CN116162149A - Method for improving expression level of synthetic recombinant human serum albumin gene in plant - Google Patents
Method for improving expression level of synthetic recombinant human serum albumin gene in plant Download PDFInfo
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Abstract
The invention relates to the field of plant synthesis biology, in particular to a method for improving the expression level of a synthetic recombinant human serum albumin gene in a plant. The method comprises the following steps: the human serum albumin gene SlrHSA after the codon and the 3' UTR of the human serum albumin are connected together to construct the plant binary expression vector. The mature recombinant human serum albumin has an amino acid sequence shown as SEQ ID NO: 4. The sequence encoding the protein includes: a) The 3' UTR sequence of HSA is shown as SEQ ID NO. 2; b) The sequence of the SlrHSA is shown as SEQ ID NO. 3. The invention utilizes tomatoes as recombinant proteins to synthesize chassis plants, and provides an excellent cell system for recombinant protein expression; can obviously improve the expression quantity of the synthesized recombinant human serum albumin gene in plants, has mature industrial system, short growth cycle and high yield, and is important to improve the quality and the yield of recombinant protein.
Description
Technical Field
The invention relates to the field of plant synthesis biology, in particular to a method for improving the expression level of a synthetic recombinant human serum albumin gene in a plant.
Background
Human serum albumin is widely existing in blood, lymph and muscle of human body, and serum albumin is the protein with the highest content in blood plasma and the most important function. Mature human serum albumin is a single chain protein consisting of 585 amino acid residues and has a molecular weight of 67kDa. Albumin has effects of maintaining blood osmotic pressure, resisting shock, transporting and removing toxic substances, and promoting liver cell repair and regeneration.
Human serum albumin is a medical protein used on a large scale, and the worldwide market demand of the human serum albumin is about 600 tons each year, and the market value of the human serum albumin is about 30 hundred million dollars. At present, medical human serum albumin is mainly obtained through blood classification and filtration, and the main social problems include the following two points: 1. human plasma products are scarce in source and expensive in price. 2. Complications are easily caused, and the most dangerous is blood-borne viral infection, such as HIV and HCV infection, of which no effective treatment measures exist at present. The development of genetic engineering and synthetic biology provides a good solution to the problems of shortage of human serum albumin, carrying potential viruses and the like.
Plants have been studied for nearly thirty years as expression and production systems for pharmaceutical proteins. Compared with single-cell microorganisms, the multicellular plant system is rich in an endomembrane system and various organelles, glandular hairs on the surface of the plant are important sites for synthesizing and storing metabolites, and the complex space-time characteristic provides the most suitable environment for synthesizing different types of enzymes and metabolites, which is beneficial to maintaining the activity and yield of the protein; the complexity of multicellular plant systems also provides an excellent model system for our synthetic biology research, plants are rich in large amounts of metabolites, and can directly provide precursors for the synthesis of plant active molecules. Therefore, the synthesis of important humanized proteins and natural active small molecules by using plants becomes a necessary path for technological development.
Currently, plant expression systems have been considered as a promising alternative to animal cell culture for large-scale production of recombinant proteins, and methods for the production of proteins in transgenic plants can be found, for example: U.S. Pat. nos. 5750871, 5565347, 5464763, 5750871, 5565347, etc. Although plants are cheaper to grow and harvest in large quantities than prokaryotic and eukaryotic cells, the expression level of foreign genes in plant cells is generally lower.
CN107641155a discloses a method for expressing recombinant human serum albumin in plants, which is capable of high level expression in plants. The method also provides nucleic acid constructs, vectors, and host cells comprising the human serum albumin genes, and further provides methods for producing transgenic plants and recombinant human serum albumin using the transgenic plants. The method greatly improves the expression level of the recombinant human serum albumin in a plant reactor, thereby enabling the low-cost production of the recombinant human serum albumin by transgenic plants. However, this method did not increase the expression level of recombinant human serum albumin in the plant reactor.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a method for improving the expression level of a synthetic recombinant human serum albumin gene in plants, which increases the expression of the human serum albumin in tomatoes and provides a solution for solving the problems of limited sources and high price of the human serum albumin at present.
In order to achieve the purpose of the invention, the invention adopts the following technical scheme:
a mature recombinant human serum albumin, the amino acid sequence of which is shown in SEQ ID NO: 4.
Further, the invention also provides a gene sequence for encoding the recombinant human serum albumin, wherein the gene sequence comprises:
a) The 3' UTR sequence of HSA is shown as SEQ ID NO. 2;
b) The sequence of the SlrHSA is shown as SEQ ID NO. 3.
Biological "central law" refers to the transfer of genetic information stored in DNA to proteins via mRNA. Wherein the mRNA comprises a Coding sequence (CDS) and an untranslated region (untranslated regions, UTR). UTRs are divided into 5'UTR and 3' UTR, upstream of the start codon and downstream of the stop codon, respectively. In general, when heterologous expression is performed in plants, only the CDS sequence is ligated into the vector, and the transcription termination of the gene is performed by using the strong terminator of the binary vector, thereby omitting the regulation of mRNA by the non-coding region of the gene itself. According to the research, the invention discovers that the AU-rich 3' UTR region can regulate gene expression and protein translation by regulating and controlling the mRNA positioning and stability of HSA and the like.
The invention utilizes the regulation and control action of a gene non-coding region (3' UTR) on mRNA to increase the expression of human serum albumin in tomatoes, and provides a solution for solving the problems of limited source and high price of the human serum albumin at present.
Further, the SlrHSA sequence is obtained by codon optimization of mature HSA sequence in human body according to codon preference of plants.
In the invention, the SlrHSA sequence is artificially designed on the human serum albumin gene sequence according to tomato codon preference, the length of the SlrHSA sequence is 1758bp, the length of the SlrHSA sequence is the same as that of the HSA gene, and the SlrHSA sequence codes the same amino acid sequence.
Further, the HSA sequence is shown as SEQ ID NO. 1.
The invention also provides an expression vector, wherein the expression vector comprises the gene sequence.
Further, the expression vector is obtained by introducing the gene sequence into a plant binary expression vector pC 1300.
The invention also provides a method for improving the expression level of the synthesized recombinant human serum albumin in plants, wherein the method comprises the following steps: the human serum albumin gene SlrHSA after the codon and the 3' UTR of the human serum albumin are connected together to construct the plant binary expression vector.
Specifically, the method comprises the following steps:
1) Human serum albumin codon optimization;
2) Construction of the slrHSA gene overexpression vectors pC1300-slrHSA and pC1300-slrHSA-3 UTR;
3) plant genetic transformation of pC1300-SlrHSA and pC1300-SlrHSA-3 UTR;
4) And (5) identifying the expression level of the human serum albumin SlrHSA gene in the transgenic plant.
Further, step 1) is:
1.1 According to the codon preference of the plant, carrying out codon optimization on the mature HSA sequence in the human body to obtain an optimized SlrHSA sequence;
1.2 The 3' UTR sequence of human serum albumin and the optimized SlrHSA sequence are connected to pGEM-T Easy vector to synthesize the amino acid sequence of the mature human serum albumin.
Further, the plant is tomato.
As a preferred embodiment, the tomato is a small fruit tomato.
As a more preferred embodiment, the small fruit tomatoes are Micro-TOM.
In the invention, the step 2) is as follows: the PCR amplified human serum albumin gene product and the linearized vector are connected by homologous recombination mode by using a seamless cloning technology.
In the invention, the step 3) is as follows: agrobacterium transformation by target vector, preparation of infection liquid and plant transformation.
Compared with the prior art, the invention has the following advantages:
(1) The invention utilizes tomatoes as recombinant protein synthesis chassis plants, has deep molecular biology and genomics, has mature gene editing and genetic transformation systems, and can provide an excellent cell system for recombinant protein expression;
(2) The invention can obviously improve the expression quantity of the synthesized recombinant human serum albumin gene in the plant;
(3) The industrialized system of the invention is mature, has shorter growth period and high yield (up to 110 tons/hectare), which is important for improving the quality and yield of the recombinant protein.
Drawings
FIG. 1 is a plasmid map of pC1300-SlrHSA expression vector;
FIG. 2 is a plasmid map of pC1300-SlrHSA-3UTR expression vector;
FIG. 3 shows analysis of the expression level of the SlrHSA gene in positive seedlings of transformed tomato.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof.
The experimental methods in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 human serum albumin codon optimization
HSA mature in humans comprises 1758bp (inclusive of the stop codon), gc= 42.95% and the sequence as set forth in SEQ ID NO: 1.
The 3 'untranslated region (3' UTR) sequence (containing polyA tail) of human serum albumin is shown in SEQ ID NO: 2.
According to codon preference in tomatoes, the optimized SlrHSA sequence is shown as SEQ ID NO:3, gc=38.91%.
The optimized SlrHSA and 3' UTR sequences were sent to gene synthesis company and ligated into pGEM-T Easy vector, named pGEM-SlrHSA (excluding 3' UTR) and pGEM-SlrHSA-3UTR (including 3' UTR), respectively. Mature human serum albumin comprises 585 amino acids, the sequence of which is shown in SEQ ID NO: 4.
EXAMPLE 2 construction of SlrHSA Gene overexpression vectors pC1300-SlrHSA and pC1300-SlrHSA-3UTR
The plant binary expression vector pC1300 vector is preserved by the applicant, bacterial resistance is Kana resistance (Kan+), plant resistance is hygromycin resistance (Hyg+), and is used for over-expression research of plant genes.
1. Primer design
The invention uses the seamless cloning technology to clone genes, the fragments are connected into pC1300, and the upstream and downstream enzyme cutting sites are respectively designed into HindIII and PstI.
| Primer name | Sequence(s) |
| SlrHSA-FP | TCTCTCTCTCAAGCTTGATGCACATAAATCAGAGGTTGCA |
| SlrHSA-RP | CGGGTCATGAGCTCCTGCAGTCACAAACCAAGAGCAGCT |
| SlrHSA-3UTR-RP | CGGGTCATGAGCTCCTGCAGGACAGGGTGTTGGCTTTACAC |
2. PCR amplification of target Gene
In order to ensure the accuracy of the products in the PCR process, the high-fidelity DNA polymerase PrimeSTAR HS DNA Polymerase is utilized for amplification, and the system and the reaction procedure are as follows:
| component (A) | Additive amount | Concentration of |
| 5×PrimeSTAR Buffer(Mg2+Plus) | 10μl | 1× |
| dNTP Mixture(2.5mM each) | 4μl | 200μM each |
| Upstream primer | 10-15pmol | 0.2-0.3μM |
| Downstream primer | 10-15pmol | 0.2-0.3μM |
| pGEM-SlrHSA/pGEM-SlrHSA-3UTR | 0.5μl | |
| PrimeSTAR HS DNA Polymerase(2.5U/μl) | 1μl | 2.5U/50μl |
| Sterilizing water | up to 50μl |
PCR reaction procedure: pre-denaturation at 98℃for 3min; denaturation at 98℃for 10sec, annealing at 58℃for 15sec, elongation at 72℃for 45sec for a total of 38 cycles; extending at 72℃for 5min.
3. Cloning of PCR products into vectors
(1) The vector is linearized, and according to the NEB double enzyme digestion method, the system is as follows:
enzyme cutting conditions: and (3) performing enzyme digestion for 30min in a water bath at 37 ℃.
(2) Electrophoresis
The PCR products and the cleavage products were electrophoresed in a 1.2% agarose gel and the DNA fragment of interest was excised under a gel imager in a 1.5ml EP tube.
(3) PCR product and enzyme digestion product recovery
The PCR products were performed according to the instructions of the Tiangen ordinary agarose gel DNA recovery kit (DP 209) and the concentration of the recovered products was determined using the NanoDrop 1000.
(4) Seamless cloning connection
Using Vazyme
II One Step Cloning Kit the PCR product was ligated into linearized pC1300 vector. The reaction system and procedure were as follows:
the optimal cloning vector used in the ClonExpII recombination reaction system was 0.03pmol, and the optimal insert was 0.06pmol (vector to insert molar ratio 1:2). The mass of DNA corresponding to these moles can be roughly calculated from the following formula:
optimal cloning vector usage= [0.02×cloning vector base pair number ] ng (0.03 pmol)
Optimal amount of insert used= [0.04×base pair number of insert ] ng (0.06 pmol)
| Component (A) | Volume/10. Mu.l |
| Linking into linearized pC1300 | X |
| Recovered PCR product | Y |
| 5×CE II Buffer | 2μl |
| Exnase II | 1μl |
The amount of vector and the amount of insert were calculated according to the formula X/Y.
The reaction procedure: reacting for 30min at 37 ℃; cooling to 4 ℃ or immediately cooling on ice.
(5) Ligation product transformation E.coli competence
In order to ensure success rate, the method of the invention immediately carries out the transformation of the escherichia coli after the connection is completed, and is a heat shock method. The procedure was as follows:
a) Melting 100 μl of Escherichia coli DH5 alpha competent cells on ice, adding 10 μl of the ligation product, and mixing with gun head;
b) After standing on ice for 30min, heat shock was conducted at 42℃for 90sec. After heat shock, placing for 2-3 min on ice, adding 800 mu l of LB liquid culture medium without resistance, and culturing for 45min at 150rpm in a 37 ℃ incubator;
c) Centrifuge at 4000rpm for 1min, leave 100. Mu.l of resuscitated culture plated (Kan+);
d) After the surface of the flat plate is dried, the flat plate is inversely cultured for 12 to 16 hours at 37 ℃.
(6) Positive clone PCR identification
After picking up the single clone in 5ml of liquid LB in a super clean bench and culturing for 16 hours at 200rpm, the bacterial liquid PCR identifies the positive clone. Mu.l of positive clone culture solution was used for glycerol sterilization, and the rest was used for plasmid extraction. And sending to a sequencing company for final sequencing identification.
(7) Positive clone restriction enzyme identification
| Component (A) | Volume of |
| Plasmid extracted from positive clone | 3μl |
| 10XrCutSmart Buffer | 2μl(1X) |
| HindIII-HF | 0.5μl(20units) |
| PstI-HF | 0.5μl(20units) |
| Nuclease-free Water | to 20μl |
Enzyme cutting conditions: and (5) carrying out enzyme digestion for 10min in a water bath at 37 ℃.
The digested products were electrophoresed in 1.2% agarose gel, and clones conforming to the expected size were picked and numbered against the DNA marker.
Clones positive in both PCR and cleavage were selected and sent to sequencing company for sequencing.
Example 3 tomato genetic transformation of pC1300-SlrHSA and pC1300-SlrHSA-3UTR
1. Preparation of Agrobacterium chemically competent cells
(1) Taking out Agrobacterium strain (LBA 4404) stored at-80deg.C, dipping a small amount of bacterial liquid with a burned sterilized inoculating loop, streaking on YEB solid culture medium plate containing 20mg/L rifampicin (Rif), and culturing at 28deg.C for about 18 hr;
(2) Picking single colony in 5mL YEB liquid culture medium containing 20mg/L Rif, and shaking and culturing at 28 ℃ and 250rpm for 16-24 h;
(3) Inoculating the activated bacterial liquid into 50mL of YEB liquid culture medium containing 20mg/L Rif according to the volume ratio of 1:100, and culturing at 28 ℃ at 250rpm until the OD600 = 0.5;
(4) Transferring the bacterial liquid after the culture to a precooled 50mL centrifuge tube, centrifuging at 4 ℃ and 5000rpm for 10min, and discarding the supernatant;
(5) 10ml of pre-chilled 0.1M CaCl was added 2 The solution was gently suspended and placed on ice for 20min. Centrifuging at 5000rpm at 4deg.C for 5min, and removing supernatant;
(6) 4ml of pre-chilled 0.1M CaCl containing 15% glycerol was added 2 Solution, lightly suspending;
(7) The Agrobacterium suspension was dispensed into sterile Eppendorf tubes, and 100. Mu.l each tube was frozen in a-80℃freezer.
2. Plasmid transformed Agrobacterium competent cells
(1) The agrobacteria competent cells are taken out from the temperature of minus 80 ℃ and placed on ice, after the agrobacteria competent cells are melted, 2 mu L of prepared pC1300-SlrHSA expression vector plasmid (the map is shown in figure 1) and pC1300-SlrHSA-3UTR expression vector plasmid (the map is shown in figure 2) are added, and the mixture is sucked and beaten by a pipetting gun and evenly mixed;
(2) Ice bath for 30min, and placing in liquid nitrogen for 5min;
(3) Water bath at 37 ℃ for 5min;
(4) Ice-bath for 5min, adding 800ml LB liquid culture medium;
(5) After 28 ℃ and 200rpm for 3hr, the solution is coated on an LB plate containing 50 mug/ml Kanamycin;
(6) The culture was carried out at 28℃until single colonies were formed.
3. Agrobacterium-mediated tomato genetic transformation
(1) Culturing aseptic seedlings: calculated on 40 seeds per petri dish. Shaking with newly prepared 10% sodium hypochlorite solution for 8min, cleaning sterilized seeds with 500mL sterile water for 5 times, placing on sterile filter paper to suck water, culturing the seeds in 1/2MS culture medium at 26deg.C under light/dark for 16/8 hr for 5-6d;
(2) Explant preparation: when cotyledon stretches and true leaves do not grow, cutting off the tip and end parts of the cotyledon to form square or rectangular explants (when the cotyledon is too long, cutting off in the middle), putting the cut cotyledon into MSO (acetosyringone-containing AS) liquid, soaking for 0.5h, transferring the cotyledon onto a D1 culture medium, facing upwards, preventing the stabbing of the cotyledon and the cut part, and culturing for 2D in the condition that the illumination/darkness is 16/8h at 26 ℃;
(3) Preparing an immersion dye liquor: transferring the constructed vector into LBA4404 agrobacterium strain, selecting positive monoclonal, culturing overnight with 3ml LB liquid medium (50 mg/L kan and 50mg/L Rif), inoculating 1:50 strain with large shake (30 ml liquid in general) on the day of infection, shaking at 28deg.C, 220rpm to make OD 600=0.6-0.8;
(4) Explant infestation: centrifuging the prepared agrobacterium for 10min at 20 ℃ and 4000rpm, discarding the supernatant, fully suspending the thalli by using MSO solution, adjusting OD600 = 0.4-0.5, shake culturing the explant and the bacterial liquid for 10min, sucking the excessive bacterial liquid by filter paper, then placing the filter paper back into a D1 culture medium, placing sterile filter paper on the culture medium, and performing dark culture at 26 ℃ with the back face upwards for 2D;
(5) Medium replacement: transferring the dark cultured explants from the D1 medium to the screening medium, culturing for 14D at 28 ℃ in 16/8h of light/dark with the right side facing upwards;
(6) Changing a new screening culture medium every two weeks;
(7) Transferring the regenerated buds growing to 2cm from the screening medium to a rooting medium for growth until rooting (about 2 weeks);
(8) And when the rooting seedlings grow to the bottle mouth, taking the leaves in the super clean bench for identification.
4. Identification of positive seedlings
(1) Extraction of plant genome DNA (CTAB method)
a) Placing plant tissues in a 2mL centrifuge tube, adding 2 sterilizing steel balls, freezing with liquid nitrogen, and grinding with a ball mill;
b) Adding 500 μl of CTAB extraction buffer, and mixing in water bath at 65deg.C for 20 min;
c) 800 μl phenol was added: chloroform: isopropanol (25:24:1), mix upside down, centrifuge at 12000rpm for 10min, transfer the upper aqueous phase to a new 2mL centrifuge tube;
d) Adding 0.6 times volume of isopropanol, and precipitating at-20deg.C for more than 30min;
e) Centrifuging at 12000rpm for 10min, discarding supernatant, and washing twice with 75% ethanol;
f) Drying residual alcohol at room temperature, dissolving in 50 μl distilled water, and preserving at-20deg.C.
(2) Identification of positive seedlings by PCR method
In order to save the design cost of the primer and the stability of PCR conditions, the invention adopts a marker gene on a carrier for identification (hygromycin). Wild-type DNA and plasmid were used as negative and positive controls, respectively. The reaction system is as follows:
| component (A) | Volume of |
| PCR mix | 10μL |
| Upstream primer | 0.5μL |
| Upstream primer | 0.5μL |
| DNA template | 1μL |
| Sterilizing water | Up to 20μL |
PCR reaction procedure: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30sec, annealing at 60℃for 30sec, elongation at 72℃for 30sec for 28 cycles total; extending at 72℃for 5min.
Example 4 identification of the expression level of human serum albumin SlrHSA Gene in transgenic tomato
Transgenic tomatoes which are transferred into overexpression vectors pC1300-SlrHSA and pC1300-SlrHSA-3UTR carrying natural genes are respectively prepared by the method of the previous example, and the protein expression quantity is identified by adopting a qRT-PCR method.
(1) Total RNA extraction and reverse transcription reaction of plants
a) Tissue: placing 50-100mg of tissue (fresh or tissue preserved in liquid nitrogen at-70deg.C) into 2ml centrifuge tube, adding steel balls, and oscillating on tissue grinder for 1min;
b) Adding 1ml Trizol, homogenizing, mixing, and standing for 5min;
c) Adding 0.2ml chloroform, oscillating for 15sec, and standing for 5min;
d) Centrifuging at 4deg.C, 12000g×15min, and collecting supernatant;
e) Adding 0.5ml of isopropanol, gently mixing the liquid in the tube, and standing at room temperature for 10min;
f) Centrifuging at 4deg.C for 12000g×10min, and discarding supernatant;
g) 1ml of 75% ethanol was added and the precipitate was gently washed. 4 ℃,7500g×5min, discarding the supernatant;
h) Air drying, adding proper amount of DEPC H 2 O dissolution (dissolution promotion at 65 ℃ for 10-15 min);
i) According to PrimeScript TM RT reagent Kit with gDNA Eraser (Perfect Real Time) to obtain cDNA;
(2) Identification of Gene expression level by fluorescent quantitation PCR (SYBR Green)
The reaction solution is prepared under weak light, and the reaction system is as follows:
| cDNA template (10X) | 1μL |
| Upstream primer | 0.8μL |
| Downstream primer | 0.8μL |
| 2 X SYBR qPCR mix | 10μL |
| DEPC water | Up to 20μL |
The reaction procedure was as follows:
amplification procedure: 95 ℃ for 2min; denaturation at 95℃for 30sec, annealing at 55℃for 30sec, elongation at 72℃for 30sec for 40 cycles.
Melting procedure:
after the amplification cycle is completed, the temperature is reduced to 60 ℃, and then the temperature is increased to 95 ℃ to denature the DNA product.
The results are shown in FIG. 3. Wherein in FIG. 3, the numbers of the positive plants of the transformed tomatoes are SlrHSA-4, slrHSA-6, slrHSA-3UTR-8 and SlrHSA-3 UTR-9. The expression levels of SlrHSA-4, slrHSA-3UTR-8 and SlrHSA-3UTR-9 were 1.08, 2.44 and 1.98, respectively, with the expression level of SlrHSA in SlrHSA-6 as a reference (the expression level was designated as 1.0). As can be seen, the expression level was significantly improved by adding 3UTR, compared with that without adding 3 UTR.
Claims (10)
1. A mature recombinant human serum albumin, the amino acid sequence of which is shown in SEQ ID NO: 4.
2. A gene sequence encoding the recombinant human serum albumin of claim 1, wherein the gene sequence comprises:
a) The 3' UTR sequence of HSA is shown as SEQ ID NO. 2;
b) The sequence of the SlrHSA is shown as SEQ ID NO. 3.
3. The gene sequence according to claim 2, wherein the slhsa sequence is obtained by codon optimization of mature HSA sequence in human body according to codon preference of the recipient plant.
4. The gene sequence of claim 3, wherein the HSA sequence is set forth in SEQ ID NO. 1.
5. An expression vector comprising the gene sequence of any one of claims 2-4.
6. The expression vector according to claim 5, wherein the expression vector is obtained by ligating the gene sequence according to any one of claims 2 to 4 into a plant binary expression vector pC 1300.
7. A method for improving the expression level of synthetic recombinant human serum albumin in plants, which is characterized by comprising the following steps: the human serum albumin gene SlrHSA after the codon and the 3' UTR of the human serum albumin are connected together to construct the plant binary expression vector.
8. The method according to claim 7, characterized in that the method comprises the steps of:
1) Human serum albumin codon optimization;
2) Construction of the slrHSA gene overexpression vectors pC1300-slrHSA and pC1300-slrHSA-3 UTR;
3) plant genetic transformation of pC1300-SlrHSA and pC1300-SlrHSA-3 UTR;
4) And (5) identifying the expression level of the human serum albumin SlrHSA gene in the transgenic plant.
9. The method according to claim 8, wherein step 1) is:
1.1 According to the preference of plant codons, carrying out codon optimization on a mature HSA sequence in a human body to obtain an optimized SlrHSA sequence;
1.2 3' UTR sequence of human serum albumin and optimized slrHSA sequence were linked to pGEM-TEasy vector by gene synthesis.
10. The method of claim 9, wherein the plant is tomato.
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| CN1896239A (en) * | 2005-07-13 | 2007-01-17 | 杨代常 | Production of recombinant human serum albumin with rice-embryo milk cell as biological reactor |
| CN107531771A (en) * | 2015-04-29 | 2018-01-02 | 意大利梅迪奥兰制药公司 | Soluble chimeric Interleukin 10 receptor and its therapeutical uses |
| CN107641155A (en) * | 2016-07-20 | 2018-01-30 | 北京泰柏生物科技有限责任公司 | A kind of method of the recombinant Human Serum Albumin Expression in plant |
| CN112118859A (en) * | 2018-05-16 | 2020-12-22 | 杰特有限公司 | Type 1 soluble complement receptor variants and uses thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1896239A (en) * | 2005-07-13 | 2007-01-17 | 杨代常 | Production of recombinant human serum albumin with rice-embryo milk cell as biological reactor |
| CN107531771A (en) * | 2015-04-29 | 2018-01-02 | 意大利梅迪奥兰制药公司 | Soluble chimeric Interleukin 10 receptor and its therapeutical uses |
| CN107641155A (en) * | 2016-07-20 | 2018-01-30 | 北京泰柏生物科技有限责任公司 | A kind of method of the recombinant Human Serum Albumin Expression in plant |
| CN112118859A (en) * | 2018-05-16 | 2020-12-22 | 杰特有限公司 | Type 1 soluble complement receptor variants and uses thereof |
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