WO2019148939A1 - Use of plant as host in expression of adalimumab antibody - Google Patents

Abstract

The present invention relates to the field of biotechnology, and in particular to use of a plant as a host in the expression of an Adalimumab antibody. In the present invention, a plant such as lettuce is used as an effective expression platform for the production of a recombinant protein, and Adalimumab monoclonal antibody (Adalimuab, Humira) is expressed by means of a simple and efficient Agrobacterium-mediated vacuum permeation method. It is determined that in the expression system, a plant exogenous protein can be collected after four days of Agrobacterium infection. Successful expression of a recombinant Adalimumab antibody is determined by using a SDS-PAGE method. The tumor necrosis factor (TNF) neutralization experiment demonstrates that the Adalimumab antibody produced by lettuce has biological activities.

Description

Application of plants as hosts in the expression of adalim antibodies

The present application claims priority to Chinese Patent Application No. 201810089048.2, entitled "Application of Plant as Host in Expression of Adamu Antibody", issued on January 30, 2018, the entire contents of which are incorporated by reference. In this application.

Technical field

The invention relates to the field of biotechnology, and in particular to the use of a plant as a host for expressing an adalimide antibody.

Background technique

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by symmetry, multiple joints, and small joints. It is known as the "undead cancer". The immune system of the sick person destroys healthy joints and triggers joints. Pain, swelling, stiffness, fatigue and weakness, late rigidity and deformity, severe impairment of function, and ultimately loss of joint function. Ankylosing spondylitis (AS) is a disease characterized by inflammation of the ankle joint and spinal attachment point. Strong association with HLA-B27. Certain microorganisms, such as Klebsiella, have a common antigen with the susceptible tissue and can trigger an abnormal immune response. Ankylosing spondylitis is a chronic inflammatory disease characterized by large joints of the extremities, as well as fibrosis and ossification of the connective tissue near the annulus fibrosus and its ankylosis.

Rheumatoid arthritis and ankylosing spondylitis are diseases with a high rate of disability. At present, adalimumab (Adalimuab, Humira, Xiomei) has a significant effect on these two types of inflammation. Adalimumab is a fully humanized monoclonal antibody and a self-injecting biotherapeutic drug. It is the world's first approved tumor developed by Cambridge Antibody Technology (CAT) and Abbott USA. Necrotic factor alpha (TNF alpha) a fully human monoclonal antibody. In 2003, the United States approved adalimumab for clinical treatment of rheumatoid arthritis and ankylosing spondylitis. Adalimumab slows the progression of joint damage in patients (X-ray display) and can improve body function. After a period of application, the patient's physical function and health-related quality of life are significantly improved.

At this stage, adalimumab is mainly produced using animal cells. However, animal cell culture requires expensive culture fluid, strict plant conditions, complicated operation, time period of at least two weeks, and low animal cell production capacity and high production cost. Sometimes the virus carried by animal cells can infect humans with low safety. Therefore, it is of great practical significance to provide an expression method of adalim antibody.

Summary of the invention

The invention provides the use of a plant as a host for the expression of an adalimide antibody. The present invention expresses adalim antibodies by using plants, especially lettuce, as an efficient platform technology for recombinant protein production. And the active isolation of the active foreign protein under mild conditions proves that the plant, especially the lettuce expression platform, can be successfully used to produce the adalima antibody protein. Short time (4d), simple purification and convenient production. Eliminate genetic pollution and eliminate potential pests and diseases that infect humans. Greatly reduce production costs and improve product safety.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides the use of a plant as a host for the expression of an adalimide antibody. Preferably, the antibody is a monoclonal antibody. The plant is selected from the group consisting of lettuce, cabbage, corn, soybean, tobacco or wheat; the organ of the plant is selected from the group consisting of a seed, a leaf, a rhizome or a whole plant.

The invention also provides an expression vector comprising a heavy chain sequence or a light chain sequence of adalima and a vector.

In some specific embodiments of the invention, the heavy chain sequence or the light chain sequence of the adalim is an optimized adamu obtained by optimizing the codons of the adalim heavy chain and the adalo light chain to a plant-preferred codon. Heavy chain sequence or optimized light chain sequence of adalim.

In some specific embodiments of the invention, the optimized heavy chain sequence of adalim is as shown in SEQ ID No. 1; the nucleotide sequence of the optimized heavy chain of adalim is set forth in SEQ ID No. 2. Show

The optimized light chain sequence of adalim is shown in SEQ ID No. 3; the nucleotide sequence of the optimized light chain of adalim is shown in SEQ ID No. 4.

In some embodiments of the invention, the vector is a binary plant vector.

In some embodiments of the invention, the method of constructing the expression vector comprises the steps of:

Step 1: Optimize the codons of the adalim heavy chain and the adalo light chain to the plant-preferred codons, respectively:

i. Optimized heavy chain sequence of adalim;

Ii. Optimized light chain sequence of adalim;

Step 2: adding a Xbal restriction enzyme site to the 5' end of the optimized adamus heavy chain sequence, and respectively adding a Sac I site at the 3' end;

Adding a Xbal restriction site at the 5' end of the optimized adamus light chain sequence and a Sac I site at the 3' end;

The cloned vector was cloned into the pUC57 vector, and the pAda-H and pAda-L cloning vectors were obtained respectively.

Step 3: The gene fragment was obtained from the cloning vector obtained in the step 2 by Xbal/Sacl, and cloned into the binary plant vector pCam35S to obtain the expression vector p35S-Ada-H, p35S-Ada-L, respectively.

Specifically, in order to provide high-efficiency expression of foreign aid proteins in plants, the present invention utilizes anti-translation software for amino acid sequences of human adamu heavy chain and light chain (https://www.drugbank.ca/drugs/DB00051) (https:/ /www.idtdna.com/CodonOpt) The nucleotide sequence was obtained and its codon was optimized to a plant-preferred codon synthesized by Kingsray (Nanjing, China). Xbal restriction sites were added to the 5' end of the optimized adalim heavy chain sequence, respectively, and the Sacl site was added to the 3' end. Xbal restriction sites were added to the 5' end of the adalim light chain sequence, respectively, and a SacI site was added to the 3' end. The cloned vector was cloned into the pUC57 vector (Fig. 1), and pAda-H and pAda-L cloning vectors were obtained, respectively. The gene fragment was isolated from the cloning vector by XbaI/Sacl and cloned into the binary plant vector pCam35S to produce the plant expression vector p35S-Ada-H, p35S-Ada-L, respectively (Fig. 2).

The invention also provides the use of the expression vector for expressing an adalimide antibody.

In addition, the present invention also provides a method for expressing an adalima antibody as a host, and the invention provides a common expression vector for transformation into Agrobacterium, and extracts and separates proteins by Agrobacterium-mediated vacuum infiltration into plant tissues. Obtained adalim antibody. The plant is selected from the group consisting of lettuce, cabbage, corn, soybean, tobacco or wheat; the organ of the plant is selected from the group consisting of a seed, a leaf, a rhizome or a whole plant.

Specifically, two plant expression vectors p35S-Ada-H, p35S-Ada-L were transformed into Agrobacterium tumefaciens GV3101 by electroporation with a Multiporator (Eppendorf, Hamburg, Germany), respectively. The resulting strain was spread evenly on selective LB plates containing kanamycin antibiotic (50 mg/L). After incubating for 2 days at 28 ° C in the dark, single colonies were picked and inoculated into 0.5 L YEB (yeast extract broth, 5 g/L sucrose, 5 g/L tryptone, 6 g/L yeast extract, 0.24 g/L MgSO ₄ , pH 7.2) and supplemented with antibiotic liquid medium (50 mg/L kanamycin). The inoculated culture was incubated at 25-28 ° C for 72 h in a shaker (220 rpm). The OD600 value was measured by adding YEB medium and adjusted to 3.5 to 4.5. The culture broth was then collected and centrifuged (4500 rpm) for 10 min. Agrobacterium cells were resuspended in osmotic medium (10 mM MES, 10 mM MgSO4) to an OD600 of 0.5.

The prepared Agrobacterium containing p35S-Ada-H and p35S-Ada-L were mixed to an O.D.600 of 0.5; The culture suspension was placed in a 2 L beaker and placed in a desiccator. The lettuce preserved in the laboratory was inverted (core up) and gently rotated into the bacterial suspension to seal the dryer. A vacuum pump (Welch Vacuum, Niles, IL, USA) was opened to evacuate and the permeate was visible in the leaf tissue. Maintain pressure for 30 to 60 seconds. The system is quickly opened to relieve pressure and allow permeate to penetrate into the space within the tissue. This process is repeated 2 to 3 times until it is clearly visible that the permeate diffuses significantly in the lettuce tissue. The lettuce tissue was then gently removed from the permeate and rinsed three times with distilled water and then transferred to a plastic film covered container. The treated samples were kept in the dark for 4 days.

In some embodiments of the invention, the Agrobacterium-mediated vacuum infiltration comprises the steps of:

Step 1: Vacuuming 25~45s;

Step 2: maintain a vacuum (-95kPa) pressure of 30 ~ 60s;

Step 3: releasing the pressure so that the permeate penetrates into the plant tissue;

Repeat the above steps 2 to 3 times, and protect from light for 4 days.

In some embodiments of the invention, the Agrobacterium is specifically Agrobacterium tumefaciens GV3101.

The pAda-H, pAda-L gene fragment was cloned according to the present invention, and two binary plant expression vectors p35S-Ada-H, p35S-Ada-L (Fig. 2) were constructed, and specific constraints were used after completion of the construct. Enzymatic digestion confirmed that the gene fragment was intact. After infiltration, most of the lettuce tissue was submerged during the vacuum infiltration process, except for the strong midrib area, which showed a yellowish brown area after 4 days of vacuum infiltration.

The protein is extracted and separated by: stirring the lettuce sample vacuum-infiltrated with Agrobacterium with a stirrer, and using a mixing buffer (100 mM KPi, pH 7.8; 5 mM EDTA; 10 mM β-mercaptoethanol) in a volume ratio of 1:1. High-speed homogenization for 1 to 2 minutes. The homogenate was adjusted to pH 8.0, filtered through gauze, and the filtrate was centrifuged at 10,000 g for 15 min at 4 ° C to remove cell debris. The supernatant was collected, mixed with ammonium sulfate (50%) and incubated for 60 min on ice. It was again separated by a centrifuge (10,000 g) at 4 ° C for 15 min. The resulting supernatant was subjected to a second round of ammonium sulfate (70%) precipitation, suspended on ice for 60 min, and again centrifuged at 10,000 g for 15 min at 4 °C. Then, the supernatant was discarded, and the treated sample precipitated protein was dissolved in 5 mL of a buffer (20 mM KPi, pH 7.8; 2 mM EDTA; 10 m Mβ-mercaptoethanol) and stored at 4 °C.

SDS-PAGE gel electrophoresis is specifically: collecting purified protein extracted from Agrobacterium vacuum infiltration lettuce, taking sample (5 μL) heat denaturation (95 ° C) loading buffer (Biorad, Hercules, CA, USA) at 4-12%

Plus SDS-denaturing gel (ThermoFisher Scientific, Waltham, MA, USA) was run for electrophoresis. Also, the affinity of the antibody is detected in non-denaturing gel electrophoresis. The gel was then photographed again after staining with Coomassie Blue G250 (Biorad).

Downstream processing of plant-derived recombinant proteins is often difficult and expensive because cellulose cell walls are difficult to lyse as well as secondary plant metabolites. We use a blender to stir the homogenate, which greatly saves the homogenization cost and process. Recombinant adalim antibodies were separated by denaturing gel SDS-PAGE. The estimated molecular weights observed in the lanes were approximately 24 kDa and 48 kDa, respectively (Fig. 3A), consistent with the protein size of the light heavy chain of the adalim antibody. A band of approximately 150 kDa (Fig. 3B) was observed in non-denaturing gel electrophoresis, demonstrating successful binding of the lettuce recombinant light heavy chain to the antibody structure, consistent with the molecular weight of the adalimide antibody protein. The protein content of the purified sample was determined to be about 0.94 mg/g based on the Bradford assay and the densitometric control group.

Tumor necrosis factor alpha (TNFα) is a naturally occurring cytokine involved in normal inflammation and immune response. Increased levels of TNF are found in the synovium of rheumatoid arthritis, including juvenile idiopathic arthritis, psoriatic arthritis, and ankylosing spondylitis. Increased levels of TNF were also found in psoriasis (Ps) plaques. Adalimumab specifically binds to TNF-α and blocks its interaction with p55 and p75 cell surface receptors. ELISA analysis of adalimumab Fab fragments can bind to TNFα. TNFα was used as a substrate-coated plate, and the HRP-labeled goat anti-human IgK-specific anti-light chain IgG specifically binds to the Fab fragment, indicating that the Fab fragment binds well to TNFα (Fig. 4). These results indicate that exogenous adalim antibodies transiently expressed by the lettuce system are biologically active and can neutralize TNFα. The results indicate that plants, especially lettuce, are a suitable bioreactor for the production of adalim antibodies.

The present invention utilizes lettuce to transiently express adalimic antibodies, and can produce high levels of protein in a short period of time (4d). Lettuce is a higher plant that can undergo a post-translational modification process, ie the expressed protein is automatically active. Moreover, this approach minimizes biosafety issues because processed lettuce tissue is typically developed in fully enclosed facilities or containers without biofouling problems. Lettuce basically contains no plant toxic substances, and its own fiber is less, which is conducive to downstream protein purification. The production of adalim monoclonal antibodies using the lettuce system can greatly shorten the production cycle and production costs.

DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art description will be briefly described below.

Figure 1 shows a schematic representation of the cloning vector pUC57;

Figure 2 shows the construction process of the adamus plant binary expression vector p35S-Ada-H (heavy chain) and p35S-Ada-L (light chain); using restriction endonuclease (Xbal/SacI) double digestion, from Figure 1 The cloning vector was ligated into the adalo H heavy chain, and ligated into the Xbal/SacI site of pCam35S to generate the plant binary expression vector p35S-Ada-H; using restriction endonuclease (Xbal/SacI) double digestion, 1 The cloning vector was ligated into the light chain of the adalim antibody, and the heavy chain was ligated into the Xbal/SacI site of pCam35S to generate the plant binary expression vector p35S-Ada-L, p35S-Ada-H;

LB and RB: the left and right borders of the Ti plasmid; 35S, the CaMV 35S promoter with the tobacco mosaic virus (TMV) 5'UTR; NPT II, the expression of the nptII gene for kanamycin resistance; Nos 3', Terminator

Figure 3 (A) shows the results of SDS-PAGE gel electrophoresis; Lane 1: adamus recombinant antibody expressed in lettuce; Lane 2: commercial adamus recombinant antibody; Figure 3 (B) shows non-denaturing gel electrophoresis results; Lane 3: Anaheim recombinant antibody expressed in lettuce; Lane 4: commercial adamus recombinant antibody;

Figure 4 shows that the experimental study by ELISA showed that the purified adalim antibody can neutralize tumor necrosis factor alpha (TNFα) and has significant biological activity.

Detailed ways

The invention discloses the application of a plant as a host in expressing an adalimic antibody, and those skilled in the art can learn from the contents of the present article and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention. The technique of the present invention is applied.

The invention finds through experiments that the plant system, especially the lettuce system, is a more economical and efficient expression platform, and is a rapid method for transient expression of recombinant protein. The vacuum Agrobacterium infiltration method described in the present invention is simple, rapid, and can increase recombinant protein production. Lettuce can increase protein production by withstanding vacuum pressure and allow for a more complete penetration of each leaf. Since lettuce is easy to grow and commercially mass-produced, it is easier to obtain and cheaper than other transiently expressed plants, such as tobacco, and the cost can be significantly reduced since no complicated special production equipment is required. In summary, the present invention can utilize the lettuce system to mass produce adalim monoclonal antibodies in a short period of time.

The materials and reagents used in the application of the plant provided by the present invention as a host in the expression of the adalima antibody are commercially available.

The present invention is further illustrated below in conjunction with the embodiments:

Example 1 Construction of Plant Transient Expression Vector

In order to provide high-efficiency expression of foreign aid proteins in plants, the amino acid sequences of human adamu antibody heavy chain, light chain, (https://www.drugbank.ca/drugs/DB00051) were translated using anti-translation software (https://www.www.www.drugbank.ca/drugs/DB00051). The nucleotide sequence was obtained from idtdna.com/CodonOpt) and its codon was optimized to a plant-preferred codon synthesized by Kingsray (Nanjing, China). Xbal restriction sites were added to the 5' end of the optimized adalim heavy chain sequence, and the SacI site was added to the 3' end. Xbal restriction sites were added to the 5' end of the adamu light chain sequence, and Sacl sites were added to the 3' end. It was cloned into the pUC57 vector by Kingsray (Fig. 1) to generate pAda-H and pAda-L cloning vectors, respectively. The gene fragments were isolated from the cloning vector by Xbal/Sacl and cloned into the binary plant vector, pCam35S, to generate the plant expression vectors p35S-Ada-H and p35S-Ada-L, respectively. Two plant expression vectors were transformed into Agrobacterium tumefaciens GV3101 by electroporation with a Multiporator (Eppendorf, Hamburg, Germany), respectively. The resulting strain was spread evenly on selective LB plates containing kanamycin antibiotic (50 mg/L). After incubating for 2 days at 28 ° C in the dark, single colonies were picked and inoculated into 0.5 L YEB (yeast extract broth, 5 g/L sucrose, 5 g/L tryptone, 6 g/L yeast extract, 0.24 g/L MgSO4, pH 7 .2) and supplemented with antibiotic liquid medium (50mg/L kanamycin). The inoculated culture was incubated at 25-28 ° C for 72 h in a shaker (220 rpm). The OD600 value was measured by adding YEB medium and adjusted to 3.5 to 4.5. The culture broth was then collected and centrifuged (4500 rpm) for 10 min. The Agrobacterium cells were resuspended in osmotic medium (10 mM MES, 10 mM MgSO ₄ ) to an OD600 of 0.5.

The pAda-H, pAda-L gene fragment was cloned (Fig. 2), and two binary plant expression vectors p35S-Ada-H, p35S-Ada-L were constructed. After completion of the construct, digestion with specific restriction enzymes confirmed that the gene fragment was intact. After infiltration, most of the lettuce tissue was submerged during the vacuum infiltration process, except for the strong midrib area, which showed a yellowish brown area after 4 days of vacuum infiltration.

Example 2 Agrobacterium-mediated vacuum infiltration

The prepared P35S-Ada-H and p35S-Ada-L Agrobacterium were mixed in an equal amount to 0.5% for O.D.600. The culture suspension was placed in a 2 L beaker and placed in a desiccator. The lettuce preserved in the laboratory was inverted (core up) and gently rotated into the bacterial suspension to seal the dryer. A vacuum pump (Welch Vacuum, Niles, IL, USA) was opened to evacuate and the permeate was visible in the leaf tissue. Maintain pressure for 30 to 60 seconds. The system is quickly opened to relieve pressure and allow permeate to penetrate into the space within the tissue. This process is repeated 2 to 3 times until it is clearly visible that the permeate diffuses significantly in the lettuce tissue. The lettuce tissue was then gently removed from the permeate and rinsed three times with distilled water and then transferred to a plastic film covered container. The treated samples were kept in the dark for 4 days.

Example 3 Protein Extraction and Separation

The lettuce sample vacuum-permeated by Agrobacterium was stirred with a stirrer and homogenized in a mixer at a volume ratio of 1:1 ratio of extraction buffer (100 mM KPi, pH 7.8; 5 mM EDTA; 10 m Mβ-mercaptoethanol). minute. The homogenate was adjusted to pH 8.0, filtered through gauze, and the filtrate was centrifuged at 10,000 g for 15 minutes at 4 ° C to remove cell debris. The supernatant was collected, mixed with ammonium sulfate (50%), and incubated for 60 minutes on ice. It was again separated by a centrifuge (10,000 g) at 4 ° C for 15 minutes. The resulting supernatant was subjected to a second round of ammonium sulfate (70%) precipitation, suspended on ice for 60 minutes, and again centrifuged at 10,000 g for 15 minutes at 4 °C. Then, the supernatant was discarded, and the treated sample precipitated protein was dissolved in 5 mL of a buffer (20 mM KPi, pH 7.8; 2 mM EDTA; 10 mM β-mercaptoethanol) and stored at 4 °C.

Downstream processing of plant-derived recombinant proteins is often difficult and expensive because cellulose cell walls are difficult to lyse as well as secondary plant metabolites. We use a blender to stir the homogenate, which greatly saves the homogenization cost and process.

Example 4 SDS-PAGE gel electrophoresis

The purified protein extracted from Agrobacterium tumefaciens vacuum was collected, and samples (5 μL) were heat-denatured (95 ° C) loading buffer (Biorad, Hercules, CA, USA) at 4-12%.

Plus SDS-denaturing gel (ThermoFisher Scientific, Waltham, MA, USA) was run for electrophoresis. Also, the affinity of the antibody is detected in non-denaturing gel electrophoresis. The gel was then photographed again after staining with Coomassie Blue G250 (Biorad). Recombinant adalim antibody was isolated by denaturing gel SDS-PAGE. We observed an estimated molecular weight of approximately 24 kDa and a 48 kDa band in the lane (Fig. 3A), consistent with the light and heavy chain protein size of the adalim antibody. A band of approximately 150 kDa (Fig. 3B) was observed in non-denaturing gel electrophoresis, demonstrating successful binding of the lettuce recombinant light heavy chain to the antibody structure, consistent with the molecular weight of the adalimide antibody protein. The protein content of the purified sample was determined to be about 0.94 mg/g based on the Bradford assay and the densitometric control group.

Example 5 Cancer Cell Inhibition Experiment

Tumor necrosis factor alpha (TNFα) is a naturally occurring cytokine involved in normal inflammation and immune response. Increased levels of TNF are found in the synovium of rheumatoid arthritis, including juvenile idiopathic arthritis, psoriatic arthritis, and ankylosing spondylitis. Increased levels of TNF were also found in psoriasis (Ps) plaques. Adalimumab specifically binds to TNF-[alpha] and blocks its interaction with p55 and p75 cell surface receptors. ELISA analysis of adalimumab Fab fragments can bind to TNFα. TNFα was used as a substrate-coated plate, and the HRP-labeled goat anti-human IgK-specific anti-light chain IgG specifically binds to the Fab fragment, indicating that the Fab fragment binds well to TNFα (Fig. 4). These results indicate that exogenous adalim antibodies transiently expressed by the lettuce system are biologically active and can neutralize TNFα. The kinetics of binding was confirmed by surface plasmon resonance. The ka binding rate constant, kd dissociation rate constant, Kd dissociation of three different batches of soluble TNFα from lettuce-derived adalimumab and commercial adalimumab The equilibrium binding constants are essentially similar (Table 1). The results indicate that plants, especially lettuce, are a suitable bioreactor for the production of adalim antibodies.

Table 1 Results of binding kinetics of lettuce adalimumab (experimental group) and commercial adalimumab (control group) to sTNFα

Group	On rateka (1/ms)	Off ratekd(1/s)	Kd(pM)
test group	6.68E+5	4.14E-5	51
test group	6.79E+5	2.93E-5	49
test group	7.25E+5	3.98E-5	50
Control group	6.35E+5	4.04E-5	52
Control group	7.44E+5	4.04E-5	49
Control group	7.62E+5	3.95E-5	52

The above results show that the combined kinetics were tested by surface plasmon resonance and three unique batches of each test monoclonal antibody were tested.

Note: ka binding rate constant, kd dissociation rate constant, Kd dissociation equilibrium binding constant, ms milliseconds, s seconds, sTNFα soluble tumor necrosis factor alpha.

Example 6

Experimental group: the plant provided by the invention produces adalim antibody;

Control group 1: production of adalim antibody using animal cells;

Experimental group 2: production of adalim antibody using tobacco leaves;

Table 2 adamu antibody

^* indicates P ≤ 0.05 compared with the control group 1; ^** shows P ≤ 0.01 compared with the control group 1;

^#示 Compared with the experimental group 2 P ≤ 0.05; ^## shows that compared with the experimental group 2 P ≤ 0.01;

As can be seen from Table 2, in the experimental group 1 and the animal system of the control group 1, the lettuce provided by the present invention transiently expressed the adalim antibody, which was extremely significant (P ≤ 0.01), shortened the production cycle, and significantly increased (P ≤ 0.01). The protein content, extremely significant (P ≤ 0.01), reduced the binding constant (Kd) with TNFα, simplified the ease of protein purification, and significantly reduced the production cost (P ≤ 0.01).

Compared with the tobacco leaf system of the experimental group 2, the experimental group 1 transiently expressed the adalimide antibody, significantly (P ≤ 0.05) shortened the production cycle, significantly (P ≤ 0.05) reduced the binding constant (Kd) with TNFα, significant (P ≤0.05) reduced the binding constant (Kd) with TNFα, which simplified the ease of protein purification, and significantly reduced the production cost (P ≤ 0.01).

Compared with the control group, the transient expression of adalimin in tobacco group was significantly higher than that in the animal system (P ≤ 0.05), which shortened the production cycle, significantly (P ≤ 0.05) reduced the binding constant (Kd) with TNFα, and simplified protein purification. The difficulty level, significantly (P ≤ 0.05) reduces production costs.

The above test results show that plant systems, especially lettuce systems, are more economical and efficient expression platforms. The ability to rapidly and transiently express recombinant proteins allows large-scale production of adalim antibody monoclonal antibodies in a short period of time.

The above application of the plant provided by the present invention as a host in expressing an adalim antibody is described in detail. The principles and embodiments of the present invention have been described with reference to specific examples, and the description of the above embodiments is only to assist in understanding the method of the present invention and its core idea. It should be noted that those skilled in the art can make various modifications and changes to the present invention without departing from the spirit and scope of the invention.

Claims (9)

1. Use of a plant as a host for the expression of an adalima antibody; the plant is selected from the group consisting of lettuce, cabbage, corn, soybean, tobacco or wheat; the organ of the plant is selected from the group consisting of a seed, a leaf, a rhizome or a whole plant.
2. An expression vector comprising a heavy chain sequence or a light chain sequence of adalima and a vector.
3. The expression vector according to claim 2, wherein the heavy chain sequence or the light chain sequence of the adamu is obtained by optimizing codons of the adalim heavy chain and the adalo light chain into plant-preferred codons. Optimized heavy chain sequence of adalim or optimized light chain sequence of adalim.
4. The expression vector according to claim 3, wherein the optimized heavy chain sequence of adalim is as shown in SEQ ID No. 1; the nucleotide sequence of the optimized heavy chain of adalim is SEQ ID No. 2;

   The optimized light chain sequence of adalim is shown in SEQ ID No. 3; the nucleotide sequence of the optimized light chain of adalim is shown in SEQ ID No. 4.
5. The expression vector according to any one of claims 2 to 4, wherein the vector is a binary plant vector.
6. The expression vector according to any one of claims 2 to 5, wherein the method of constructing comprises the steps of:

   Step 1: Optimize the codons of the adalim heavy chain and the adalo light chain to the plant-preferred codons, respectively:

   i. Optimized heavy chain sequence of adalim;

   Ii. Optimized light chain sequence of adalim;

   Step 2: adding a Xbal restriction enzyme site to the 5' end of the optimized adamus heavy chain sequence, and respectively adding a Sac I site at the 3' end;

   Adding a Xbal restriction site at the 5' end of the optimized adamus light chain sequence and a Sac I site at the 3' end;

   Cloning into pUC57 vector, respectively obtaining pAda-H, pAda-L cloning vector;

   Step 3: Gene fragments were obtained from the cloning vector obtained in Step 2 by Xbal/Sacl, and cloned into the binary plant vector pCam35S to obtain expression vectors p35S-Ada-H and p35S-Ada-L, respectively.
7. Use of an expression vector according to any one of claims 2 to 6 for expression of an adalimide antibody.
8. A method for expressing an adalima antibody as a host, wherein the expression vector according to any one of claims 2 to 6 is transformed into Agrobacterium, and is introduced into a plant tissue by Agrobacterium-mediated vacuum infiltration. , separating proteins, obtaining adalim antibodies;

   The plant is selected from the group consisting of lettuce, cabbage, corn, soybean, tobacco or wheat; the organ of the plant is selected from the group consisting of a seed, a leaf, a rhizome or a whole plant.
9. The method of claim 8 wherein said Agrobacterium-mediated vacuum infiltration comprises the steps of:

   Step 1: Vacuuming 25~45s;

   Step 2: maintain a vacuum (-95kPa) pressure of 30 ~ 60s;

   Step 3: releasing the pressure so that the permeate penetrates into the plant tissue;

   Repeat the above steps 2 to 3 times, and protect from light for 4 days.

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