CN107236759A - Application of the romaine lettuce as host in expressing protein and/or polypeptide - Google Patents

Abstract

The present invention relates to biological technical field, more particularly to application of the romaine lettuce as host in expressing protein and/or polypeptide.Using romaine lettuce, come transient expression, (4d) can produce the protein of high content to the present invention in the short period of time.This method reduces bio-safety problem to greatest extent, and romaine lettuce is substantially free of plant noxious material, and itself fiber is few, beneficial to the protein purification in downstream.

Description

Application of the romaine lettuce as host in expressing protein and/or polypeptide

Technical field

The present invention relates to biological technical field, more particularly to romaine lettuce as host in expressing protein and/or polypeptide should With.

Background technology

Expression and production system of the plant as pharmaceutical protein, have studied nearly 30 years.Except low cost and production Outside the high advantage of amount, the expression system based on plant also reduces humans and animals pathogen and passed during produce protein It is multicast to the risk of the mankind.In addition, plant eukaryotic albumen inner membrance expression system and secretory pathway are similar to mammalian cell.Plant Expression system can largely produce macromolecule and multi-subunit pharmaceutical protein, and be significantly better than prokaryotic expression system, Such as escherichia expression system.If desired for posttranslational modification, glycosylated albumen, and need the monoclonal assembled to resist Body, it can not be realized with prokaryotic system.Pharmaceutical protein by the use of plant production has been commercialized as biological reagent, or uses writer The vaccine additive of fowl.2012, food and drug administration (FDA) have approved to be thanked for treating the type dagger-axe of genetic disease 1 The albumen ELELYSO of disease^TM(taliglucerase alfa), and this albumen is produced using carrot.Past 10 years In, people sharply increase to the demand of pharmaceutical protein, so FDA ratifies the number of the plant medical protein for clinical test Amount is also being continuously increased.

Green fluorescent protein is repaiied et al. in 1962 in a kind of scientific name Aequorea victoria water by Xia Cun earliest Found in mother.Protein produced by its gene, in the case where the light of blue wavelength region is excited, can send green fluorescent.This Cold light a-protein equorin help, and this cold light protein and calcium ion (Ca are also needed in luminous process²⁺) can produce Raw reciprocation.

Earthworm is utilized as conventional medicament thousands of years in China, Japan and other Far Eastern countries.Oral dry earthworm powder quilt Think to be conducive to blood circulation system.Lumbrokinase (Lumbrokinase) is one group for separating and purifying from different types of earthworm Enzyme.These enzymes are considered to be useful for treating the fibrinolytic agent of the various illnesss related to thrombosis.A lot of Lumbrokinases Gene has been cloned.The progress of genetic technique provide production restructuring Lumbrokinase ability, and have been used to clinical test with And for oral health care product.So far position, GenBank has stored 24 lumbrokinase gene sequences.In Escherichia coli, mountain Lumbrokinase is successfully expressed in sheep mammar gland, yeast P. pastoris and plant.Because it is easily handled and fast fast-growing Long, escherichia expression system has with the potentiality of the production Lumbrokinase of inexpensive high yield.However, being produced in Escherichia coli Restructuring LKs be generally inclusion body.Therefore, renaturation process is recovery and rebuild necessary to enzymatic activity.Because prokaryotic can not Posttranslational modification is carried out, Escherichia coli possibly can not carry out appropriate folding, process and glycosylate to express eucaryon Lumbrokinase egg In vain.Surface is studied, plant is had become except bacterium, the replacement of the convenience and economy of yeast or mammalian cell production medicine Product.Plant has the mechanism needed for posttranslational modification necessary to realize protein stability and bioactivity.Albumen in plant Matter route of synthesis is also closely similar with zooblast.The cost ratio for producing pharmaceutical protein using botanical system uses mammal Cell culture and microbial fermentation system considerably cheaper.In general, recombinant human pharmaceutical protein is produced using botanical system Matter and its cost of derived product are only the 2%- of 0.1% and microflora using mammalian cell culture system 10%.Importantly, plant is not the host of human pathogen.Therefore, the recombinant protein from plant unlikely will Pathogenic agent is broadcast to the mankind.

Recombinant protein is expressed in plant two kinds of main methods：Develop the Transgenic Plant Lines of stable heredity or in plant Middle Transient Expression exogenous proteins.First strategy is will to encode the protein DNA to be cloned into expression vector, and is passed In the nucleus or plastid genome of delivering to plant.Stable integration of the exogenous DNA in plant causes the albumen in the table of offspring Reach, and can in the wild or greenhouse large-scale production.However, stablizing expressing protein labor intensive material resources in plant.And sieve Select expression quantity high, inheritance stability, the good homozygote strain of economical character usually requires several years.In addition, on bio-safety Problem, such as genetic drift, pollen contamination and the prevention and control of plant diseases, pest control can also cause the concern of biotechnology regulator.And second of side Method is instant expression of exogenous protein.Foreign gene is not integrated directly into Plant Genome, only the transient expression in host cell Required protein.Compared with stable conversion, transient expression can produce the albumen of high content (in one week) in the short period of time Matter.This method reduces bio-safety problem to greatest extent, because treated plant or tissue is typically to seal completely Developed in the facility or container that close, in the absence of biological pollution problem.

Plant transient expression system can also be used to production recombinant protein to tackle emergency.2014, it is uniquely used for It is effective against the Antybody therapy medicine of Ebola virus outburst, ZMapp^TM, it is exactly in tobacco leaf using Agrobacterium permeating method Middle production.ZMapp effect and security opens road for the industry of promotion plant pharmacy industry.At present, tobacco is moment egg Most common host plant is expressed in vain, and has developed various carriers and Agrobacterium permeating method, in a short time Mass produced.However, tobacco has a high microsteping content and potential toxic compounds, such as alkaloid nicotine, significantly The cost in downstream purification process is added, the further development of plant foreign protein medicine is greatly hindered.

Have at this stage and produce vaccine, albumen using genetically modified plants.But genetically modified plants production process is long, general two Year, and the problem of there is gene contamination in genetically modified plants, and contain a large amount of albumen in plant, starch, polysaccharide etc., to downstream Purification requirements are higher.Have at present using tobacco leaf come the technology of Transient Expression pharmaceutical protein, general 7 days of cycle.Tobacco contains There is nicotine, nicotine, various poisonous aldehydes matters cause downstream purification difficulty in process.

Compared with tobacco leaf system, less phenols and toxic compounds are contained in romaine lettuce, therefore provide a kind of romaine lettuce system Expression medical protein or polypeptide have important practical significance.

The content of the invention

In view of this, the present invention provides application of the romaine lettuce as host in expressing protein and/or polypeptide.The present invention is utilized Two kinds of albumen green fluorescent proteins (GFP) and Lumbrokinase prove that romaine lettuce expression platform can be for life under mild conditions Produce other allogenic polypeptides and protein.

In order to realize foregoing invention purpose, the present invention provides following technical scheme：

Application the invention provides romaine lettuce as host in expressing protein and/or polypeptide.

In some specific embodiments of the present invention, the albumen is medical protein.

In some specific embodiments of the present invention, the medical protein is selected from antibody, enzyme, growth factor or vaccine.

In some specific embodiments of the present invention, the albumen is green fluorescent protein or Lumbrokinase.

Present invention also offers a kind of expression vector, including binary plant carrier and albumen in the application and/or The nucleotide sequence of polypeptide.

In some specific embodiments of the present invention, the albumen is Lumbrokinase, the construction method of the expression vector Comprise the following steps：

The present invention have selected Lumbrokinase PI239 as candidate gene, and it is cloned from Bifidobacterium (L.bimastus). PI239 previously has excellent antithrombus formation feature in Escherichia coli and Yeast system are expressed and are proved to.

Step 1：Xbal restriction enzyme sites are separately added into 5 ' ends of Lumbrokinase nucleotide sequence, in 3 ' ends point Not Jia Ru BamH1 restriction enzyme sites, be cloned into pUC57 carriers, obtain pUC-Lum cloning vectors；Lumbrokinase PI239cDNA sequences (GenBank:AF433650.1) as shown in SEQ ID No.1；The ammonia of Lumbrokinase albumen PI239 complete sequences Base acid sequence is as shown in SEQ ID No.2；The amino acid sequence of ripe Lumbrokinase albumen PI239 sequences such as SEQ ID No.3 institutes Show；The PI239DNA sequences of restriction enzyme site are added as shown in SEQ ID No.4；

Step 2：Genetic fragment PI239 is obtained from the pUC-Lum cloning vectors by Kpnl/Sacl, is cloned into double First plant vector pCam35S, obtains expression vector p35-Lum.

The present invention some specific embodiments in, the albumen be green fluorescent protein, the expression vector be containing There is GFP plant expressing vector pCambia1302.

Present invention also offers application of the described expression vector in expressing protein and/or polypeptide.

Present invention also offers a kind of method of romaine lettuce as host expresses albumen and/or polypeptide, described expression is carried Body is transformed into Agrobacterium, is entered by agriculture bacillus mediated vacuum infiltration after romaine lettuce tissue, extracting and developing protein, obtains albumen And/or polypeptide.Preferably, the Agrobacterium is selected from agrobacterium tumefaciens；It is furthermore preferred that agrobacterium tumefaciens are LBA 4404.

In some specific embodiments of the present invention, the agriculture bacillus mediated vacuum infiltration comprises the following steps：

Step 1：Vacuumize 25~45s；

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

Step 3：Release pressure causes penetrating fluid to penetrate into the plant tissue；

Repeat the above steps 2~3 times, lucifuge processing 4d.

The growth time of the tobacco plant permeated for vacuum Agrobacterium was usually 4 to 6 weeks.And this invention removes plant Growth cycle, greatlys save the time that early stage cultivates plant.The present invention using romaine lettuce come transient expression in the short period of time (4d) can produce the protein of high content.This method reduces bio-safety problem to greatest extent, because treated life Dish tissue is typically to be developed in completely enclosed facility or container, in the absence of biological pollution problem.Romaine lettuce is substantially free of plant Thing noxious material, and itself fiber is few, beneficial to the protein purification in downstream.

The present invention proves romaine lettuce under mild conditions using two kinds of albumen green fluorescent proteins (GFP) and Lumbrokinase Expression platform can be for producing other allogenic polypeptides and protein.

Brief description of the drawings

In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing There is the accompanying drawing used required in technology description to be briefly described.

Fig. 1 shows plant binary pCambia1302；

Fig. 2 (A) shows the method demonstration of romaine lettuce Agrobacterium vacuum infiltration；

Fig. 2 (B) shows Lettuce in Soilless Culture；

Fig. 2 (C) shows that whole system airtight vacuum is handled in penetrating fluid of the reversing immersion containing Agrobacterium；

After Fig. 3 (A) shows Agrobacterium vacuum infiltration 4 days；

Fig. 3 (B) shows that under ultraviolet light most romaine lettuce, which are organized in after vacuum infiltration 4d, shows strongly green glimmering Light (GFP)；On the contrary, non-Agrobacterium infiltration romaine lettuce is then shown red (chloroplaset) under ultraviolet light；

Fig. 4 shows the visualization that GFP is expressed in crude extract (A), final purifying (B)；Wherein WT:Impermeable romaine lettuce；GFP：Agriculture Bacillus vacuum infiltration romaine lettuce；

Fig. 5 shows that SDS-PAGE is analyzed；Swimming lane 1：GFP from Escherichia coli；The homogenate of wild type romaine lettuce is (swimming lane 2,3) or pure GFP albumen (4) after change；Arrow shows GFP (27kD) size

Fig. 6 shows that GFP concentration is 0.37mg/g leaf weights by fluorescence spectrum method for measuring GFP yield；

Fig. 7 shows plant Transient Expression carrier schematic diagram；Wherein, 35S, with ' the UTR CaMV of tobacco mosaic virus (TMV) (TMV) 5 35S promoter；NPT II, the expression for the coding nptII genes of kalamycin resistance；Nos 3 ', terminator；

Fig. 8 shows SDS-PAGE (A) and western traces (B) analysis restructuring PI239；-CK：Feminine gender from impermeable leaf Control；

Fig. 9 shows that fibrin plate is determined；Fig. 9 (A) tests the eluent of purifying on fibrin plate；Fig. 9 (B) is cracked Haloing diameter；Wherein-CK：The elution (negative control) of impermeable leaf；WT：PI239；LK：Standard Lumbrokinase (18,000u/mg)； Value represents average value ± SE；Different letters represents significant difference (p≤0.05；N=4；One-way analysis of variance is tested)；

Figure 10 shows external human blood Clot Lysis Test；Figure 10 (A) restructuring PI239 dissolving human body clots；Figure 10 (B) clot Dissolve percentage；PBS：1 × phosphate buffered saline (PBS)；-CK：The false elution (negative control) of impermeable；LK：Standard Lumbrokinase (18,000u/mg)；Value represents average value ± SE；Different letters represents significant difference (p≤0.01；N=3；Unidirectional variance point Analysis test).

Embodiment

Application the invention discloses romaine lettuce as host in expressing protein and/or polypeptide, those skilled in the art can To use for reference present disclosure, technological parameter realization is suitably modified.In particular, all similar replacements and change are to this It is it will be apparent that they are considered as being included in the present invention for art personnel.The method of the present invention and application are It is described by preferred embodiment, related personnel substantially can be not departing from present invention, in spirit and scope to herein Described methods and applications are modified or suitably change is with combining, to realize and apply the technology of the present invention.

Result of the present invention shows that romaine lettuce system can be more effective expression platform, be quick, transient expression recombinant protein Matter provides method.Vacuum Agrobacterium permeating method described herein is simple, quickly, reduces blade necrosis, and can improve Recombinant protein yield.Romaine lettuce can increase protein output by bearing vacuum pressure, and allow every leaf more complete Infiltration.Due to romaine lettuce be easy to growth and can commercial a large amount of productions, therefore than other transient expression plants, such as tobacco is more held Easily obtain and less expensive.And due to not needing special installation or liquid nitrogen, cost benefit is higher.This research proves that this method can For large-scale production of recombinant proteins matter in the short time.

The romaine lettuce that the present invention is provided is equal as raw materials used and reagent in application of the host in expressing protein and/or polypeptide It can be bought by market.

With reference to embodiment, the present invention is expanded on further：

Embodiment 1 utilizes romaine lettuce system production green fluorescent protein (GFP)

Tissue and Agrobacterium tumdfaciens prepare：

Ripe romaine lettuce (red autumnal leaves, greenery and come into leaves) is have purchased from local greengrocer's, is placed under standard growth desk lamp and cultivates 12h；Or carry out romaine lettuce kind shown in soilless culture such as Fig. 2 (B).(it will contain containing binary plant carrier pCambia1302 GFP, Fig. 1) agrobacterium tumefaciens bacterial strain LBA 4404 be inoculated into 0.5L YEB (yeast extract meat soup, 5g/L sucrose, 5g/L pancreases Peptone, 6g/L yeast extracts, 0.24g/L MgSO₄, pH7.2) and supplement antibiotic liquid culture medium (that is mould for 50mg/L cards Element).The culture of inoculation is incubated 72h in oscillator (220rpm) with 25-28 DEG C.Measured by adding YEB culture mediums OD600 values are simultaneously adjusted to 3.5~4.5.Then nutrient solution is collected, (4500 rotating speed) 10min is centrifuged.Agrobatcerium cell is resuspended in Osmotic medium (10mM MES, 10mM MgSO₄) in O.D.600 be 0.5.

Agriculture bacillus mediated vacuum infiltration：

Optimize the method (Fig. 2) of Agrobacterium vacuum infiltration.The Agrobacterium culture suspension prepared is placed in 2L beakers, It is placed in drier.Preceding 10% romaine lettuce is cut off with knife, (core is upward) is inverted and lightly rotates on bacterium and hanged In supernatant liquid, drier is sealed, shown in such as Fig. 2 (C).Vavuum pump (Welch Vacuum, Niles, IL, USA) is opened to take out Sky, keeps 30~60s of pressure state.Then open the system to discharge pressure, the space for making penetrating fluid penetrate into tissue.The mistake Cheng Chongfu 2~3 times, until high-visible penetrating fluid has obvious diffusion in romaine lettuce tissue.Then by romaine lettuce tissue from penetrating fluid In gently take out, and with distilled water continuous flushing three times, in the container for being then transferred into plastic foil covering.The sample of processing is existed 4d is kept in dark, monitors and takes pictures under UV light (395nm).Experiment is repeated at least 5 times.

After infiltration, most romaine lettuce flood during being organized in vacuum immersion, in addition to firm middle rib region, remaining Part shows strong green fluorescence (Fig. 3) after vacuum infiltration 4d.Observed by test of many times, the system is proved to very Effectively.Present invention optimizes agroinfiltration, reduction blade necrosis increases protein expression, extracted, reclaims and purge process.

Protein Extraction and separation：

Romaine lettuce sample through Agrobacterium vacuum infiltration is stirred with agitator, and is 1 with volume ratio:The extraction buffering of 1 ratio Liquid (100mM KPi, pH7.8；5mM EDTA；10m M beta -mercaptoethanols) 1~2min of mixer high speed homogenate.By homogenate Regulation is 8.0 to pH, and with filtered through gauze, filtrate with 10,000g centrifuges 15min to remove cell fragment at 4 DEG C.Collect supernatant Liquid, is mixed with ammonium sulfate (50%), and is incubated 60min shaking on ice.Divided again at 4 DEG C by centrifuge (10,000g) From 15min.Obtained supernatant is carried out second and takes turns ammonium sulfate (70%) precipitation, suspension 60min is shaken on ice, again at 4 DEG C Under with 10,000g centrifuge 15min.Then, abandoning supernatant, 5mL buffer solutions (20mM is dissolved in by processing sample pellet protein KPi, pH7.8；2mM EDTA；10mM beta -mercaptoethanols) in and at 4 DEG C store.GFP yield passes through spectrofluorimeter (PerkinElmer Enspire 2300Multilabel Reader, MA, USA) is quantitative, emission peak 470~506nm it Between.

The Downstream processing of the recombinant protein of plant origin is typically difficult to and expensive, because cellulose cell wall is difficult to split Solution and secondary plant metabolites.The fluorescence naked eyes visible (Fig. 4) of GFP in sample are extracted in mixer mixing.Use SDS-PAGE Gel confirms to detect restructuring GFP bands (Fig. 5) from the romaine lettuce of vacuum infiltration.In addition, pure using light splitting fluorescence analysis (Fig. 6) The protein content for changing sample is 0.37mg.

GFP is visualized and SDS-PAGE analyses：

In order to confirm that GFP expresses necessary being, sample (5 μ of raw plant extract and the final protein of purifying are collected L), and in dark interior show under w light.By room temperature or thermal denaturation (95 DEG C) GFP loading buffer solution (Biorad, Hercules, CA, USA) 4~12%Bis-Tris Plus SDS- gels (ThermoFisher Scientific, Waltham, MA, USA) run electrophoresis.Then gel is entered again after being dyed with Coomassie blue G250 (Biorad) Row is taken pictures.

GFP is carried out by sepectrophotofluorometer to quantify：

Bacterial controls GFP and romaine lettuce are measured using PerkinElmer Enspire 2300Multilabel Reader The GFP of conversion, with the albumen quality that relatively time method is obtained.Standard is developed using the GFP of the known quantity from commercial sample Curve.By to the comparative analysis from romaine lettuce (wild type WT) sample of the same race, except removal background egg from the sample containing GFP White fluorescence.The RFU of the background proteins concentration of each sample is plotted on standard curve, to determine that GFP's in each sample is near Like amount.Relative fluorescence units are calculated using from the transmitting data of the single excitation peak between 406-460nm, emission peak is in 470- Between 506nm.Duplicate measurements (n=12) is carried out in these parameters.

Embodiment 2 utilizes romaine lettuce system production Lumbrokinase

Plant expression constructs：

Based on GenBank (Accession no：AF433650 total length PI239DNA sequences), we are by restriction site XbaI sites are connected to PI239 5' ends, BamH1 sites are added in the 3' ends of fragment, by GenscriptInc (Piscataway, NJ, USA) is synthesized, and is cloned into pUC57 carriers, is obtained cloning vector pUC-Lum.By KpnI/SacI from Fragment PI239 is isolated in pUC-Lum, and is cloned into plant expressing vector pCam35S (preservation of this laboratory), to generate plant Expression vector p35-Lum.Then by plant expression vector p35-Lum by Multiporator (Eppendorf, Hamburg, Germany) Electroporation Transformation is determined into agrobacterium tumefaciens BA4404 for transient expression then in romaine lettuce.Utilize The size of PAGE gel electrophoresis observation purification of Recombinant Lumbrokinase albumen.

FA is determined：

Agarose (0.2g) is placed in 40mL phosphate buffered saline (PBS) (1xPBS), by being boiled then in microwave It is cooled to about 40 DEG C.Before gel solidification, by 32mg human fibrinogens (Sigma-Aldrich, St.Louis, MO, USA), 10 unit human plasminogens (rPeptide LLC, Bogart, GA, USA) and 10 unit human prothrombin (BioPharm Laboratories LLC, Bluffdale, UT, USA) it is added in gel and slightly rotates mixing to prevent from introducing bubble.So After pour the mixture into culture dish and it is solidified at room temperature.Hole (3mm diameters) is then made in curing gel.By 10 μ L concentrating samples (1 μ g/ μ L) are diluted in 40 μ L 1xPBS buffer solutions, are fitted into gel pore, and be incubated overnight at room temperature. Using the Lumbrokinase (Doctor's Best Inc, Irvine, CA, USA) of standard as positive control (10 μ g), it will come from non- The romaine lettuce blade eluent of Agrobacterium infiltration conversion is used as negative control.Measure the actual diameter (mm) of clear haloing.With standard LK products (18,000U/mg) are compared, and calculate restructuring PI239 FA.Average value ± SE (n=4) passes through list Analysis of variance is analyzed.

The present invention has cloned binary plant expression vector p35-Lum.After the completion of structure, digested and demonstrate,proved with specific restriction enzyme Real genetic fragment is complete (Fig. 7).PI239 is recombinated by SDS-PAGE separation (Fig. 8 A).We observe estimation point in swimming lane Son amount is about 37kDa band.Without significantly corresponding band in Negative control lanes.In addition, passing through HisProbe- The Western blot analysis of HRP methods also detects that about 37kDa band (Fig. 8 B).It was observed that molecular weight (37kDa) it is big In the prediction size of PI239-His6 chains (32kDa), this species diversity is most likely to be because the glycosylation of PI239 albumen causes 's.Obtained protein content is calculated based on the band intensity on Bradford determination methods and SDS-PAGE and is about 135 μ g/g Leaf fresh weight (LFW).

Blood clotting block analysis is determined：

Carry out extracorporeal blood clot lysis measure.By the clot of purchase (Interstate Blood Bank, Memphis, TN, USA) it is sliced into small pieces, and with 1xPBS buffer solution thoroughly cleanings.Then, blood clot (about 50mg/ grumeleuses) is transferred to 24 In orifice plate, and with 1xPBS wash buffers twice.40 μ L concentrating samples (1 μ g/ μ L) are diluted in 460 μ L 1xPBS buffer solutions In, and add in the hole containing clot.After being incubated overnight at 37 DEG C, undissolved sample is measured, and determine afterwards before treatment The weight differential of sample.The eluent of PBS and non-transfection blade is used as negative control.Standard Lumbrokinase (40 μ g) is used as Positive control.Clot lysis is calculated with the percentage of weight difference/untreated samples.Average value ± SE (n=3) passes through single factor test side Difference analysis is analyzed.

Lumbrokinase can be by direct solution fibrin come clot of degrading.It can also strengthen endogenous t-PA activity, Plasminogen is converted into fibrinolysin, causes fibrinolysis.In our current research, fibrin plate, which is determined, shows that plant comes The Lumbrokinase in source has obvious solution fibrin (Fig. 9).Show translucent around commercial Lumbrokinase and recombinant protein Haloing region (Fig. 9 A).The cracking diameter for recombinating Lumbrokinase PI239 is respectively 8.8mm, activity less than business Lumbrokinase (18, 000U/mg), it dissolves a diameter of 12.25mm (Fig. 9 B).Restructuring PI239 relative fiber protein dissolution activity is calculated as about 13, 400U/mg.The Lumbrokinase PI239 of plant origin can dissolve people's blood clot (Figure 10 A) in vitro.By grumeleuse autolytic process, Slight cracking is observed in PBS holes and impermeable leaf extract.Observed when with standard Lumbrokinase and restructuring PI239 processing grumeleuses To obvious cracking.Standard Lumbrokinase and restructuring Lumbrokinase show 92% and 76% clot lysis (Figure 10 B) respectively.With standard Lumbrokinase is compared, and restructuring Lumbrokinase PI239 relatively low activity is probably due to the plant component or residual in purge process Eluent.In the research reported in the past, the significant interference Fibrinolysis egg of residual plant pollutant present in final eluent The enzymatic activity of white enzyme.Therefore it may only be necessary to which further optimized purification strategy obtains the restructuring Lumbrokinase PI239 of high-purity, you can have Effect ground increase fibrinolysis activity.

Embodiment 3

Control group：Utilize leaf tobacco production green fluorescent protein and Lumbrokinase；

Experimental group：Romaine lettuce production green fluorescent protein and Lumbrokinase that the present invention is provided；

The green fluorescent protein of table 1 and Lumbrokinase

^*Show P≤0.05 compared with control group；^#Show P≤0.01 compared with control group；

As shown in Table 1, compared with the tobacco leaf system of control group, romaine lettuce transient expression green fluorescent protein and Lumbrokinase show Write (P≤0.05) and shorten the production cycle, notable (P≤0.05) improves protein content, notable (P≤0.05) improves albumen Activity, simplifies the complexity of protein purification, and extremely significantly (P≤0.01) reduces production cost.

Summary result of the test shows that botanical system especially romaine lettuce system is more economical, efficiently expresses platform. Can quick transient expression recombinant protein, green fluorescent protein and Lumbrokinase can be mass produced in a short time.

Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should It is considered as protection scope of the present invention.

SEQUENCE LISTING

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<120>Application of the romaine lettuce as host in expressing protein and/or polypeptide

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<170> PatentIn version 3.3

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Leu Ser Val Lys Asp Gly Ser Gly Ile Phe Ser Leu Ile Gly Ile Val

195 200 205

Ser Trp Gly Ile Gly Cys Ala Ser Gly Tyr Pro Gly Val Tyr Ala Arg

210 215 220

Val Gly Ser Gln Thr Gly Trp Ile Thr Asp Ile Ile Thr Asn Asn

225 230 235

<210> 4

<211> 866

<212> DNA

<213>Add the PI239 DNA sequence dnas of restriction enzyme site

<400> 4

tctagaatgt tacttctcgc tcttgcatcg ttggtagcgg tgggctttgc gcaaccacca 60

gtctggtacc ccggtggtca atgcggtgtc agccagtact cagatgctgg tgacatggaa 120

cttcctcccg gaacaaaaat tgtcggagga attgaagcca gaccatacga gttcccatgg 180

caggtgtccg tccgaaggaa gtcttccgat tcccatttct gcggaggtag catcatcaac 240

gatcgttggg ttgtctgcgc tgctcactgc atgcagggag agagccctgc cctggtttca 300

ttggtcgtcg gtgagcacga tagcagcgct gcgagtacag tacgtcagac tcatgacgtt 360

gacagcatct tcgtccacga ggactacaac ggaaatacct ttgagaacga cgtttctgtc 420

atcaagacag ttaacgccat cgccatcgac atcaacgttg ggccaatctg cgctccagat 480

ccagccaacg attacgtcta ccgtaagagc cagtgctccg gatggggaac tatcaactca 540

ggtggagtct gctgccccaa cgttctgcga tatgtgacac tgaacgtcac aaccaacgcc 600

ttctgcgatg atatctacag cccattatat acaattacca gcgacatgat ctgcgccacg 660

gacaacaccg gacagaacga gagagactct tgccagggtg actctggcgg ccctctgagc 720

gtcaaggatg gcagcggaat cttcagcctc attggtattg tgtcttgggg aatcggttgc 780

gcatctggat atccaggagt ctacgcccgc gtcgggtccc aaactggatg gatcacagac 840

atctgagagc tcttaattaa ggatcc 866

Claims (10)

1. application of the romaine lettuce as host in expressing protein and/or polypeptide.

2. application according to claim 1, it is characterised in that the albumen is medical protein.

3. application according to claim 2, it is characterised in that the medical protein is selected from antibody, enzyme, growth factor or epidemic disease Seedling.

4. the application according to any one of claims 1 to 3, it is characterised in that the albumen is green fluorescent protein or earthworm Kinases.

5. a kind of expression vector, it is characterised in that should including binary plant carrier and as described in any one of Claims 1-4 The nucleotide sequence of albumen and/or polypeptide in.

6. expression vector according to claim 5, it is characterised in that the albumen is Lumbrokinase, the expression vector Construction method comprises the following steps：

Step 1：Xbal restriction enzyme sites are separately added into 5 ' ends of Lumbrokinase nucleotide sequence, are added respectively in 3 ' ends Enter BamH1 restriction enzyme sites, be cloned into pUC57 carriers, obtain pUC-Lum cloning vectors；

Step 2：Genetic fragment PI239 is obtained from the pUC-Lum cloning vectors by Kpnl/Sacl, double base plant is cloned into Thing carrier pCam35S, obtains expression vector p35-Lum.

7. expression vector according to claim 5, it is characterised in that the albumen is green fluorescent protein, the expression Carrier is the plant expressing vector pCambia1302 containing GFP.

8. application of the expression vector in expressing protein and/or polypeptide according to any one of claim 3 to 7.

9. a kind of romaine lettuce is used as the method for host expresses albumen and/or polypeptide, it is characterised in that will be as any such as claim 3 to 7 Expression vector described in is transformed into Agrobacterium, is entered by agriculture bacillus mediated vacuum infiltration after romaine lettuce tissue, extracting and developing egg White matter, obtains albumen and/or polypeptide.

10. method according to claim 9, it is characterised in that the agriculture bacillus mediated vacuum infiltration comprises the following steps：

Step 1：Vacuumize 25~45s；

Step 2：Keep 30~60s of vacuum -95kPa pressure；

Step 3：Release pressure causes penetrating fluid to penetrate into the plant tissue；

Repeat the above steps 2~3 times, lucifuge processing 4d.