CN109679985B

CN109679985B - Application of plant as host in expression of coagulation nine factor

Info

Publication number: CN109679985B
Application number: CN201710979577.5A
Authority: CN
Inventors: 王跃驹; 马洁
Original assignee: Sagacity Faithful Convergence Health Technology Ltd
Current assignee: Sagacity Faithful Convergence Health Technology Ltd
Priority date: 2017-10-19
Filing date: 2017-10-19
Publication date: 2022-08-02
Anticipated expiration: 2037-10-19
Also published as: CN109679985A

Abstract

The invention relates to the technical field of biology, in particular to application of a plant as a host in expressing coagulation nine factors. The invention utilizes a recombinant vector and an agrobacterium-mediated vacuum infiltration method to express human coagulation nine factors (AHF, FIX). The expression system can collect the plant exogenous protein after confirming that the agrobacterium is infected for 4 d. The successful expression of recombinant FIX was confirmed by SDS-PAGE and immunoblotting (Western). Blood coagulation experiments prove that FIX produced by lettuce has biological activity. The invention provides a method for producing recombinant human FIX with activity in a large scale with low cost and convenience.

Description

Application of plant as host in expression of coagulation nine factor

Technical Field

The invention relates to the technical field of biology, in particular to application of a plant as a host in expressing coagulation nine factors.

Background

Human coagulation factor nine (FIX) is known under the scientific name Prothrombin Complex (PCC), and is also known under the general name of freeze-dried human Prothrombin Complex, commonly known as factor nine, and is a human plasma product mainly used for treating hemophilia b. FIX is a non-enzymatic plasma protein that is essential for abnormal blood coagulation. Insufficient activity of human factor FIX is associated with congenital bleeding disorders known as hemophilia B. Patients bleed more and more due to the lack of coagulation factors (most patients lack "eight factors", and a few patients lack "nine factors" or "eleven factors"), and if timely treatment is not available, the symptoms of bleeding are intolerable pain at the bleeding site, disability left, and even death. Hemophilia b is a X-linked recessive hereditary disease that accounts for approximately 10% of hemophilia. The blood coagulation factor IX replacement therapy is the fundamental measure for treating the disease, and common substitutes include fresh blood, blood plasma, concentrated blood coagulation factor IX and the like. The administration of prophylactic replacement therapy to severe patients can prevent spontaneous bleeding and also delay the progression of chronic arthropathy.

At present, the coagulation nine factors in the market of China are all obtained from blood, and are extracted by a two-step inactivation production process. Due to insufficient blood source supply, the yield of the coagulation nine factor is far from clinical demand, and the price of the coagulation nine factor is rapidly increased. Since coagulation factors VII, II, etc. are proteins having properties similar to factor IX, obtaining pure factor IX requires a further isolation from the thrombocyte complex and a more complicated purification process. Moreover, there are various risks associated with potential pathogens in plasma, such as hepatitis and HIV, causing coagulation factor nine from current sources in plasma. In order to eliminate the potential risk of plasma pathogen and virus contamination, the use of genetic engineering techniques to produce recombinant factor nine (rFIX) can be used to replace plasma-derived factor nine by producing a safer and lower cost FIX on a large scale.

Plants have been studied for nearly thirty years as a system for the expression and production of pharmaceutical proteins. In addition to the advantages of low cost and high yield, plant-based expression systems reduce the risk of transmission of human and animal pathogens to humans from the process of protein production. In addition, plant eukaryotic protein inner membrane expression systems and secretory pathways are similar to those of mammalian cells. The plant expression system can produce large amount of high molecular weight and subunit medicine protein and is superior to prokaryotic expression system, such as colibacillus expression system. Proteins that require post-translational modification, glycosylation, and monoclonal antibodies that require assembly, which cannot be achieved with prokaryotic systems. The use of plant-produced pharmaceutical proteins as biological agents has been commercialized or used as vaccine additives for poultry. In 2012, the united states Food and Drug Administration (FDA) approved the protein eleyso for the treatment of the genetic disease type 1 gaucher disease ^TM (taliglucerase alfa), and this protein is produced from carrot. Over the past decade, the demand for pharmaceutical proteins has increased dramatically, so the number of plant pharmaceutical proteins approved by the FDA for clinical trials has increased.

The plant transient expression system can produce recombinant proteins in large quantities for clinical studies or to cope with paroxysmal diseases. In 2014, the only antibody therapeutic drug used to effectively resist ebola virus outbreaks, ZMappTM, was produced in tobacco leaves by the agrobacterium infiltration method. The efficacy and safety of ZMapp opens the way for the industry to advance the plant pharmaceutical industry. Currently, tobacco transient protein expression is the most common host plant, and various vectors and agroinfiltration methods have been developed for large-scale production in a short time. However, tobacco has a high fiber content and potentially toxic compounds, such as the alkaloid nicotine, significantly increasing the cost of downstream purification processes, greatly impeding the further development of plant foreign protein pharmaceuticals. Compared with a tobacco leaf system, the lettuce contains less phenols and toxic compounds, so that the lettuce has important practical significance in expressing the human coagulation nine factor by taking the lettuce as a host.

Disclosure of Invention

In view of the above, the present invention provides the use of plants as hosts for expressing factor nine. The invention utilizes lettuce as an effective platform for recombinant protein production, eliminates the growth cycle of plants and greatly saves the time for cultivating plants in the early stage. The invention uses lettuce system to express coagulation nine Factor (FIX), and successfully separates active exogenous protein under mild condition, thus proving that lettuce expression platform can be used for producing recombinant human coagulation nine factor.

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

the invention provides an application of a plant as a host in expressing coagulation nine factors; the plant is selected from lettuce, tobacco, Chinese cabbage, rice, corn, soybean or wheat; the plant organ is selected from the group consisting of seed, leaf, rhizome, or whole plant.

In some embodiments of the invention, the coagulation factor nine is human FIX.

The invention also provides an expression vector, which comprises the nucleotide sequence of the coagulation nine factor and a binary plant vector.

In some embodiments of the invention, the factor ix in the expression vector comprises human FIX.

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

step 1: adding an Xbal restriction site at the 5 'end and a Sacl restriction site at the 3' end of the nucleotide sequence of factor IX; the coagulation nine factor is human FIX;

step 2: cloning into a vector pUC57 to obtain a cloning vector pUC 57-FIX;

and step 3: the gene fragment FIX was obtained from the cloning vector obtained in step 2 by Xbal/Sacl and cloned into the binary plant vector pCam35S to obtain the expression vector p 35S-FIX.

Specifically, the construction method of the expression vector provided by the invention comprises the following steps: codon optimization of human FIX (GenBank Accession No.: A22493.1) to plant-preferred codons by GeneArt ^TM GeneOptimizer ^TM (ThermoFisher) was designed and synthesized by Kinsley. Xbal restriction sites were added to the 5 'end of the optimized FIX sequence, Sacl sites were added to the 3' end, and the resulting product was cloned into pUC57 vector by Kinsley to obtain the cloning vector pUC57-FIX (FIG. 1). The human coagulation nine factor gene fragment FIX was isolated from pUC57-FIX by Xbal/Sacl and cloned into the binary plant vector pCam35S, yielding the transient expression vector p35S-FIX, respectively (FIG. 2). The plant expression constructs were transformed into Agrobacterium tumefaciens GV3101 by electroporation with a multipolator (Eppendorf, Hamburg, Germany), respectively. The resulting strain was spread evenly on selective LB plates containing kanamycin antibiotic (50 mg/L). After incubation in the dark at 28 ℃ for 2 days, a single colony was picked and inoculated into 0.5L YEB (yeast extract broth, 5g/L sucrose, 5g/L tryptone, 6g/L yeast extract, 0.24g/L MgSO4, pH7.2) and supplemented with antibiotic liquid medium (50mg/L kanamycin). The inoculated culture was incubated in a shaker (220rpm) for 72h at 25-28 ℃. OD600 values were measured by addition of YEB medium and adjusted to 3.5-4.5. The culture broth was then collected and centrifuged (4500 rpm) for 10 min. The Agrobacterium cells were resuspended in osmotic medium (10mM MES, 10mM MgSO ₄ ) The neutral to o.d.600 is 0.5.

In the present invention, the FIX cDNA sequence (GenBank: A22493.1) is shown in SEQ ID No. 1; the FIX amino acid sequence is shown as SEQ ID No. 2; the sequence of FIX cDNA after codon optimization is shown in SEQ ID No. 3.

The invention also provides application of the expression vector in expressing coagulation nine factors. In some embodiments of the invention, the coagulation factor nine is human FIX.

The invention also provides a method for expressing the coagulation nine factor by using the plant as a host, which comprises the steps of transforming the expression vector into agrobacterium, penetrating the agrobacterium into plant tissues through agrobacterium-mediated vacuum, extracting and separating protein to obtain the human coagulation nine factor.

In some embodiments of the invention, the factor nine is human FIX.

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

step 1: vacuumizing for 25-45 s;

step 2: keeping the vacuum (-95kPa) pressure for 30-60 s;

and step 3: releasing the pressure such that the permeate permeates the plant tissue;

repeating the steps for 2-3 times, and carrying out light-proof treatment for 4 d.

In some embodiments of the invention, the agrobacterium is agrobacterium tumefaciens GV 3101.

Specifically, the method for agrobacterium-mediated vacuum infiltration comprises the following steps: the prepared agrobacterium culture suspension was placed in a 2L beaker and placed in a desiccator. Lettuce kept in this laboratory was inverted (core up) and gently swirled into the bacterial suspension and the desiccator was sealed. The Vacuum pump (Welch Vacuum, Niles, IL, USA) was turned on to evacuate and the permeate was visible in the leaf tissue. Keeping the pressure state for 30-60 s. The system is rapidly opened to release pressure and allow the permeate to penetrate into the space within the tissue. The process is repeated for 2-3 times until the clear visible penetrating fluid is obviously diffused in the lettuce tissues. The lettuce tissues were then gently removed from the permeate and rinsed three times in succession with distilled water before being transferred to a plastic film covered container. The treated sample was kept in the dark for 4 d.

After infiltration, most lettuce tissues were submerged during vacuum infiltration, except for the firm intercostal areas, which all showed a light tan area 4 days after vacuum infiltration. To increase the number of Agrobacterium tumefaciens that are immersed in the leaf tissue, 10% of the lettuce leaves were cut off from the head with scissors so that the lettuce leaf tissue was as infiltrated in the permeate as possible and released. This method reduces leaf tissue necrosis compared to longer vacuum exposure times.

In some embodiments of the invention, the extracting and isolating of proteins is in particular:

the lettuce samples permeated by the agrobacterium under vacuum are stirred by a stirrer, and are homogenized for 1-2 min at high speed in an extraction buffer (100mM KPi, pH 7.8; 5mM EDTA; 10mM beta-mercaptoethanol) stirrer with the volume ratio of 1: 1. The homogenate was adjusted to pH 8.0, filtered through gauze, and the filtrate was centrifuged at 10,000g for 15min at 4 ℃ to remove cell debris. The supernatant was collected, mixed with ammonium sulfate (50%) and incubated on ice for 60min with shaking. The separation was again carried out by means of a centrifuge (10,000g) at 4 ℃ for 15 min. The resulting supernatant was subjected to a second round of ammonium sulfate (70%) precipitation, suspended with shaking on ice for 60min, and centrifuged again at 10,000g for 15min at 4 ℃. Then, the supernatant was discarded, and the treatment sample precipitated protein was dissolved in 5mL of a buffer (20mM KPi, pH 7.8; 2mM EDTA; 10 m. beta. -mercaptoethanol) and stored at 4 ℃.

The purified protein from vacuum infiltrated lettuce of Agrobacterium was collected and a sample (5uL) was heat denatured (95 ℃) loading buffer (Biorad, Hercules, Calif., USA) at 4-12%

The Plus SDS-gel (ThermoFisher Scientific, Waltham, MA, USA) was run for electrophoresis and then the gel was photographed again after staining with Coomassie blue G250 (Biorad). For Western Blot Western Blot hybridization of recombinant FIX, 10ul of recombinant sample and human FIX standard (Biovision) were 10-20%

The fractions were separated on a Plus polyacrylamide gel, electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane, immunoreactive with an anti-FIX antibody (Abcam), diluted 1: 10000 and goat anti-rabbit IgG labeled with horseradish peroxidase (HRP) (Beyotime), dilution 1:20000, respectively, and visualized using ECL plus (Amersham Biosciences), and the display image was photographed.

Downstream processing of recombinant proteins of plant origin is often difficult and expensive because of the difficulty of lysis of the cellulose cell wall and secondary plant metabolites. The invention uses the stirrer to stir and homogenize, thereby greatly saving the homogenization cost and the process. Recombinant FIX was separated by SDS-PAGE we observed a band with an estimated molecular weight of approximately 46kDa in the lane (fig. 3A, lane 1) and no corresponding band was evident in the stealth control lane (fig. 3A, lane 2). The protein content of the purified sample was determined to be 0.48mg/kg based on the Bradford assay and densitometry controls. In addition, a band of approximately 46kDa was also detected by Western blot analysis (FIG. 3B, lane 1), and the observed protein molecular weight (46kDa) was consistent with the predicted protein molecular weight.

Human coagulation factor nine 5ug was added to fresh plasma. Plasma coagulation was checked after 5 minutes. Examination of the plasma results indicated that the plasma without any treatment did not have any clotting reaction. In contrast, clotting was good with purified recombinant FIX or equivalent positive control FIX standard treatment (fig. 4). These results indicate that exogenous FIX expressed transiently by the lettuce system is biologically active, and the lettuce system may be a suitable bioreactor for the bulk production of biologically active recombinant drug proteins.

The growth time of tobacco plants for vacuum agroinfiltration is typically 4 to 6 weeks. The invention utilizes lettuce as an effective platform for recombinant protein production, eliminates the growth cycle of plants and greatly saves the time for cultivating plants in the early stage. According to the invention, the lettuce system is used for expressing FIX, and active exogenous protein is successfully separated out under mild conditions, so that the lettuce expression platform can be used for producing human coagulation nine factors.

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 description of the embodiments or the prior art will be briefly described below.

FIG. 1(A) shows FIX cloning vector (constructed and synthesized by Kingsry);

FIG. 1(B) shows FIX gene fragment restriction (XbaI/SacI) identification;

FIG. 2 shows the construction process of plant transient expression vector p 35S-FIX; cutting FIX fragments from the cloning vector in the figure 1 by using restriction endonuclease (XbaI/SacI) for double digestion, and connecting the cut FIX fragments into XbaI/SacI sites of pCam35S to generate a plant transient expression vector p 35S-FIX;

wherein 35S is the CaMV 35S promoter with Tobacco Mosaic Virus (TMV) 5' UTR; NPT II, the expression of the nptII gene encoding for kanamycin resistance; nos 3', terminator;

FIG. 3(A) shows the detection of purified recombinant human recombinant growth Factor (FIX) by polyacrylamide gel electrophoresis (SDS-PAGE); lane 1 purified recombinant FIX (5. mu.g); lane 2: negative control of non-vacuum osmotic leaf eluent;

FIG. 3(B) shows Western blot hybridization detection of purified recombinant human coagulation factor nine; lane 1 purified recombinant FIX (5. mu.g); lane 2: negative control of non-vacuum osmotic leaf eluent;

figure 4 shows that factor nine significantly promotes clotting experiments; plasma was treated with purified FIX and plasma coagulation was observed; after 5 minutes, the recombinant human coagulation factor nine significantly promoted plasma coagulation.

Detailed Description

The invention discloses application of a plant as a host in expressing coagulation nine factors, and a person skilled in the art can realize the expression by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The research of the invention shows that the lettuce system can be an effective expression platform and provides a method for quickly and instantaneously expressing recombinant protein. The vacuum agrobacterium infiltration method is simple and rapid, reduces the leaf necrosis and can improve the yield of recombinant protein. Lettuce can increase protein production by withstanding vacuum pressure and allow for more complete penetration of each leaf. Lettuce is easier to grow and cheaper to produce commercially in large quantities than other transiently expressing plants, such as tobacco and the like. And is more cost-effective as no special equipment or liquid nitrogen is required. The invention proves that the method can be used for producing FIX recombinant protein in a large scale in a short time.

The invention provides a method for using plant, especially lettuce as host to express blood coagulation factor nine, wherein the raw material and reagent can be purchased from market.

The invention is further illustrated by the following examples:

example 1 construction of plant transient expression vectors

In order to provide high-efficiency expression of foreign proteins in plants, the invention optimizes the codon of human FIX (GenBank Accession number: A22493.1) into the codon preferred by plants and uses GeneArt ^TM GeneOptimizer ^TM (ThermoFisher) was designed and synthesized by Kinsley. Xbal restriction sites were added to the 5 'end of the optimized FIX sequence, Sacl sites were added to the 3' end, and the vector was cloned into pUC57 vector by Kinsley to obtain the cloning vector pUC57-FIX (FIG. 1). The human coagulation nine factor gene fragment FIX was isolated from pUC57-FIX by Xbal/Sacl and cloned into the binary plant vector pCam35S, yielding the transient expression vector p35S-FIX, respectively (FIG. 2). The plant expression constructs were transformed into Agrobacterium tumefaciens GV3101 by electroporation with a multipolator (Eppendorf, Hamburg, Germany), respectively. The resulting strain was spread evenly on selective LB plates containing kanamycin antibiotic (50 mg/L). After incubation in the dark at 28 ℃ for 2 days, a single colony was picked and inoculated into 0.5L YEB (yeast extract broth, 5g/L sucrose, 5g/L tryptone, 6g/L yeast extract, 0.24g/L MgSO4, pH7.2) and supplemented with antibiotic liquid medium (50mg/L kanamycin). The inoculated culture was incubated in a shaker (220rpm) for 72h at 25-28 ℃. OD600 values were measured by addition of YEB medium and adjusted to 3.5-4.5. The culture broth was then collected and centrifuged (4500 rpm) for 10 min. The Agrobacterium cells were resuspended in osmotic medium (10mM MES, 10mM MgSO ₄ ) The neutral to o.d.600 is 0.5.

Example 2 Agrobacterium-mediated vacuum infiltration

The invention optimizes the method for vacuum infiltration of agrobacterium tumefaciens. The prepared agrobacterium culture suspension was placed in a 2L beaker and placed in a desiccator. Lettuce stored in this laboratory was inverted (core up) and gently swirled into the bacterial suspension and the desiccator was sealed. The Vacuum pump (Welch Vacuum, Niles, IL, USA) was turned on to evacuate and the permeate was visible in the leaf tissue. Keeping the pressure state for 30-60 s. The system is rapidly opened to release pressure and allow the permeate to penetrate into the space within the tissue. The process is repeated for 2-3 times until the clear visible penetrating fluid is obviously diffused in the lettuce tissues. The lettuce tissues were then gently removed from the permeate and rinsed three times in succession with distilled water before being transferred to a plastic film covered container. The treated sample was kept in the dark for 4 d.

Example 3 protein extraction and isolation

The lettuce samples permeated by the agrobacterium in vacuum are stirred by a stirrer, and are homogenized for 1-2 min at high speed in an extraction buffer (100mMKPi, pH 7.8; 5mM EDTA; 10mM beta-mercaptoethanol) stirrer with the volume ratio of 1: 1. The homogenate was adjusted to pH 8.0, filtered through gauze, and the filtrate was centrifuged at 10,000g for 15min at 4 ℃ to remove cell debris. The supernatant was collected, mixed with ammonium sulfate (50%) and incubated for 60 minutes on ice with shaking. The separation was again carried out by means of a centrifuge (10,000g) at 4 ℃ for 15 min. The resulting supernatant was subjected to a second round of ammonium sulfate (70%) precipitation, suspended with shaking on ice for 60min, and centrifuged again at 10,000g for 15min at 4 ℃. Then, the supernatant was discarded, and the treatment sample precipitated protein was dissolved in 5mL of a buffer (20mM KPi, pH 7.8; 2mM EDTA; 10 mM. beta. -mercaptoethanol) and stored at 4 ℃.

Example 4SDS-PAGE gel electrophoresis and Western Blot hybridization

Collect from the farm poleThe bacteria were vacuum infiltrated with the purified protein from lettuce, and a sample (5uL) was heat denatured (95 ℃) loading buffer (Biorad, Hercules, Calif., USA) at 4-12%

The Plus SDS-gel (ThermoFisher Scientific, Waltham, MA, USA) was run for electrophoresis and then the gel was photographed again after staining with Coomassie blue G250 (Biorad). For Western Blot Western Blot hybridization of recombinant FIX, 10ul of recombinant sample and hFIX and standard (Biovision) were 10-20%

The fractions were separated on a Plus polyacrylamide gel, electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane, immunoreactive with an anti-hFIX antibody (Abcam), diluted 1: 10000 and goat anti-rabbit IgG labeled with horseradish peroxidase (HRP) (Beyotime), dilution 1:20000, respectively, and visualized using ECL plus (Amersham Biosciences), and the display image was photographed.

Downstream processing of recombinant proteins of plant origin is often difficult and expensive because of the difficulty of lysis of the cellulose cell wall and secondary plant metabolites. A stirrer is used for stirring and homogenizing, so that the homogenizing cost and the homogenizing process are greatly saved. Recombinant FIX was separated by SDS-PAGE we observed a band with an estimated molecular weight of approximately 46kDa in the lane (fig. 3A, lane 1) and no corresponding band was evident in the stealth control lane (fig. 3A, lane 2). The protein content of the purified sample was determined to be 0.48mg/kg based on the Bradford assay and densitometry controls. In addition, a band of approximately 46kDa was also detected by Western blot analysis (FIG. 3B, lane 1), and the observed protein molecular weight (46kDa) was consistent with the predicted protein molecular weight.

Example 5 coagulation nine factor coagulation assay

Human coagulation factor nine 5ug is added to fresh plasma. After 5 minutes the plasma coagulation was checked. Examination of the plasma results indicated that the plasma without any treatment did not have any clotting reaction. In contrast, clotting was good with purified recombinant FIX or equivalent positive control FIX standard treatment (fig. 4). These results indicate that exogenous FIX, transiently expressed by the lettuce system, is biologically active, and the lettuce system may be a suitable bioreactor for the mass production of biologically active recombinant pharmaceutical proteins.

Example 6

Control group: producing human coagulation factor IX by using tobacco leaves;

experimental groups: the lettuce provided by the invention produces human coagulation nine factors;

TABLE 1 human coagulation nine factors

^* Shows that P is less than or equal to 0.05 compared with the control group; ^# shows that P is less than or equal to 0.01 compared with the control group;

as can be seen from Table 1, compared with the tobacco leaf system of the control group, the lettuce instantly expresses human coagulation factor nine (FIX) obviously (P is less than or equal to 0.05), the production period is shortened, the protein content is obviously (P is less than or equal to 0.05), the protein activity is obviously (P is less than or equal to 0.05), the difficulty of protein purification is simplified, and the production cost is greatly reduced (P is less than or equal to 0.01).

The comprehensive test results show that the plant system, especially the lettuce system, is a more economic and efficient expression platform. Can express recombinant protein rapidly and transiently, and can produce human coagulation factor nine in a large scale in a short time.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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aaccaaaaat cttgcgagcc tgctgttccc ttcccatgtg gtagagttag cgtaagccaa 540

acctctaaac ttacccgtgc tgaagccgtc tttcccgatg tcgactacgt aaacagcacc 600

gaagctgaga ctattctgga taacatcacg caaagcaccc aatctttcaa cgactttact 660

cgtgtcgtgg gaggtgaaga tgcaaagcca ggtcaattcc catggcaagt cgtgctcaac 720

ggaaaggttg atgctttctg cgggggctct attgtgaatg agaagtggat agttactgcc 780

gcacactgcg tagagactgg ggtgaaaata acagtggtag caggcgaaca caatattgag 840

gaaaccgagc atacagagca aaagaggaac gtgatcagga taatcccaca tcataattat 900

aacgcagcta tcaacaaata taaccatgac attgcactgt tagagctcga tgaacccctc 960

gttctcaata gctatgtaac tcctatttgt atagcagata aggagtacac caatatcttc 1020

ttaaagttcg gcagtggtta tgtatctggg tggggtcgtg tcttccacaa aggaagaagc 1080

gcccttgtcc tgcagtactt gcgagtcccg cttgtagaca gagctacgtg cttgaggtca 1140

acaaagttta caatatacaa caatatgttt tgtgctgggc tccacgaggg ggctagagac 1200

agctgccaag gagacagtgg tggcccacat gttactgaag tagaaggaac gtcattcctc 1260

actgggatca tcagttgggg ggaagaatgc gccatgaagg gcaaatacgg tatatacaca 1320

aaagttagca gatacgttaa ttggattaag gagaaaacta agttgactta a 1371

Claims

1. A method for expressing coagulation nine factor by using plant as host is characterized by that the expression vector is transferred into agrobacterium, and after it is passed through agrobacterium mediated vacuum infiltration into plant tissue, the protein can be extracted and separated so as to obtain coagulation nine factor;

the coagulation nine factor is human FIX;

the agrobacterium-mediated vacuum infiltration comprises the following steps:

step 1: vacuumizing for 25-45 s;

step 2: keeping the vacuum at-95 kPa for 30-60 s;

repeating the steps for 2-3 times, and carrying out light-proof treatment for 4 d;

the plant is lettuce;

the expression vector comprises a nucleotide sequence of the coagulation nine factor and a binary plant vector;

the coagulation nine factor is FIX;

the construction method of the expression vector comprises the following steps:

step 1: adding an Xbal restriction site at the 5 'end and a Sacl restriction site at the 3' end of a nucleotide sequence of coagulation factor nine (FIX);

step 2: cloning into a vector pUC57 to obtain a cloning vector pUC 57-FIX;

and step 3: obtaining a gene fragment FIX from the cloning vector obtained in the step 2 through Xbal/Sacl, cloning to a binary plant vector pCam35S, and obtaining an expression vector p 35S-FIX;

the codon optimized FIXcDNA sequence of the coagulation nine factor is shown in SEQ ID No. 3.