequence encoding the recombinant human type III collagen described in claim 1 is constructed into the multiple cloning site position on the plant expression vector pCAMBIA-COR47-HSPT878 by the PmeI / Mlu I enzyme digestion and ligation method to obtain the plant expression vector pCAMBIA-COR47-1059-HSPT878 expressing the recombinant human type III collagen.

[0053] The embodiments of the present application provide a high-yield plant suspension cell line expressing recombinant human type III collagen, comprising any of the above-mentioned plant expression vectors expressing recombinant human type III collagen.

[0054] In one embodiment, the plant is tobacco, rice or lettuce suspension cells.

[0055] Plant suspension cells are usually grown in a specific culture medium and developed in a completely closed container. There is no problem of biological contamination or genetic contamination, which eliminates the risk of potential pathogens that infect humans and immunogenicity, and improves the safety of the product; therefore, this method minimizes biosafety issues. Plant suspension cells basically do not contain plant toxic substances, and they have less fiber, which is conducive to downstream protein purification. It also has the advantages of short production cycle, simplified purification process, convenient production operation, and significantly reduced production costs.

[0056] In this embodiment, the high-yield plant suspension cell strain expressing recombinant human type III collagen is obtained by screening plant suspension cells mediated by Agrobacterium transformed with an expression vector.

[0057] Preferably, the plant expression vector expressing the recombinant human type III collagen is transformed into Agrobacterium, and the plant suspension cells are infected through Agrobacterium-mediated infection, and the suspension cells with stable expression are screened using a suspension cell screening medium.

[0058] In one embodiment, the Agrobacterium is Agrobacterium GV3101.

[0059] Further preferably, the suspension cell screening medium is K427 basal medium + 30 g / L sucrose + 1 mg / L 6-BA + 0.5 mg / L NAA + hygromycin 50 ug / L + cephalosporin 200 mg / L + carbenicillin 200 mg / L.

[0060] In one embodiment, the suspension cells are prepared by subculturing the plant body after callus induction.

[0061] In this embodiment, callus induction is carried out in a callus induction medium. The callus induction medium is preferably MS basic medium + 30 g / L sucrose + 1 mg / L 6-BA + 0.1 mg / L 2,4-D + 7.5 g / L agar, pH = 5.8. The culture conditions are 25° C. and dark culture.

[0062] In this embodiment, the subculture is performed in a suspension cell culture medium, which is K427 basal medium + 30 g / L sucrose + 1 mg / L 6-BA + 0.5 mg / L NAA; the culture conditions are 25° C., 120 r / min, and subculture is performed once a week.

[0063] In one embodiment, a plant expression vector expressing recombinant human type III collagen is transformed into Agrobacterium, and the plant suspension cells are infected through Agrobacterium-mediated infection to perform suspension cell infection and co-cultivation.

[0064] The transformation of Agrobacterium is carried out by using a plant expression vector expressing recombinant human type III collagen according to existing methods. The transformed Agrobacterium is cultured in YEB medium, centrifuged and resuspended in Agrobacterium suspension medium to a specific OD value. In this embodiment, the OD value is 0.8.

[0065] Suspension cell infection and co-cultivation is to mix plant suspension cells with Agrobacterium suspension culture medium for infection. After infection, the cells are cultured in co-culture medium to obtain transgenic suspension cell lines.

[0066] The present invention provides a method for expressing recombinant human type III collagen in plant suspension cells, comprising the following steps:

[0067] Transforming any of the above-mentioned plant expression vectors expressing recombinant human type III collagen into Agrobacterium, infecting plant suspension cells through Agrobacterium-mediated infection, and screening suspension cells with stable expression using a suspension cell screening medium;

[0068] The stably expressed suspension cells are cultured with expression medium, the medium is removed, and the cells are resuspended with extraction buffer; the cell suspension is crushed, and the supernatant is collected by centrifugation; the protein is separated to obtain recombinant human type III collagen.

[0069] The suspension cell line is screened by using a suspension cell screening medium, wherein the suspension cell screening medium may be a solid screening medium or a screening liquid medium.

[0070] In one embodiment, the suspension cell liquid screening medium is K427 basal medium + 30g / L sucrose + 1mg / L 6-BA + 0.5mg / L NAA + hygromycin 50ug / L + cephalosporin 200mg / L + carbenicillin 200mg / L;

[0071] The suspended cell solid screening culture medium is MS basic culture medium + 30g / L sucrose + 1mg / L 6-BA + 0.5mg / L NAA + hygromycin (hyg) 50ug / L + cephalosporin (cefo) 200mg / L + carbenicillin (Carb) 200mg / L.

[0072] In one embodiment, the suspension cell expression medium is SH basal medium + 30 g / L sucrose + 1 mg / L 6-BA + 0.5 mg / L NAA.

[0073] In one embodiment, the extraction buffer consists of 100 mM phosphate buffer at pH 7, 5 mM EDTA and 1% PVP.

[0074] In one embodiment, the mass volume ratio of the suspended cells to the extraction buffer is 1:2.

[0075] In one embodiment, a purification process is also included, wherein the purification uses a Ni column affinity chromatography method.

[0076] The following is a further description with reference to specific embodiments.

[0077] Example 1

[0078] According to the amino acid sequence of recombinant human type III collagen as shown in SEQ ID NO: 1, the corresponding gene sequence encoding human type III collagen was codon optimized. The Pme I restriction site GTTTAAAC, the start codon and the 6*His sequence were added to the front end of the gene sequence encoding human type III collagen, and the stop codon TAG and the Mlu I restriction site ACGCGT were added to the back end of the sequence to obtain the optimized gene encoding recombinant human type III collagen, the sequence of which is shown in SEQ ID No: 2; and it was synthesized by GenScript (Nanjing, China).

[0079] SEQ ID NO:1

[0080] GKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCCGGVGAAAIAGIGGEKAGGFAPYYGDEPMDFKINTDEIMTSLKSVNGQIESLISPDGSRKNPARNCRDLKFCHPELKSGEYWVDPNQGCKLDAIKVFCNMETGETCISANPLNVPRKHWWTDSSAEKKHVWFGESMDGGFQFSYGNPELPEDVLDVHLAFLRLLSSRASQNITYHCKNSIAYMDQASGNVKKALKLMGSNEGEFKAEGNSKFTYTVLEDGCTKHTGEWSKTVFEYRTRKAVRLPIVDIAPYDIGGPDQEFGVDVGPVCFL。

[0081] SEQ ID NO:2

[0082]

[0083] Example 2

[0084] The embodiment of the present application provides a plant expression vector for expressing human collagen, wherein the plant expression vector for expressing human collagen comprises a recombinant gene fragment;

[0085] The recombinant gene fragment includes the promoter CaMV 35S promoter, the cis-regulatory element COR47-5'UTR, the gene nucleotide sequence encoding the recombinant human type III collagen described in claim 1, and the terminator HSPT878, which are connected in sequence.

[0086] The specific construction method is as follows:

[0087] (1) Construction of recombinant plasmid pCAMBIA-COR47-MCS-HSPT878

[0088] The universal constitutive expression promoter CaMV 35S promoter (enhanced) in plants, the cis-acting element COR47-5′UTR for efficient regulation of translation, the multiple cloning site sequence, and the HSPT878 terminator for efficient regulation of expression were constructed and placed upstream of the right border of the T-DNA region of the pCAMBIA1300 plant expression vector; the expression cassette element sequence was synthesized and constructed by GenScript (Nanjing, China) using full gene synthesis. After optimizing the expression elements, the plant expression vector pCAMBIA-COR47-MCS-HSPT878 was finally obtained. The optimized pCAMBIA-COR47-MCS-HSPT878 vector expression cassette is as follows Figure 1 shown.

[0089] (2) Construction of recombinant plasmid pCAMBIA-COR47-1059-HSPT878.

[0090] The synthesized gene nucleotide sequence encoding the recombinant human type III collagen (SEQ ID No: 2) was constructed into the multiple cloning site of the plant expression vector pCAMBIA-COR47-MCS-HSPT878 by PmeI / Mlu I digestion and ligation to obtain the optimized expression vector pCAMBIA-COR47-1059-HSPT878 recombinant plasmid; the recombinant plasmid map is shown in Figure 2 As shown;

[0091] The recombinant plasmid was transformed into Escherichia coli T1, and the positive strains were identified by double restriction digestion and gene sequencing; the nucleotide sequence of pCAMBIA-COR47-1059-HSPT878 is shown in SEQ ID No: 3 (see ST.26 standard sequence nucleotide or amino acid sequence table).

[0092] In this embodiment, the method for constructing the expression vector pCAMBIA-COR47-1059-HSPT878 by the PmeI / Mlu I enzyme ligation method is to perform an enzyme digestion reaction in the enzyme digestion system in Table 1.

[0093] Table 1 Enzyme digestion system

[0094]

[0095]

[0096] The enzyme digestion reaction conditions were: 25°C for 30 min and heat inactivation at 65°C for 20 min.

[0097] Example 3 Establishment of tobacco suspension cell system

[0098] Callus induction: Using Nicotiana benthamiana varieties, leaves were used as explants. Sterile tobacco leaves were cut into squares of about 5 mm and evenly inoculated on the best callus induction medium (MS basic medium + 30 g / L sucrose + 1 mg / L 6-BA + 0.1 mg / L 2,4-D + 7.5 g / L agar, pH = 5.8); 25°C, dark culture; the culture process is as follows Figure 4 .

[0099] Establishment of suspension cell line: After successful callus induction of suspension cell line, loose and well-dispersed callus tissue that has been induced and cultured for more than one month was picked up with forceps, crushed with forceps, and inoculated into liquid culture medium in a conical flask at a ratio of about 1:10 (C:V), cultured at 25°C, 120r / min, and subcultured once a week ( Figure 5 ). The liquid culture medium was K427 basal medium + 30 g / L sucrose + 1 mg / L 6-BA + 0.5 mg / L NAA.

[0100] Example 4: Agrobacterium-mediated transformation of tobacco suspension cells

[0101] Agrobacterium transformation: The recombinant expression vector pCAMBIA-COR47-1059-HSPT878 in Example 2 was transformed into Agrobacterium GV3101 according to the existing method. The transformed Agrobacterium was inoculated in YEB medium at 28°C, 200 rpm, cultured overnight, centrifuged and resuspended in Agrobacterium suspension medium to an OD value of 0.8; Agrobacterium suspension medium: K427 basal medium + 30 g / L sucrose + 1 mg / L 6-BA + 0.5 mg / L NAA + 100 μM acetosyringone, pH = 5.8.

[0102] Suspension cell infection and co-culture: Select vigorous and healthy tobacco suspension cells and mix them with the above culture medium in a ratio of 1:1, pipette 100 times, and then shake and infect at 28°C for 10 minutes; filter the suspension cells with a sterile filter, spread them on a culture dish containing sterile filter paper to absorb excess bacterial liquid, dry them, transfer them to a co-culture medium covered with a layer of filter paper at 25°C, and inoculate them on the screening medium after dark culture for 3 days to obtain transgenic suspension cell lines. Co-culture medium: MS basic medium + 30g / L sucrose + 1.0mg / L 6-BA + 0.5mg / L NAA + 7.5g / L agar + 100μM acetosyringone, pH = 5.8.

[0103] Example 5: Construction of a suspension cell line that stably expresses collagen

[0104] Suspended cell resistance screening: The co-cultured suspended cells are inoculated into a liquid screening medium for screening culture and sterilization. One week later, the suspended cells are filtered with a sterile filter and inoculated into a suspended cell solid screening medium. The liquid screening medium is K427 basal medium + 30g / L sucrose + 1mg / L6-BA + 0.5mg / L NAA + hygromycin (hyg) 50ug / L + cephalosporin (cefo) 200mg / L + carbenicillin (Carb) 200mg / L; the solid screening medium is MS basal medium + 30g / L sucrose + 1mg / L6-BA + 0.5mg / L NAA + hygromycin (hyg) 50ug / L + cephalosporin (cefo) 200mg / L + carbenicillin (Carb) 200mg / L;

[0105] After 2-3 weeks of screening, obvious single cell clusters can be seen. The cell clusters are subcultured and shaken for expression. The cell clusters are positive for expression by WB detection ( Figure 6 ), the cell is the transgene positive cell.

[0106] Collagen expression: The resistant single cell group is expressed in a shake flask; the suspension cell expression medium is SH basic medium + 1 mg / L 6-BA + 0.5 mg / L NAA; the transgenic plant suspension cells can be collected after 7 days of culture in a shake flask or bioreactor.

[0107] Example 6: Recombinant collagen extraction and separation

[0108] The transgenic tobacco suspension cell culture fluid with high expression of human collagen obtained by screening is purified using a protein purifier to obtain recombinant human collagen, specifically:

[0109] 1) Take the suspended cells in Example 5 and filter them through a 500-mesh nylon filter bag to remove the culture medium, and resuspend them in an extraction buffer with a mass-to-volume ratio of 1:2, and preliminarily homogenize them in a blender at medium-high speed for 1-2 minutes; adjust the homogenate to pH = 7.5. The extraction buffer is composed of pH = 7 100mM phosphate buffer, 5mM EDTA and 1% PVP;

[0110] 2) After the initial homogenization, the suspension is crushed using a high-pressure homogenizer; wherein the high-pressure homogenizer is first crushed with a pressure of 200 bas and then with 500 bas;

[0111] 3) The suspension after disruption was filtered through a 2000-mesh nylon filter bag to remove cell debris, and then centrifuged at 4°C and 12,000 rpm for 30 min to collect the supernatant;

[0112] 4) The supernatant after centrifugation is filtered using a suction filter device and filtered through a 0.22 μm filter membrane to obtain a recombinant human collagen stock solution;

[0113] Recombinant human collagen was purified by Ni column affinity chromatography, the chromatographic filler was NiSeplife FF (TED), the equilibration buffer A was a mixture of 20 mM Tris, 500 mM NaCl and 20 mM imidazole, and the elution buffer B was a mixture of 20 mM Tris, 500 mM NaCl and 500 mM imidazole; step gradient elution was used, and the elution gradient was shown in Table 4:

[0114] Table 2

[0115] Flow rate Proportion Column volume Remark 1ml / min 0％B 5 balance 1ml / min 5％B 3 Wash 1ml / min 12％B 4 Wash 1ml / min 50％B 4 Target product 1ml / min 100％B 4 -

[0116] Comparative Example 1:

[0117] The gene sequence encoding human type III collagen was added with the BglII restriction site AGATCT, the start codon and the 6*His sequence at the front, and the termination codon TAG and the BtE II restriction site GGTNACC at the rear to obtain the optimized gene sequence of human collagen. The pCAMBIA1300 was constructed into the original vector pCAMBIA1300 by BglII / BtE II restriction enzyme ligation to obtain pCAMBIA1300-1059. Different from the embodiment, the pCAMBIA1300-1059 expression vector does not have the cis-acting element COR47-5′UTR for efficient regulation of translation and the HSPT878 terminator for efficient regulation of expression; Figure 3 shown.

[0118] The plant expression vector was transformed according to the experimental conditions in the example. Except for the different vectors, the other conditions were the same. After the suspension cells of the transgenic plants in the comparative example and the example were cultured in a shake flask for 7 days, samples were centrifuged to remove the culture medium and the cell lysis solution was added for grinding; at the same time, the operation was carried out according to the standard WB experiment. Figure 6 shown.

[0119] pass Figure 6 The WB experimental results show that pCAMBIA-COR47-1059-HSPT878 transgenic tobacco suspension cells express collagen more efficiently than pCAMBIA1300-1059 expression vector.

[0120] In summary, the present application further constructs a plant expression vector expressing human collagen after codon optimization of the gene nucleotide sequence encoding recombinant human type III collagen, obtains a high-yield plant suspension cell line expressing recombinant human collagen, and successfully expresses it in plant suspension cells, and the expression level is significantly enhanced.

[0121] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples, unless they are contradictory.

[0122] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of this application, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0123] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any technician familiar with the technical field can easily think of various changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A gene encoding 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; the nucleotide sequence of the gene encoding the recombinant human type III collagen is shown in SEQ ID NO:

2.

2. A plant expression vector for expressing recombinant human type III collagen, characterized in that: The plant expression vector expressing recombinant human type III collagen comprises a recombinant gene fragment; The recombinant gene fragment includes the promoter CaMV 35S promoter, the cis-regulatory element COR47-5'UTR, the gene nucleotide sequence encoding the recombinant human type III collagen described in claim 1, and the terminator HSPT878, which are connected in sequence.

3. The plant expression vector for expressing recombinant human type III collagen according to claim 2, characterized in that: The plant expression vector for expressing recombinant human type III collagen is a pCAMBIA1300 plant expression vector; The recombinant gene fragment is constructed and placed upstream of the right border of the T-DNA region of the pCAMBIA1300 plant expression vector.

4. The plant expression vector for expressing recombinant human type III collagen according to claim 2 or 3, characterized in that: The nucleotide sequence of the plant expression vector expressing recombinant human type III collagen is shown in SEQ ID NO.

3.

5. The method for constructing a plant expression vector for expressing recombinant human type III collagen according to any one of claims 2 to 4, characterized in that: The steps include: The promoter CaMV 35S promoter, cis-regulatory element COR47-5′UTR, multiple cloning site sequence, and terminator HSPT878 were constructed and placed upstream of the right border of the T-DNA region of the pCAMBIA1300 plant expression vector to obtain the plant expression vector pCAMBIA-COR47-HSPT878; The gene nucleotide sequence encoding the recombinant human type III collagen described in claim 1 is constructed into the multiple cloning site position on the plant expression vector pCAMBIA-COR47-HSPT878 by the PmeI / MluI enzyme digestion and ligation method to obtain the plant expression vector pCAMBIA-COR47-1059-HSPT878 expressing the recombinant human type III collagen.

6. A high-yield plant suspension cell line expressing recombinant human type III collagen, characterized in that: A plant expression vector for expressing recombinant human type III collagen comprising any one of claims 2 to 5.

7. The high-yield plant suspension cell line expressing recombinant human type III collagen according to claim 5, characterized in that: The plant is tobacco, rice or lettuce suspension cells.

8. A method for expressing recombinant human type III collagen in plant suspension cells, characterized in that: The following steps are involved: Transforming the plant expression vector expressing recombinant human type III collagen according to any one of claims 2 to 5 into Agrobacterium, infecting plant suspension cells through Agrobacterium-mediated infection, and screening the suspension cells with stable expression using a suspension cell screening medium; The stably expressed suspension cells are cultured with expression medium, the medium is removed, and the cells are resuspended with extraction buffer; the cell suspension is broken, and the supernatant is collected by centrifugation; the protein is separated to obtain recombinant human type III collagen.

9. The method for expressing recombinant human type III collagen in plant suspension cells according to claim 8, characterized in that: The suspension cell screening medium is K427 basal medium + 30g / L sucrose + 1mg / L 6-BA + 0.5mg / L NAA + hygromycin 50ug / L + cephalosporin 200mg / L + carbenicillin 200mg / L; The suspension cell expression medium is SH basic medium + 30g / sucrose + 1mg / L6-BA + 0.5mg / L NAA; The extraction buffer consists of 100 mM phosphate buffer at pH=7, 5 mM EDTA and 1% PVP; the mass volume ratio of the suspended cells to the extraction buffer is 1:

2.

10. Use of the gene encoding recombinant human type III collagen as described in claim 1, the plant expression vector expressing recombinant human type III collagen as described in any one of claims 2-5, the high-yield plant suspension cell strain expressing recombinant human type III collagen as described in claim 6 or 7, and the recombinant human type III collagen prepared by the method of expressing recombinant human type III collagen in plant suspension cells as claimed in claim 8 or 9 in the fields of food, cosmetics, health products or pharmaceuticals.

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