DNA fragment and application thereof in improving expression quantity of foreign protein in liverwort

Abstract

The invention provides a DNA (deoxyribonucleic acid) fragment and application thereof in improving the expression quantity of foreign protein in liverwort. The nucleotide sequence of the DNA fragment provided by the invention is as shown in SEQ ID NO: 1, and the DNA fragment is obtained by optimizing a signal peptide coding sequence of xyloglucan hydrolase according to a liverwort codon. Experiments prove that the gene can guide secretory expression of foreign proteins in liverwort cells, the expression quantity of the foreign proteins in the liverwort is remarkably improved, and the gene has important significance on large-scale production of the foreign proteins, especially medicinal proteins in the liverwort.

Description

Technical Field

[0001] The invention relates to the technical field of gene engineering, and in particular to a DNA fragment and an application thereof in increasing the expression amount of foreign protein in Marchantia polymorpha. Background Art

[0002] Marchantia polymorpha L. is also known as a cloud, Bagulong, rice seaweed, pus scab grass, ground thrush, ground duckweed, lichen, longan grass, etc. It is a spore plant of the genus Marchantia in the family Mosses. The plant body is light green or dark green, and the fronds are flat and ribbon-shaped. Marchantia belongs to the common group of Mosses plants, widely distributed all over the world, and is also an emerging model plant. Compared with classic model plants such as tobacco, Marchantia polymorpha has more unique advantages: (1) The growth cycle of Marchantia polymorpha is relatively shorter. It takes about one month for tobacco to obtain mature plants through sexual reproduction, while it only takes half a month for Marchantia polymorpha to obtain usable experimental materials through asexual reproduction using embryos; (2) Marchantia polymorpha already has a relatively mature and stable genetic transformation system. By using embryos for Agrobacterium-mediated transformation, a large number of transgenic strains can be obtained from one culture dish, which is easier to obtain than tobacco; (3) Marchantia polymorpha is a haploid plant with a simpler and clearer genetic background. Haploid plants can better ensure the purity of the transgenic strains of the offspring. Using embryo reproduction, a large number of transgenic strains with consistent genetic background can be obtained, which can provide rich experimental materials for subsequent research. Tetraploid plants such as tobacco have the problem of genetic heterozygosity during the genetic transformation process, making it more difficult to obtain pure transgenic strains; (4) Experimental materials can be preserved for a long time through embryo cold storage and spore freezing technology. There are also advantages in using Marchantia polymorpha as a bioreactor for protein expression: (1) Marchantia polymorpha is a bryophyte, which belongs to lower plants in the plant kingdom, and its organ differentiation is relatively simple. Therefore, the modification of the glycosylation pathway in Marchantia polymorpha has little effect on its growth; (2) Marchantia polymorpha has low environmental requirements for growth. One embryo cup of a vigorously growing Marchantia polymorpha can produce more than 100 embryos. Through asexual reproduction of embryos, a large number of pure and progeny with the same genetic background as the mother plant can be quickly obtained, which can ensure the mass production of antibodies; (3) Marchantia polymorpha is a haploid plant with a simple genetic background. The modification of the glycosylation pathway is relatively convenient, and it is easier to obtain humanized protein products. The expression of proteins in Marchantia polymorpha has great research prospects. The improvement of protein expression in Marchantia polymorpha is the prerequisite for the Marchantia polymorpha bioreactor. Therefore, elements that can improve the expression of proteins in Marchantia polymorpha are of great significance for the expression and production of proteins in Marchantia polymorpha. Summary of the invention

[0003] The technical problem to be solved by the present invention is how to increase the expression amount of exogenous protein in Marchantia polymorpha.

[0004] In a first aspect, the present invention provides a DNA fragment having a nucleotide sequence as shown in SEQ ID NO:1.

[0005] The second aspect of the present invention provides a fusion protein, wherein the fusion protein is formed by fusion of an exogenous protein and a signal peptide located at the N-terminus of the exogenous protein, and the amino acid sequence of the signal peptide is shown in SEQ ID NO:2.

[0006] Furthermore, the signal peptide is obtained by translating a DNA fragment having a nucleotide sequence as shown in SEQ ID NO:1.

[0007] The third aspect of the present invention provides a recombinant expression vector for increasing the expression level of foreign protein in Marchantia polymorpha, comprising the above DNA fragment and the coding sequence of the foreign protein, wherein the DNA fragment is located at the 5' end of the coding sequence of the foreign protein.

[0008] Furthermore, it also includes the CaMV35S promoter and terminator derived from cauliflower mosaic virus.

[0009] Furthermore, resistance screening genes are also included.

[0010] Furthermore, the CaMV35S promoter and terminator and resistance screening gene are obtained according to conventional techniques in the art.

[0011] The fourth aspect of the present invention provides a recombinant transformant comprising the above-mentioned recombinant expression vector.

[0012] Furthermore, the transformant may be Agrobacterium tumefaciens.

[0013] The fifth aspect of the present invention provides the use of the above-mentioned DNA fragment or the above-mentioned recombinant expression vector or the above-mentioned recombinant transformant in increasing the expression amount of foreign protein in Marchantia polymorpha.

[0014] A sixth aspect of the present invention provides a method for increasing the expression level of exogenous protein in Marchantia polymorpha, comprising the following steps:

[0015] Constructing any of the above-mentioned recombinant expression vectors;

[0016] The recombinant expression vector is introduced into Marchantia polymorpha, so that the recombinant expression vector is expressed in Marchantia polymorpha to obtain foreign protein, thereby increasing the expression amount of foreign protein in Marchantia polymorpha.

[0017] Furthermore, the exogenous protein is FGF-2 protein derived from human.

[0018] A seventh aspect of the present invention provides a method for producing protein, comprising:

[0019] Constructing any of the above-mentioned recombinant expression vectors, wherein the recombinant expression vector comprises the above-mentioned DNA fragment and the coding sequence of the protein to be produced, and the DNA fragment is located at the 5' end of the coding sequence of the protein to be produced;

[0020] The recombinant expression vector is introduced into Marchantia polymorpha, so that the recombinant expression vector is expressed in Marchantia polymorpha to obtain the protein to be produced, and the protein to be produced is extracted from the Marchantia polymorpha.

[0021] In order to increase the expression level of exogenous proteins in Marchantia polymorpha, the present invention optimizes the xyloglucan hydrolase signal peptide coding sequence according to the Marchantia polymorpha codon. The optimized sequence is shown in SEQ ID NO: 1, which can guide the secretory expression of exogenous proteins in Marchantia polymorpha cells, significantly increase the expression level of exogenous proteins in Marchantia polymorpha, and is of great significance for the large-scale production of exogenous proteins, especially medicinal proteins, in Marchantia polymorpha. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The expression of FGF-2 protein in total protein was detected by western blot experiment, and four transgenic Marchantia polyphylla plants with higher expression of FGF-2 protein were screened out, which were numbered 4 / 7 / 16 / 20 respectively;

[0023] Figure 2 This is the western blot result of FGF-2 protein expression in Marchantia polymorpha using XTH signal peptide and GBA signal peptide;

[0024] Figure 3 This is a comparison of the grayscale values ​​of the western blot results of FGF-2 protein expression in Marchantia polymorpha using XTH signal peptide and GBA signal peptide. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] The experimental methods in the following examples where specific conditions are not specified are generally performed under conventional conditions, such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer.

[0027] The GoldenBraid kit used in the following examples, including PUPD2 vector, pDGB3-α1, pDGB3-α2, pDGB3-Ω1 vector, etc., can be purchased publicly on the market. For details on vector usage and connection methods, please refer to the GoldenBraid kit manual and product website https: / / gbcloning.upv.es.

[0028] The Agrobacterium used in the following examples is Agrobacterium tumefaciens, and the strain is GV3101, which can be purchased from the market.

[0029] In the following examples, FGF-2 protein is used as an example to illustrate the effect of XTH signal peptide on the expression level of exogenous proteins in Marchantia polymorpha.

[0030] Example 1: Expression of human FGF-2 protein in Marchantia polymorpha

[0031] 1. Construction of plant expression vector containing XTH-FGF-2 protein

[0032] 1.1. The amino acid sequence of the XTH signal peptide (shown in SEQ ID NO: 2) was fused to the N-terminus of the amino acid sequence of the human FGF-2 protein, and labeled as XTH-FGF-2; the entire amino acid sequence of XTH-FGF-2 was optimized according to the codons of Marchantia polymorpha, and the nucleotide sequence obtained after optimization was artificially synthesized by Sangon Biotech (Shanghai) Co., Ltd., and was ligated by the goldengate method while being digested, and was connected to the BsmbI double restriction site of the PUPD2 vector in the GoldenBraid kit by the BsmbI restriction endonuclease and T4 ligase;

[0033] 1.2. Use BsmbI restriction endonuclease and T4 ligase to connect the CaMV35S promoter from cauliflower mosaic virus into the BsmbI double restriction site of the PUPD2 vector in the GoldenBraid kit;

[0034] 1.3. Use BsmbI restriction endonuclease and T4 ligase to connect the CaMV35S terminator from cauliflower mosaic virus into the BsmbI double restriction site of the PUPD2 vector in the GoldenBraid kit;

[0035] 1.4. Use BsaI restriction endonuclease and T4 ligase to connect the XTH-FGF-2 nucleotide sequence, CaMV35S promoter and CaMV35S terminator into the BsaI double restriction site of the pDGB3-α1 vector in the GoldenBraid kit to construct an α1-XTH-FGF-2 expression vector;

[0036] 1.5. Connect the hygromycin HYG gene nucleotide sequence together with the CaMV35S promoter and CaMV35S terminator into the BsaI double restriction site of the pDGB3-α2 vector in the GoldenBraid kit to construct the α2-HYG expression vector;

[0037] 1.6. Use BsmbI restriction endonuclease and T4 ligase to connect the α1-XTH-FGF-2 vector and α2-HYG vector into the BsmbI double restriction site of the pDGB3-Ω1 vector in the GoldenBraid kit to construct the Ω1-XTH-FGF-2-HYG plant expression vector.

[0038] 2. Genetic transformation of Marchantia polymorpha with XTH-FGF-2 protein expression vector mediated by Agrobacterium tumefaciens to obtain transgenic Marchantia polymorpha plants

[0039] 2.1. Obtaining Agrobacterium tumefaciens containing XTH-FGF-2 protein expression vector

[0040] The Ω1-XTH-FGF-2-HYG plant expression vector was transferred into Agrobacterium tumefaciens GV3101 by freeze-thaw method and verified by PCR. The results showed that the plant expression vector containing XTH-FGF-2 protein gene had been successfully constructed into Agrobacterium tumefaciens strain.

[0041] 2.2. Agrobacterium tumefaciens-mediated transformation of Marchantia polymorpha with XTH-FGF-2

[0042] 2.2.1 Pre-culture of Marchantia polyphylla embryos

[0043] Liquid pre-culture: Pour about 20 mL of 1 / 2 B5 liquid culture medium into a conical flask, take the embryo of Marchantia polymorpha and place it in the liquid culture medium for static pre-culture, culture at 21°C and 16h / 8h (light / dark) light for 6 days, and it can be used for Marchantia polymorpha transformation.

[0044] 2.2.2 Co-cultivation of Agrobacterium and embryos

[0045] The embryos of Marchantia polyphylla were pre-cultured and added to the activated MES-MgCl2 suspension of the Agrobacterium tumefaciens engineered bacteria containing the XTH-FGF-2 protein expression vector, so that the embryos were fully in contact with the bacterial solution, and were allowed to stand for infection in a vacuum environment for 10 minutes. Excess bacterial solution was sucked off, and the embryos were washed with sterile water for 3 times. The washed embryos were evenly spread on 1 / 2B5 solid transformation medium and cultured in the dark at 21°C for 2 days. The embryos added with the MES-MgCl2 suspension of Agrobacterium tumefaciens without the target gene were used as controls.

[0046] 2.2.3. Screening of resistant regenerated plants

[0047] Wash the co-cultured embryos on 1 / 2B5 solid medium with sterile water several times to wash off the Agrobacterium propagated during dark culture until the washing liquid is clear. Then add 5mL of screening solution (50μg / mL hygromycin + 2000μg / mL cephalosporin), shake slightly to evenly distribute the screening solution in the culture dish, suck away 4mL of screening solution and leave 1mL, let it dry naturally, and place it at 21°C and 16h / 8h light conditions to continue culturing for 2-3 weeks. Move the well-growing resistant regenerated embryos to the screening medium (1 / 2B5 + 5μg / mL hygromycin + 100μg / mL cephalosporin) for culture, so as to obtain hygromycin-resistant regenerated Marchantia plants.

[0048] 2.3 PCR detection of transgenic Marchantia polyphylla plants

[0049] According to the gene sequence of XTH-FGF-2, forward detection primers (XTH-FGF-2-F: 5'-ATGGGATTCAACCGCGGATT-3') and reverse detection primers (XTH-FGF-2-R: 5'-TCAGGACTTAGCAGACATAG-3') were designed, and positive detection was performed on overexpression plants. The results showed that the specific PCR detection primers designed could amplify specific DNA fragments. However, no fragments were amplified when non-transformed Marchantia polymorpha genomic DNA was used as a template.

[0050] 3. Detect the expression of FGF-2 protein in transgenic Marchantia polyphylla using western blot

[0051] 3.1. Extraction of total protein from plant tissues

[0052] Take 0.1g of PCR-positive transgenic Marchantia leaves, grind them into powder in liquid nitrogen, add an equal volume of pre-cooled lysis buffer (PBS+0.1% Triton X100+cocktail+pefabloc), quickly vortex mix, put on ice for 20min, centrifuge at 12000rpm at 4℃ for 10min, and take the supernatant into a new 1.5mL centrifuge tube. Repeat the centrifugation several times, and take the supernatant, which is the total protein extract.

[0053] 3.2 SDS-PAGE gel electrophoresis

[0054] Take 60 μL of total protein extract and add it to 20 μL of 4× loading buffer, mix well, boil in 100℃ water bath for 10 min, centrifuge at 12000 rpm for 2 min. Take 20 μL of boiled protein extract and add it to the well of denatured protein precast gel (Yisheng Biotechnology Shanghai Co., Ltd.), keep constant voltage at 100V, and perform SDS-PAGE electrophoresis for 1h.

[0055] 3.3. Transfer (wet transfer)

[0056] Prepare two 3M filter papers (10cm×7cm) and a PVDF membrane (9cm×6cm), and place the PVDF membrane in methanol to wake up for 30s. In 1×Transfer buffee, clamp the film in the order of sponge-3M filter paper-protein glue-PVDF membrane-3M filter paper-sponge, put it in the transfer tank, and transfer the membrane for 50min at a constant current of 200mA.

[0057] 3.4. Closure

[0058] Soak the transferred membrane in 1×PBS for 5 minutes, discard the PBS, add blocking solution (5% skim milk powder + PBST) for 1 hour, and wash three times with 1×PBST (1L PBS + 1mL Tween20), each time for 5 minutes.

[0059] 3.5. Primary Antibody Incubation

[0060] Anti-FGF2 antibody (Abimat Pharmaceutical Technology Shanghai Co., Ltd.) was added to 4 mL 3% BSA solution (3% BSA + PBST) at a ratio of 1:2000, and the antibody solution was poured into the blocked and washed membrane and incubated at 4°C overnight.

[0061] 3.6. Secondary Antibody Incubation

[0062] The membrane was washed three times with 1×PBST for 10 min each time, rabbit secondary antibody was added to 5% skim milk powder at a ratio of 1:10,000, and incubated at room temperature for 2 h. The membrane was washed three times with 1×PBST for 10 min each time.

[0063] 3.7. Development

[0064] The washed membrane was placed in a black developing box, developer was added dropwise, and the membrane was developed for 1 min in the dark. The membrane was then developed and photographed using the Tanon 4600SF fully automatic chemiluminescence image analysis system.

[0065] The results are as follows Figure 1 As shown, it can be seen that the expression level of FGF-2 protein in transgenic Marchantia polymorpha was detected by western blot experiment. Compared with the wild type (WT), the xyloglucan hydrolase XTH signal peptide can significantly increase the expression level of FGF-2 protein in Marchantia polymorpha.

[0066] Example 2

[0067] The same method as in Example 1 was used to construct a recombinant expression vector, except that the signal peptide used was human β-glucocerebrosidase GBA signal peptide, whose amino acid sequence was MEFSSPSREECPKPLSRVSIMAGSLTGLLLLQAVSWASG, and whose nucleotide sequence was ATGGAGTTCTCCTCCCCTTCCCGCGAGGAGTGCCCTAAGCCTTTGTCCC GCGTGTCCATCATGGCCGGATCCTTGACCGGATTGTTGTTGTTGCAGGC CGTGTCCTGGGCCTCCGGA; and the recombinant expression vector was introduced into Marchantia polyphylla, and the expression level of FGF-2 protein was detected by the same method.

[0068] Test results such as Figure 2-3 As shown, it can be seen that compared with the GBA signal peptide, the XTH signal peptide used in Example 1 of the present invention can significantly increase the expression level of FGF-2 protein in Marchantia polymorpha.

[0069] In summary, the present invention provides a signal peptide coding sequence that can increase the expression amount of foreign protein in Marchantia polymorpha, laying a solid foundation for large-scale expression and production of foreign proteins, especially medicinal proteins, in Marchantia polymorpha using the signal peptide sequence.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A DNA fragment having a nucleotide sequence as shown in SEQ ID NO:

1.

2. A fusion protein, characterized in that: The fusion protein is formed by fusing a foreign protein with a signal peptide located at the N-terminus of the foreign protein, and the amino acid sequence of the signal peptide is shown in SEQ ID NO:

2.

3. A recombinant expression vector for increasing the expression level of exogenous protein in Marchantia polymorpha, characterized in that: It comprises the DNA fragment according to claim 1 and the coding sequence of the foreign protein, wherein the DNA fragment is located at the 5' end of the coding sequence of the foreign protein.

4. The recombinant expression vector according to claim 3, characterized in that Also included is the CaMV35S promoter and terminator derived from the cauliflower mosaic virus.

5. The recombinant expression vector according to claim 3, characterized in that: Also included are resistance selection genes.

6. A recombinant transformant comprising the recombinant expression vector according to any one of claims 3 to 5.

7. Use of the DNA fragment according to claim 1, the recombinant expression vector according to any one of claims 3 to 5, or the recombinant transformant according to claim 6 in increasing the expression level of foreign protein in Marchantia polymorpha.

8. A method for increasing the expression of exogenous protein in Marchantia polymorpha, characterized in that: The steps include: Constructing the recombinant expression vector according to any one of claims 3 to 5; The recombinant expression vector is introduced into Marchantia polymorpha, so that the recombinant expression vector is expressed in Marchantia polymorpha to obtain foreign protein, thereby increasing the expression amount of foreign protein in Marchantia polymorpha.

9. The method according to claim 8, characterized in that The exogenous protein is FGF-2 protein derived from human.

10. A method for producing a protein, characterized in that: include: Constructing the recombinant expression vector according to any one of claims 3 to 5, wherein the recombinant expression vector comprises the DNA fragment according to claim 1 and a coding sequence of a protein to be produced, and the DNA fragment is located at the 5' end of the coding sequence of the protein to be produced; The recombinant expression vector is introduced into Marchantia polymorpha, so that the recombinant expression vector is expressed in Marchantia polymorpha to obtain the protein to be produced, and the protein to be produced is extracted from the Marchantia polymorpha.

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