CN110229237A

CN110229237A - Plant production takes orally the application of EPO and transferrin fusion protein capsule

Info

Publication number: CN110229237A
Application number: CN201910550550.3A
Authority: CN
Inventors: 王跃驹
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-06-24
Filing date: 2019-06-24
Publication date: 2019-09-13
Also published as: WO2020259109A1

Abstract

The present invention relates to field of biotechnology, the in particular to application of plant production oral EPO and transferrin fusion protein capsule.The present invention produces bioactive substance using plant such as romaine lettuce as the effective expression platform of recombinant protein production, using simple and efficient expression system.Then the blade for producing the active material is lyophilized, capsule is made through special producing technique.This capsule can keep bioactivity being stored at room temperature.Anemicus therapeutic activity albumen successful expression is determined using Western Blot protein hybridization method.Biological activity test is the result shows that be obviously improved the erythrocyte number of mouse using the anemicus treatment capsule that the platform technology produces.

Description

Plant production takes orally the application of EPO and transferrin fusion protein capsule

Technical field

The present invention relates to field of biotechnology, in particular to plant production takes orally EPO and transferrin fusion protein capsule Application.

Background technique

Hematopoietin (EPO) is also known as red blood cell stimulating factor, is glycoprotein, molecular weight is about 2.5 ten thousand~4.5 ten thousand Between, it is an important factor for stimulating RBC acceptor garland rate.Animal is under the stimulation of body anoxic, the erythrogenin of kidney generation (Erythrogenin) erythropoitinogen generated in blood plasma by liver is acted on, it is made to be converted into promoting erythrocyte generation Element.

Hematopoietin acts on hemogonia in marrow, is allowed to be converted into pronormoblast (Proerythroblast)；Promote the mitosis of erythroblast and the synthesis of hemoglobin；Networking is knitted red in promotion marrow The release of cell and mature erythrocyte.A large amount of mature erythrocytes carry more oxygen to tissue, and oxygen abundance is as a kind of negative-feedback Information can inhibit liver again and generate erythropoietin(EPO) and kidney generation erythrogenin, adjust the generation of red blood cell in this way Amount.

EPO belongs to saliva glycoprotein hormone, was most found earlier than 1906, is a kind of human endogenous's property compound, point Son amount is 34 000, is made of 165 amino acid.It is mainly derived from kidney (on a small quantity from liver), around cortex pipe Interstitial cell synthesis.It is 30400 by the rhEPO molecular weight that gene recombination technology synthesizes, physicochemical property and biological activity Identical as native endogenous erythropoietin(EPO), it is glycoprotein that difference, which is gene loci in No. 7 chromosomes,.

The physiological action of EPO is by red thin with normoblast shape colony forming unit (CFu-E) or its early young stage Born of the same parents are the receptor combination that explosion forms unit (BFuE), and promote it to erythroid precursors (granulophilocyte) and pronormoblast point Change, ultimately produces red blood cell (RBC).When serious damage occurs for the renal function of people, as in acute or chronic renal failure or the nephrectomy In the case of, the generation of EPO is reduced, and anaemia occurs.Therefore EPO has good curative effect to most renal anemia patients, by There is affirmative curative effect in clinic in EPO, product has been put into China " national basic medical insurance catalogue ".

Although natural EPO has many advantages, such as that the Related product listed at present has rush red on treatment anemic disorders Archusia etc..Due to the various barriers that the property and human body of polypeptide drug itself generate it, conventional administration way Through always with injection based on.EPO also achieves oral administration by special preparation way, and the present invention by EPO and turns iron egg White amalgamation and expression, same realize are being administered orally, and mitigate sufferer long term frequent injection bring pain.

Summary of the invention

In view of this, the present invention provides the application of a kind of plant production oral EPO and transferrin fusion protein capsule.This Invention carries out structure of modification and modification to the hematopoietin (EOP) with anemicus therapeutic effect, lead to its acquisition can It crosses enteron aisle and absorb and reach in vivo the characteristic of effective treatment concentration, and produce the active material by plant.This hair The bright efficient platform technology produced using plant especially romaine lettuce as recombinant protein expresses merging for EPO and transferrins Albumen.And it is made and takes orally anemicus treatment capsule.

In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:

The present invention provides the fusion proteins of EPO and transferrins, include

(I), the amino acid sequence as shown in SEQ ID No.1；Or

(II), the amino acid sequence as described in (I) is substituted, lacks or adds the amino that one or more amino acid obtain Acid sequence, and with the amino acid sequence functionally identical or similar of amino acid sequence shown in (I)；Or

(III), with the amino acid sequence of (I) or (II) described sequence at least 80% homology.

The present invention also provides the nucleotide of encoding said fusion protein, have

(I), the nucleotide sequence as shown in SEQ ID No.2；Or

(II), the complementary nucleotide sequence of the nucleotide sequence as shown in SEQ ID 2；Or

(III), with the nucleotide sequence coded same protein of (I) or (II), but due to the degeneracy of genetic code with (I) or the different nucleotide sequence of the nucleotide sequence of (II)；Or

(IV), one or more nucleosides are substituted, lack or added with nucleotide sequence shown in (I), (II) or (III) Acid sequence obtain nucleotide sequence, and with nucleotide sequence nucleosides functionally identical or similar shown in (I), (II) or (III) Acid sequence；Or

(V), with the nucleotide sequence of (I), (II), (III) or (IV) described nucleotide sequence at least 80% homology.

On the basis of the studies above, the present invention also provides expression vector, including the nucleotide and to be transformed Carrier.

In some specific embodiments of the invention, the carrier to be transformed is chloroplast expression vector.

In addition, the present invention also provides the construction methods of the expression vector, which comprises the steps of:

Step 1: being respectively the password of favorite plant by the codon optimization of the EPO and the fusion protein of transferrins Son, nucleotide sequence is as shown in SEQ ID No.2；

Step 2: the nucleotide sequence being cloned into pUC57 carrier, pEPO is obtained.

The present invention also provides the expression vectors or plant in fusion protein or the preparation for expressing EPO and transferrins Application in drug comprising the fusion protein；The plant is selected from romaine lettuce, spinach, tomato, radish, Chinese cabbage, corn, big Beans, wheat or tobacco；The organ of the plant is selected from seed, leaf, rhizome or whole plant.

In some specific embodiments of the invention, the drug is the oral preparation for treating anaemia.

The present invention also provides host, conversion has the plant or microorganism of the expression vector；The plant be selected from romaine lettuce, Spinach, tomato, radish, Chinese cabbage, corn and soybean, wheat or tobacco；The organ of the plant is selected from seed, leaf, rhizome or whole strain Plant.

The present invention also provides drugs, including the fusion protein and pharmaceutically acceptable auxiliary material.

A kind of method the present invention also provides plant as host expresses EPO and the fusion protein of transferrins, by institute The expression vector stated biolistic bombardment blade obtains regeneration plant after expressing in plant chloroplast, plant leaf blade is lyophilized It crushes, extract, obtain the fusion protein of EPO and transferrins.

In some specific embodiments of the invention, the biolistic bombardment includes the following steps:

Step 1: preparing conversion carrier；

Step 2: preparing particle bullet；

Step 3: biolistic bombardment；

Step 4: being cultivated after conversion, be regenerated as plant.

The present invention also provides a kind of plants, and the method for treating the drug of anaemia is prepared as host, and the expression is carried Body biolistic bombardment blade obtains regeneration plant after expressing in plant chloroplast, and plant leaf blade freeze-drying is crushed, is extracted, The fusion protein of EPO and transferrins are obtained, it is filling.

Step 1: preparing conversion carrier；

Step 2: preparing particle bullet；

Step 3: biolistic bombardment；

Step 4: being cultivated after conversion, be regenerated as plant.

Plant chloroplast expression technology be by biolistic bombardment, homologous recombination in the way of by the plasmid containing target protein It is transferred in plant chloroplast, obtains the technology of high efficient expression in the gene plant chloroplaset.With animal cell expression system phase Than the cost of plant expression system is very low, only its one thousandth to 2/1000ths.

The present invention utilizes plant leaf blade, and production takes orally anemicus treatment capsule.The anemicus treatment product does not need to inject, Mitigate the pain of sufferer.Romaine lettuce does not contain plant noxious material, and this product is not required to protein purification process, can greatly shorten Production cycle and production cost.

The present invention is found through experiments that, botanical system especially romaine lettuce system be it is more economical, efficiently express platform, leaf Green body can efficient expression activity albumen.Due to romaine lettuce be easy to grow and can commercial mass production, than other plants Object, such as tobacco are easier to obtain and cheaper, and due to not needing complicated special producing equipment, cost can significantly drop It is low.In conclusion the present invention can use the fusion protein of romaine lettuce system large-scale production EPO and transferrins.

Detailed description of the invention

In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below There is attached drawing needed in technical description to be briefly described.

Fig. 1 shows carrier pEPO schematic diagram；

Fig. 2 shows western-blot result.

Specific embodiment

The invention discloses the applications that a kind of plant production takes orally EPO and transferrin fusion protein capsule.This field skill Art personnel can use for reference present disclosure, be suitably modified realization of process parameters.In particular, it should be pointed out that all similar replacements and Change apparent to those skilled in the art, they are considered as being included in the present invention.Method of the invention and Using being described by preferred embodiment, related personnel can obviously not depart from the content of present invention, spirit and scope It is interior that method described herein and application are modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.

The present invention provides plant as host expression EPO and transferrins fusion protein application.Preferably, institute It states plant and is selected from romaine lettuce, spinach, tomato, radish, Chinese cabbage, corn and soybean, wheat or tobacco；The organ of the plant is selected from kind Son, leaf, rhizome or whole plant.The present invention also provides a kind of expression vectors, the fusion protein sequence including EPO and transferrins Column and carrier.

In some specific embodiments of the invention, the fusion protein codon optimization of the EPO and transferrins is The codon of favorite plant.

In some specific embodiments of the invention, the fusion protein sequence of the EPO of the optimization and transferrins is such as Shown in SEQ ID No.1；The nucleotide sequence of the fusion protein of the EPO and transferrins of the optimization such as SEQ ID No.2 institute Show.

In some specific embodiments of the invention, the carrier is plant chloroplast carrier.

In some specific embodiments of the invention, the construction method of the expression vector includes the following steps:

Step 1: being the codon of favorite plant by the codon optimization of EPO and the fusion protein of transferrins；

Step 2: gene chemical synthesis being carried out by Jin Sirui and is cloned into pUC57 carrier, pEPO carrier is obtained；

Specifically, in order to provide high efficient expression of the foreign protein in plant, the present invention is merged EPO and transferrins Protein amino acid sequence utilizes anti-translation software

(https: //www.ebi.ac.uk/Tools/st/emboss_backtranseq/) obtains nucleotide sequence, and It is the codon of favorite plant by its codon optimization, by Jin Sirui company (Nanjing, China) synthesis.And by golden Stryker it is grand to In pUC57 carrier, obtain pEPO carrier (Fig. 1).

The present invention also provides application of the expression vector in the fusion protein of expression EPO and transferrins.

By expression vector provided by the invention biolistic bombardment plant leaf blade, plant leaf blade is harvested simultaneously after being regenerated as plant It is made and takes orally anemicus treatment capsule.

Plant production provided by the invention takes orally raw materials used in the application of EPO and transferrin fusion protein capsule and examination Agent is available on the market.

Below with reference to embodiment, the present invention is further explained:

The building of 1 chloroplast expression vector of embodiment

For the high efficient expression by foreign protein in plant, by the fusion protein amino acid sequence of EPO and transferrins Nucleotide is obtained using anti-translation software (https: //www.ebi.ac.uk/Tools/st/emboss_backtranseq/) Sequence, and be the codon of favorite plant by its codon optimization, by Jin Sirui company (Nanjing, China) synthesis.

2 converting material of embodiment prepares

By vegetable seeds sterile water soaked overnight, used aseptic water washing 1 time after being impregnated 1 minute with 70% ethyl alcohol；It uses again 2%NaClO (adding 0.1%Tween-20) is handled 15 minutes, soft mixing in every 5 minutes 1 time, aseptic water washing 4-5 times；With sterile Plantation is placed in illumination box in (containing 3% sucrose, 0.7% agar powder, pH value 5.8) on 1/2MS culture medium after filter paper blots In 25 DEG C, 16h illumination 8h dark culturing can be used for converting for about 3 weeks.

3 particle gun of embodiment prepares

50-60mg bronze (0.6 μm) is weighed in dry 1.5mL sterilizing EP centrifuge tube.1mL dehydrated alcohol, vortex 2 is added Minute.1mL sterile water is added, is vortexed 1 minute, is placed at room temperature for 1 minute, supernatant is removed in 10,000rpm centrifugations 2 minutes.1mL is added Bronze is resuspended in 50% glycerol, and -20 DEG C freeze.

The bronze suspension that glycerol saves state is vortexed 5 minutes and bronze is resuspended.Take 50 μ L bronze suspensions in sterile 1.5mL from Heart pipe is vortexed 1 minute.10 μ g Plasmid DNA are added, are vortexed 30 seconds.50 μ L 2.5M CaCl are added₂, it is vortexed 30 seconds.20 μ L are added 0.1M spermidine, mixture are vortexed 5 minutes, stand 2 minutes on ice.The dehydrated alcohol for adding 60 μ L to be pre-chilled, finger, which flicks, is allowed to weight Outstanding, 14,000rpm centrifugations 10 seconds are removed supernatant, are repeated once.The resuspension of 50 μ L dehydrated alcohols is added, it is spare.

4 biolistic bombardment of embodiment

A certain number of carrier films are measured according to sample number, can split that film, stopping net, (note: carrier film can split film and need every rifle Replacement, stopping to net same sample can share) it is impregnated 15 minutes in dehydrated alcohol, with aseptic water washing 2 times, naturally dry, It is spare.The carrier film dried is put into sterile iron hoop, is flattened.The bullet prepared vortex is mixed well, 10 μ L bullets are taken In carrier film center, naturally dry.Corpuscular emission device is removed bombardment room, lid is screwed off, is added and stops net, particle slide glass It is mounted in fixing groove (fine-grained one down), screws on lid, corpuscular emission device is put back into bombardment room.

Embodiment 5 is cultivated after converting and screening

1. dark culture: the romaine lettuce blade after bombardment being cut, the leaf dish for being cut into 10~20mm2 is placed in RMOL culture medium (no Added with antibiotic) in 25 DEG C dark culture 2 days.

2. screening and culturing: the material that dark culture is terminated is transferred in screening and culturing medium (antibiotic concentration is 50 μ g/mL) Carry out screening and culturing.

3. culture of rootage: bud is transferred to root induction in root media (antibiotic concentration is 100 μ g/mL).

Embodiment 6Western blot testing goal protein expression situation

Vegetable protein is extracted using liquid nitrogen grinding, denatured lysis, cracking 5 × sample-loading buffer of supernatant (is added using preceding Enter beta -mercaptoethanol to it is final concentration of 5%) in 4:1 ratio mixing (such as 200 μ l protein cleavage supernatants and 50 μ 5 × loadings of l delay Fliud flushing mixing), it mixes, 95 DEG C of heating 6min, while handling negative control and positive control；Electrophoretic voltage spacer gel 80V, separation Glue 120V is run after destination protein to separation gel middle position, stops electrophoresis, is recycled lower slot electrophoresis liquid, is dismantled electrophoretic apparatus, press According to cathode (black), sponge, filter paper, gel, pvdf membrane (1 × transfer is soaked in after being washed in advance with methanol activation 15s, ddH2O In buffer) or NC film (being not required to activate), filter paper, sponge, anode (transparent) sequence place, exhaust bubble after assemble, be put into electricity Swimming slot (note black corresponds to electrophoresis tank black and is put on one side), fills it up with transfering buffering liquid, entire electrophoresis tank is put into ice water mixed liquor In, 90V electrophoresis 1.0h；5% skimmed milk power (confining liquid) is prepared at the end of electrophoresis is fast, and the film after transfer is put into room in confining liquid Temperature closing at least 1h, 4 DEG C of incubation primary antibodies are overnight (primary antibody is diluted in 5% skimmed milk power, thinner ratio reference book)；It uses PBST or TBST washs 15min × 3 time, and incubation at room temperature 1~2h of secondary antibody, PBST or TBST are washed 15min × 3 time, tried using DAB Agent box develops the color, and takes pictures, and analyzes destination protein expression, the results showed that, there is epo protein expression in transgenic lettuce, sees Fig. 2.

The fusion protein Activity determination of 7 EPO of embodiment and transferrins

Adult male mice is randomly divided into Liang Ge treatment group, every group 10, receives contain anemicus treatment albumen respectively (fusion protein of EPO made from the embodiment of the present invention 5 and transferrins) and be free of two kinds of experiment capsules of anemicus treatment albumen One of, it does and repeats for the first time.It mouse successive administration 7 days, is administered once a day.Blood sampling measurement periphery is blood red after the test Cell (RBC), hemoglobin (Hb), granulophilocyte (RET).With rhEPO, (recombinant epo, you are rich for ring, 5000IU/ml, Fourth Ring Biology Pharmacy Co., Ltd) it is positive control.

The result shows that romaine lettuce expression EPO is suitable with the activity of street drug, the effect for the anaemia that all improves significantly.

Influence of the 1 EPO-Tf fusion protein of table to mouse blood

Note: * shows with significant difference (P < 0.05)；* shows with extremely significant difference (P < 0.01).

8 animal toxicity test of embodiment

The experimental white mouse of 7 weeks sizes is randomly divided into three treatment groups, every group 10, receives contain hypoglycemic egg respectively White (according to weight feeding 500ng/g) (fusion protein of EPO and transferrins that the present invention obtains), and be free of blood sugar reducing proteins two One of kind experiment capsule, receives identical experimental diet.Continuous feeding 10 days is observed after each feeding, daily in fact It needs to be observed continuously 6 hours or more, does not see that mouse is in excitatory state or holddown, do not occur being slow in action Also there is not situations such as diarrhea in phenomenon.Prove that the fusion protein oral administration safety of EPO and transferrins is high.

The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.

Sequence table

<110>Wang Yueju

<120>plant production takes orally the application of EPO and transferrin fusion protein capsule

<130> MP1907673

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<170> SIPOSequenceListing 1.0

<210> 1

<211> 887

<212> PRT

<213>fusion protein (Fusion protein of EPO and transferrin) of EPO and transferrins

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Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu

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Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu

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Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu

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Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg

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Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu

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Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser

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Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly

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Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu

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Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile

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Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu

165 170 175

Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp

180 185 190

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

195 200 205

Val Pro Asp Lys Thr Val Arg Trp Cys Ala Val Ser Glu His Glu Ala

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Thr Lys Cys Gln Ser Phe Arg Asp His Met Lys Ser Val Ile Pro Ser

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Asp Gly Pro Ser Val Ala Cys Val Lys Lys Ala Ser Tyr Leu Asp Cys

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Ile Arg Ala Ile Ala Ala Asn Glu Ala Asp Ala Val Thr Leu Asp Ala

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Gly Leu Val Tyr Asp Ala Tyr Leu Ala Pro Asn Asn Leu Lys Pro Val

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Val Ala Glu Phe Tyr Gly Ser Lys Glu Asp Pro Gln Thr Phe Tyr Tyr

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Ala Val Ala Val Val Lys Lys Asp Ser Gly Phe Gln Met Asn Gln Leu

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Arg Gly Lys Lys Ser Cys His Thr Gly Leu Gly Arg Ser Ala Gly Trp

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Asn Ile Pro Ile Gly Leu Leu Tyr Cys Asp Leu Pro Glu Pro Arg Lys

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Pro Leu Glu Lys Ala Val Ala Asn Phe Phe Ser Gly Ser Cys Ala Pro

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Cys Ala Asp Gly Thr Asp Phe Pro Gln Leu Cys Gln Leu Cys Pro Gly

370 375 380

Cys Gly Cys Ser Thr Leu Asn Gln Tyr Phe Gly Tyr Ser Gly Ala Phe

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Lys Cys Leu Lys Asp Gly Ala Gly Asp Val Ala Phe Val Lys His Ser

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Gly Gly Lys Glu Asp Leu Ile Trp Glu Leu Leu Asn Gln Ala Gln Glu

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His Phe Gly Lys Asp Lys Ser Lys Glu Phe Gln Leu Phe Ser Ser Pro

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His Gly Lys Asp Leu Leu Phe Lys Asp Ser Ala His Gly Phe Leu Lys

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Val Pro Pro Arg Met Asp Ala Lys Met Tyr Leu Gly Tyr Glu Tyr Val

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Thr Ala Ile Arg Asn Leu Arg Glu Gly Thr Cys Pro Glu Ala Pro Thr

530 535 540

Asp Glu Cys Lys Pro Val Lys Trp Cys Ala Leu Ser His His Glu Arg

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Leu Lys Cys Asp Glu Trp Ser Val Asn Ser Val Gly Lys Ile Glu Cys

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Val Ser Ala Glu Thr Thr Glu Asp Cys Ile Ala Lys Ile Met Asn Gly

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Glu Ala Asp Ala Met Ser Leu Asp Gly Gly Phe Val Tyr Ile Ala Gly

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Lys Cys Gly Leu Val Pro Val Leu Ala Glu Asn Tyr Glu Lys Ser Asp

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Asn Cys Glu Asp Thr Pro Glu Ala Gly Tyr Phe Ala Val Ala Val Val

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Lys Lys Ser Ala Ser Asp Leu Thr Trp Asp Asn Leu Lys Gly Lys Lys

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Ser Cys His Thr Ala Val Gly Arg Thr Ala Gly Trp Asn Ile Pro Met

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Gly Leu Leu Tyr Asn Lys Ile Asn His Cys Arg Phe Asp Glu Phe Phe

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Ser Glu Gly Cys Ala Pro Gly Ser Lys Lys Asp Ser Ser Leu Cys Lys

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Leu Cys Met Gly Ser Gly Leu Asn Leu Cys Glu Pro Asn Asn Lys Glu

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Gly Tyr Tyr Gly Tyr Thr Gly Ala Phe Arg Cys Leu Val Glu Lys Gly

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Asp Val Ala Phe Val Lys His Gln Thr Val Pro Gln Asn Thr Gly Gly

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Leu Leu Cys Leu Asp Gly Thr Arg Lys Pro Val Glu Glu Tyr Ala Asn

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Cys His Leu Ala Arg Ala Pro Asn His Ala Val Val Thr Arg Lys Asp

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Lys Glu Ala Cys Val His Lys Ile Leu Arg Gln Gln Gln His Leu Phe

805 810 815

Gly Ser Glu Val Thr Asp Cys Ser Gly Asn Phe Cys Leu Phe Arg Ser

820 825 830

Glu Thr Lys Asp Leu Leu Phe Arg Asp Asp Thr Val Cys Leu Ala Lys

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Leu His Asp Arg Asn Thr Tyr Glu Lys Tyr Leu Gly Glu Glu Tyr Val

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Lys Ala Val Gly Asn Leu Arg Lys Cys Ser Thr Ser Ser Leu Leu Glu

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Ala Cys Thr Phe Arg Arg Pro

885

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atgggggtgc acgaatgtcc tgcctggctg tggcttctcc tgtccctgct gtcgctccct 60

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aggtacctct tggaggccaa ggaggccgag aatatcacga cgggctgtgc tgaacactgc 180

agcttgaatg agaatatcac tgtcccagac accaaagtta atttctatgc ctggaagagg 240

atggaggtcg ggcagcaggc cgtagaagtc tggcagggcc tggccctgct gtcggaagct 300

gtcctgcggg gccaggccct gttggtcaac tcttcccagc cgtgggagcc cctgcagctg 360

catgtggata aagccgtcag tggccttcgc agcctcacca ctctgcttcg ggctctggga 420

gcccagaagg aagccatctc ccctccagat gcggcctcag ctgctccact ccgaacaatc 480

actgctgaca ctttccgcaa actcttccga gtctactcca atttcctccg gggaaagctg 540

aagctgtaca caggggaggc ctgcaggaca ggggacagag gtggtggtgg atctggtggc 600

ggaggttctg gcggtggtgg ttctgttcct gataagactg ttaggtggtg cgctgtttca 660

gagcatgagg ctactaagtg ccagagcttc agggaccata tgaagtctgt gatccctagc 720

gacggtcctt ctgttgcttg tgtgaagaag gctagctacc tggattgcat cagggctatt 780

gctgctaacg aggctgatgc tgtgactctt gatgctggtc ttgtgtacga tgcttacctg 840

gctcctaaca accttaagcc tgttgtggct gagttctacg gcagcaaaga agatcctcag 900

accttctact acgctgtggc tgtggttaag aaggacagcg gctttcagat gaaccagctg 960

aggggtaaga agtcttgcca tactggtctt ggtaggtccg ctggttggaa tatccctatt 1020

ggtctgctgt actgcgatct gcctgaacct agaaagcctc ttgagaaggc tgtggccaac 1080

ttcttctctg gatcttgtgc tccttgcgct gatggcactg attttccaca gctttgtcag 1140

ctttgccctg gttgcggttg ctctactctt aaccagtact tcggttacag cggcgctttc 1200

aagtgcctta aggatggtgc tggtgatgtg gcattcgtga agcactctac catcttcgag 1260

aacctggcta acaaggccga tagggatcag tacgagcttc tgtgccttga caacaccaga 1320

aagcctgtgg atgagtacaa ggattgccac cttgctcagg tgccatctca tactgtggtg 1380

gctagatcca tgggtggcaa agaggatctt atctgggagc ttctgaacca ggctcaagag 1440

cacttcggca aggacaagtc taaagagttc cagctgttca gcagccctca cggtaaggat 1500

ctgctgttca aggattctgc tcacggcttc cttaaggtgc cacctagaat ggacgctaag 1560

atgtacctgg gctacgagta cgttaccgcc attaggaatc ttagagaggg gacttgtcca 1620

gaggctccta ctgatgaatg caagccagtt aagtggtgtg ccttgtctca tcacgagagg 1680

ctgaagtgtg atgagtggtc tgtgaacagc gtgggcaaga ttgagtgtgt gtctgctgaa 1740

actaccgagg actgcattgc caagatcatg aacggtgagg ctgacgctat gtctctggat 1800

ggtggattcg tgtacattgc tggtaagtgc ggtcttgtgc ctgtgcttgc tgagaactac 1860

gagaagtctg ataactgcga ggatacccct gaggctggtt actttgctgt tgcagtggtg 1920

aagaagtccg cttctgatct gacctgggat aacctgaagg gcaagaagtc atgtcacacc 1980

gctgttggta gaactgctgg ctggaatatc ccaatgggcc tcctgtacaa caagatcaac 2040

cactgcag 2048

Claims

The fusion protein of 1.EPO and transferrins, which is characterized in that it is included

(I), the amino acid sequence as shown in SEQ ID No.1；Or

(II), the amino acid sequence as described in (I) is substituted, lacks or adds the amino acid sequence that one or more amino acid obtain Column, and with the amino acid sequence functionally identical or similar of amino acid sequence shown in (I)；Or

(III), with the amino acid sequence of (I) or (II) described sequence at least 80% homology.
2. encoding the nucleotide of fusion protein as described in claim 1, which is characterized in that have

(I), the nucleotide sequence as shown in SEQ ID No.2；Or

(II), the complementary nucleotide sequence of the nucleotide sequence as shown in SEQ ID2；Or

(III), with the nucleotide sequence coded same protein of (I) or (II), but due to the degeneracy of genetic code with (I) or (II) the different nucleotide sequence of nucleotide sequence；Or

(IV), one or more nucleotides sequences are substituted, lack or added with nucleotide sequence shown in (I), (II) or (III) Arrange obtain nucleotide sequence, and with nucleotide sequence nucleotides sequence functionally identical or similar shown in (I), (II) or (III) Column；Or

(V), with the nucleotide sequence of (I), (II), (III) or (IV) described nucleotide sequence at least 80% homology.
3. expression vector, which is characterized in that including nucleotide as claimed in claim 2 and carrier to be transformed.
4. expression vector as claimed in claim 3, which is characterized in that the carrier to be transformed is chloroplast expression vector.
5. the construction method of expression vector as described in claim 3 or 4, which comprises the steps of:

Step 1: it is respectively the codon of favorite plant by the codon optimization of the EPO and the fusion protein of transferrins, Nucleotide sequence is as shown in SEQ ID No.2；

Step 2: the nucleotide sequence being cloned into pUC57 carrier, pEPO is obtained.
6. expression vector or plant as described in claim 3 or 4 is wrapped in the fusion protein or preparation of expression EPO and transferrins Application in drug containing the fusion protein；The plant be selected from romaine lettuce, spinach, tomato, radish, Chinese cabbage, corn and soybean, Wheat or tobacco；The organ of the plant is selected from seed, leaf, rhizome or whole plant.
7. application as claimed in claim 6, which is characterized in that the drug is the oral preparation for treating anaemia.
8. host, which is characterized in that conversion has the plant or microorganism of the expression vector as described in claim 3 or 4；The plant Selected from romaine lettuce, spinach, tomato, radish, Chinese cabbage, corn and soybean, wheat or tobacco；The organ of the plant be selected from seed, leaf, Rhizome or whole plant.
9. drug, which is characterized in that including fusion protein as described in claim 1 and pharmaceutically acceptable auxiliary material.
10. drug as claimed in claim 9, which is characterized in that the drug is the oral preparation for treating anaemia.