CN1372001A - Human SP-A1 expression in pichia pastoris - Google Patents

Abstract

The present invention discloses the expression of human SP-Al in Pichia pastoris. Said method is characterized by that it clones human SP-Al gene into expression vector, and utilizes electric shock transformation technique to transfer in into pichia pastoris, and screens out multicopy transformant from transformed strain, and utilizes high-expression methanol oxidase 1 (AOXI) to gene promotor, and uses methanol as unique carbon source to make induction expression of recombinant P. pastoris, in the described inductive expression the concentration of methanol is 0.1-1.0% (g/ml). Said ivnention can produce lots of high-activity human SP-Al, its maximum expression amount can be up to 150 mg/L,l and the expressed object protein can possess opsonic action for making macrophage to kill pathogen.

Description

The expression of human SP-A 1 in Pichia pastoris

The present invention relates to the recombinant technology of a kind of DNA, particularly relate to the expression of people's pulmonary surfactant associated protein human SP-A 1 in Pichia pastoris.

Pulmonary surfactant (pulmonary surfactant, PS) be that lung II type cell is synthetic, a kind of lipid-protein complex of excretory, its major function is to reduce alveolar surface tension, keep alveolar ectasia, prevent the collapse of lung, keep the eupnea physiological activity, this mixture is made up of phosphatide and protein, wherein 90% is phosphatide, and based on DPPC (DPPC) and phosphatidyl glycerol (PG), 8～10% is pulmonary surfactant associated protein (surfactant-associated protein, SP), be divided into SP-A, SP-B, SP-C and SP-D etc., the abundantest with SP-A content, account for 50% of total protein concentration.SP-A is a kind of multi-functional glycoprotein, has a very important role aspect immunity and the inflammatory reaction in the metabolism of regulating pulmonary surfactant, SP genetic expression, lung.

SP-A is a kind of hydrophilic glycoprotein, and the monomer molecule amount is approximately 32Ku, can fluctuate between 28～36Ku isoelectric pH 4～5 because of its glycosylation number difference.Monomer SP-A is divided into 4 discontinuous zones: short aminoterminal ball-like structure district, be rich in oxyproline collagen sample district (CLR), with the hydrophobic region and the carboxyl lectin sugar cog region (CRD) of phospholipids incorporate.Human SP-A gene is positioned at the long-armed middle part of karyomit(e) No. 10, and the about 5kb of SP-A full length gene contains 7 exons, and wherein 4 exons is encoding sequence partly or entirely.The SP-A gene is comprising gene family-SP-AI, an II and pseudogene, with two corresponding cDNA of functional gene be 6A and 1A, both have 94% homology in nucleic acid level, at amino acid levels 96% homology are arranged, and are closely similar.Each functional gene a kind of SP-A amyloid protein precursor of all encoding, molecular weight is about 200～250Ku, needs obtain sialoglycoprotein through a series of posttranslational modifications.Sophisticated people SP-A peptide chain has 2 types (SP-AI, SP-AII), and both reach 96% at homology, and 6 amino-acid residue differences are only arranged.SP-AI and SP-AII are wound in a subunit with 2: 1 ratio, and 6 triple-helix structure subunits link together by covalent linkage and non covalent bond again, form the macromole that a molecular weight is about the biologically active of 700Ku.At the alveolar internal surface, SP-A is many, and form with 18 aggressiveness exists, and is a branch of flower-shaped, and stem is made of collagen sample district, and floral leaf then is phytohemagglutinin sample district.

Clinical employed SP-A is animal lung tissue or people's amniotic fluid extract, because the imperfection of extraction process, water-soluble SP-A loses in a large number, has restricted the function of PS and the application of SP-A.On the other hand, because proteinic immunogenicity, the SP-A medicine can not be extracted by animal lung tissue as the PS phospholipid fraction, is extracted also in quantity and obviously limited ethically by human lung tissue and amniotic fluid.

Up to now, all effort at the active SP-A of procaryotic cell expression system expression all fail, though the research of expressing inhuman SP-A at mammalian cell expression system has the report of success, the still gene engineering product of unmanned SP-A report.

The objective of the invention is to utilize eukaryotic expression system Pichia pastoris to produce a large amount of, highly active human SP-A 1.

Human SP-A 1 gene among the present invention is to obtain according to the content clone who is put down in writing in the paper " conjugated protein AcDNA clone of pulmonary surfactant and sequential analysis ", and the document is derived from " Chinese paediatrics " magazine 2000,38 (3), P153.

Expression vector among the present invention is pPIC9K, also can be in the corresponding yeast born of the same parents and secretion expression carrier.

Host cell among the present invention is Pichia pastoris.

The present invention at first with the human SP-A 1 gene clone to expression vector, change P.pastoris over to by the electric shock transformation technology, and from transform bacterial strain, filter out the multiple copied transformant, with methanol oxidase 1 gene (AOX1) promotor of high expression level, can utilize methyl alcohol to induce the P.pastoris that recombinates is expressed as sole carbon source.Its concrete steps are:

1. human SP-A 1 gene clone.

2. cut etc. the positive colony of method screening reorganization by conversion, plasmid extraction, enzyme, and carry out sequential analysis.

3. the structure of expression vector and sequencing are translated the correct of frame with assurance.

4. the evaluation of gene integration.

5. the evaluation of expression product.

6. the determination of activity of expression product.

Because the present invention adopts methyl alcohol nutritional type yeast expression system, therefore, has following advantage: (1) uses the AOX promotor, transcribes the efficient height, is easy to bring out regulation and control; (2) expression plasmid is readily integrated into genome, is difficult for losing, and is suitable for high density fermentation, the output height; (3) expression product letter sorting enters peroxysome etc. and helps purifying; (4) in the time of translating/after modification.

By to the evaluation of expression product as can be known, human SP-A 1 successfully obtains to express in P.Pastoris, and the target protein mass-energy of expressing is killed and wounded pathogenic agent performance opsonization to scavenger cell.

The invention will be further described below in conjunction with drawings and Examples.

Fig. 1 is the restriction map of pPIC9K/SP-A1 and pPIC9K.

Fig. 2 is the PCR of pPIC9K/SP-A1 and pPIC9K.

Fig. 3 is cloned into T carrier T7 and SP6 primer sequencer map.

Fig. 4 is the expression 12%SDS-PAGE of SP-A1 in Saccharomyces cerevisiae S-78 and Pichia pastoris GS115.

Fig. 5 is the purpose peak of SP-A1 through the HPLC purifying.

Fig. 6 is that the Western hybridization of SP-A1 detects.

Fig. 7 is the kill rate of SP-A conditioning pulmonary macrophage to intestinal bacteria J5.

Embodiment

Related plasmid pPIC9K, yeast strain Pichia postoris GS115, KM71, SMD1168, GS115 Albumin, GS115 β-Gal are all available from Invitrogen company in the present embodiment.

Related being used for amplifying target genes SP-A and identifying that the PCR primer of transformant is synthetic by the biological company limited of the living worker in Shanghai in the present embodiment.

Main enzyme and reagent related in the present embodiment comprise DNA restriction restriction endonuclease, T ₄Dna ligase, Taq enzyme are available from precious biotechnology company limited in Dalian and magnificent biological company limited, and yeast lyase, G418, granulated glass sphere are available from Sigma company.

The operation steps of present embodiment is as follows:

1. substratum

YPD, MGY, MGYH, RD, RDH, RDB, RDHB, MD, MDH, MM, MMH, BMG, BMM, BMGY, the required reagent of BMMY are commercially available, and its concentration is by the explanation preparation.

2.SP-A1 the structure design of amplification primers of expression vector

(+)5’-GATACGTAATGTGGCTGTGCCCTCTG-3’

(-)5’-GCGGAATTCGAACTCACAGATGGTCAG-3’

As template amplification human SP-A 1 gene, the PCR product is cut back purifying purpose fragment with SnaB I with EcoR I enzyme and is connected with the pPIC9K that same enzyme is cut, and makes up pPIC9K/SP-A1 with recombinant plasmid pUC19/SP-A1.

3.E.coli competent making of JM109 and conversion

In the LB of 2ml, 37 ℃, 250rpm shaking culture 10～12 hours is got among the LB that the 0.5ml seed liquor is inoculated in 50ml at dull and stereotyped picking E.coli JM109 or the single bacterium colony of TOP10F ', and 37 ℃, it is 0.6 that 250rpm is cultured to A600, needs 1.5～2.0h approximately; Put culture in 10min on ice, move in the 50ml centrifuge tube, 4 ℃, the centrifugal 5min of 4000rpm; Abandon supernatant, adding 15ml concentration is precooling (4 ℃) CaCl of 0.1mol/L ₂Suspension cell, ice bath 30min; 4000rpm, 4 ℃ of centrifugal 10min reclaim cell, abandon supernatant, add 2ml precooling CaCl ₂Suspension cell, ice bath is standby; Get 100 μ l competent cells, add 10 μ l and connect liquid, mixing, ice bath 30min; 42 ℃ of heat-shockeds 90 seconds are put back to 2min in the ice rapidly, add 500 μ l LB nutrient solutions and cultivate 1h for 37 ℃; Get 150 μ l nutrient solutions and coat the LB flat board that contains penbritin 50～100 μ g/ml, be inverted for 37 ℃ and cultivated 12 hours.Screening purpose recon wherein contains recombinant plasmid pPIC9K/SP-A1.

4. the evaluation of recombinant plasmid

Place the LB that contains penbritin 50-100 μ g/ml, shaking culture 10～12 hours at the dull and stereotyped picking list of the LB that contains penbritin 100 μ g/ml bacterium colony; Alkaline lysis method or purification kit extract plasmid routinely; Carry out enzyme with restriction enzyme SnaB I and EcoR I and cut evaluation; Enzyme is cut correct plasmid alpha factor primer

(+)5’TACTATTGCCAGCATTGCTGC3’

Sequential analysis is carried out in (-) 5 ' CAAATGGCATTCTGACATCC3 ' amplification, guarantees that frame and sequence are correct.

5. the preparation of plasmid

The recombinant plasmid pPIC9K/SP-A1 and the pPIC9K of parental plasmid that correctly make up are increased in e. coli jm109 or TOP10F ', extract required plasmid with the plasmid extraction test kit, carry out restriction enzyme digestion with Sal I then, make it complete linearizing, reclaim test kit recovery linearization plasmid with glue after 0.8% agarose electrophoresis and be dissolved among 1 * TE.

6. the conversion of yeast strain

The single bacterium colony of picking P.pastoris GS115, SMD1168 and KM71 respectively, the YPD that is inoculated in 5ml places the 50ml triangular flask, cultivates 10 hours for 30 ℃, gets 50～100 μ l and is inoculated among the 50mlYPD, and shaking culture 16 hours is to A600=1.3～1.5; 4 ℃ of centrifugal 1500g * 5min, the precipitation sterilization ddH of 50ml precooling ₂O is resuspended; Ditto centrifugal, the precipitation sterilization ddH of 25ml precooling ₂O is resuspended; Ditto centrifugal, precipitation is resuspended with the 1M sorbyl alcohol of 5ml precooling; Ditto centrifugal, precipitation is resuspended standby with the 1M sorbyl alcohol of 1.5ml precooling; The yeast competent cell is packed as 80 μ l/ parts; The linearizing DNA of 5～20 μ g adds in the competence yeast strain, is transferred to the pole cup of 0.2cm, puts 5 minutes on ice; With the Bio-Rad GenePulser conversion of shocking by electricity, its parameter is 1500V, 25 μ F, 400 Ω, adds the 1M sorbyl alcohol 1ml of precooling immediately, and 200～600 μ l/ parts are coated with MD and RDB and select dull and stereotypedly, put 28 ℃ and cultivate 48h and occur to transformant.

7. the screening of multi-copy integration incident

(1) adopt aseptic technique to add 200 μ l YPD in every hole of 96 well culture plates, transformant of every hole inoculation, and thalline is suspended in the YPD substratum, place 30 ℃ to cultivate 2 days; (2) get seed liquor 10 μ l respectively and be inoculated in another 96 well culture plate and respectively contain the corresponding culture hole of 200 μ l YPD, cultivated 1 day for 30 ℃; (3) repeating step (2) once; (4) getting 10 μ l is inoculated in and contains the G418 final concentration and be respectively 0.5,0.75,1.0,1.5,2.0,3.0mg/ml YPD or MD solid culture plate, treat that liquid adsorbs fully after, put 30 ℃ of cultivations, in inoculation back 2,3, observed the growth bacterium colony in 4,5 days, resistance is high more, and the copy number of integration is many more.

8. screen GS115His ⁺Mut ⁺/ His ⁺Mut ^sPhenotype

Transform GS115 or SMD1168 bacterial strain with Sal I linearization plasmid, can there be two kinds of phenotype: His in the recombinant bacterial strain of generation ⁺Mut ⁺/ His ⁺Mut ^sPhenotype uses GS115 Albumin and GS115 β-Gal respectively as His ⁺Mut ^s, His ⁺Mut ⁺Contrast, and KM71 only has His ⁺Mut ^sPhenotype.

(1) picking is seeded in respectively on MM and the MD flat board successively at the single bacterium colony on MD or the RDB flat board, with GS115/ β-Gal and GS115/Albumin in contrast.Cultivated 36～48 hours for 30 ℃.

(2) different its phenotypes of judgement of speed of growing at MM and MD according to bacterium colony, the dull and stereotyped well-grown of MM and MD is His ⁺Mut ⁺Phenotype, the dull and stereotyped well-grown of MD and the dull and stereotyped poor growth of MM or be not grown to His ⁺Mut ^sPhenotype.

9. the extraction of the total DNA of yeast

Nutrient solution (reorganization bacterium and parent GS115, KM71, the SMD1168) 1.5ml that cultivated 16 hours is moved to the Eppendorf pipe, centrifugal 30 seconds, abandon supernatant; It is resuspended to add 45 μ l lyase damping fluids in the precipitation, adds 5 μ l yeast lyase, and the vibration back vibrates frequently in 37 ℃ of hatching 1h; Add 10 μ l Proteinase Ks, vibration several seconds, 55 ℃ of hatching 15min, vibrations frequently; Add 5 μ l RNase A, normal temperature is placed 15min down; Add 500 μ l extracts, vibration, normal temperature is placed 10min, vibration frequently down; Centrifugal, 12000～16000r/min * 2min, crisp of sedimentation cell, supernatant is moved in the GFX post, and 5000g * 1min outwells liquid in the collection tube, again adsorption column is put to collection tube, add 500 μ l extracts, 8000r/min * 1min outwells liquid in the collection tube, adds 500 μ l washing solns, 12000～16000r/min * 3min; Remove collection tube, adsorption column is put to aseptic Eppendorf pipe, add elution buffer or the sterilization ddH of 200 μ l ₂O, room temperature is placed 1min; 8000r/min * 1min, the DNA of collection purifying, 0.8% agarose gel electrophoresis detects.

10. the pcr analysis integrated of yeast

Design following reaction system

10×PCR?buffer 5μl

Genomic?DNA(～1μg) 5μl

100mM?dNTP(25mM?each) 1μl

5’AOX1?Primer(0.1μg/μl) μl

3’AOX1?Primer(0.1μg/μl) μl

Taq?Polymerase(5U/μl) 0.25μl

DdH ₂It is 50 μ l that O adds to cumulative volume

Recombinant plasmid is as positive control, and corresponding empty plasmid contrasts as another, and its thermal circulation parameters is as shown in table 1.

Table 1 thermal circulation parameters

72 ℃ of 7min 1 of step temperature-time cycle number Hot Start 94 ℃ of 1min Annealing of 94 ℃ of 2min, 1 Denaturation 55 ℃ of 1min, 25 Extension, 72 ℃ of 1min Final Extension

Get 10 μ l samples and carry out 1.5% agarose gel electrophoresis.

11. the expression of reorganization P.pastoris

Picking His ⁺Mut ⁺Single bacterium colony, place the 250ml of the BMG that fills 25ml to shake bottle, 28 ℃, 250r/min are cultured to A600=2～6 (about 16～18h), 1500～3000g under the normal temperature * 5min centrifugation thalline, remove supernatant, to A600=1.0 (about 200ml), place that 1000ml's shake bottle 96h with BMMY resuspended thalline, respectively at 0h, 6h, 12h, 24h, 36h, 48h, 60h, 72h, 84h, the 96h 1ml that takes a sample, the centrifuging and taking supernatant stores-80 ℃ and treats further electrophoretic analysis.Every 24h adds 100% methyl alcohol, and to make final concentration to the substratum be 0.5%, keeps abduction delivering.

12.SDS-PAGE analyze

The sample of getting each time point melts and places on ice, and rotary evaporation or ultrafiltration and concentration sample are to 1/5～1/10 of original volume, and equal-volume adds the electrophoresis sample-loading buffer, boils 10min, and last sample carries out the 12%SDS-PAGE electrophoresis.Surplus sample place-20 ℃ standby, supernatant places-80 ℃ to wait until further analysis.

13.SP-A1 expression in P.pastoris and product purification

The supernatant that above-mentioned expression obtains is through rotary evaporation (lyophilize Sentry ^TMVirTis company) is concentrated into 1/5～1/10, after transferring that pH to 6.5 is centrifugal and remove sedimentary foreign protein with solid Tris,, collects albumen elutriant (about 25ml), through Sepharose 4B and Sephadex G-75 chromatography through the desalination in batches of Sephadex G-25 chromatography column.

14. high performance liquid chromatography (HPLC) purified product through the C-8 column purification, with the warm and fine water elution of second, obtains protein peak in the 20min place.

15.Western hybridization

Connecing ordinary method handles protein example and carries out SDS-PAGE; After electrophoresis was finished, dismounting gel interlayer was removed spacer gel.With an amount of transfering buffering liquid equilibrium at room temperature gel 30min.; Preparation nitrocellulose filter (NC film) is pressed the gel size but than the about 1mm of gel, distilled water is slowly put into gel in angle at 45, and water will infiltrate in the film and moistening whole surface.Balance 10～15min in transfering buffering liquid then; Base in the plastics transfer box, successively with following article assembling transfer printing interlayer: sponge, several are sheared as the filter paper of gel size, and in advance with the moistening gel of transfering buffering liquid, NC film, several moistening filter paper, sponge, assembling process notes getting rid of all bubbles; In transfer groove, add transfering buffering liquid, transfer box is put in the electroporation, connect power supply by correct polar orientation; 100V electrotransfer 30～60min under cooling conditions, the dismounting transfer device takes out the NC film, and cuts Yi Xiaojiao serve as a mark on film; Gel uses coomassie brilliant blue staining to determine transfer efficiency; Film is put into heat-sealable plastics bag, add the 5ml confining liquid, sealed plastic bag shakes 30～60min in rotary shaker or oscillating platform; With confining liquid dilution mouse-anti people SP-A (1: 3000); The liquid of blockading that inclines adds the first antibody of dilution, and 4 ℃ are spent the night; Be with gloves from plastics bag, to take out the NC film, be placed in the plastics casing and shake, with 20ml PBST washing 4 times, each 10～15min; With PBS dilution HRP mark sheep anti-mouse igg (1: 5000); Put the NC film into new plastic bag, 37 ℃ of 1h take out the NC film, wash film 15min with PBS under the room temperature and remove excessive phosphoric acid salt and Tween20; Film is put into chromophoric substrate colour developing liquid, and band occurs in 10～30min; With the distillation washing film, termination reaction is taken pictures or is directly taken pictures after drying.

16.SP-A1 opsonization

(1) separation of mouse macrophage: pulmonary macrophage is pressed document Journal of Infectiousdiseases 1995; Separate in the male rat of reported method by specific-pathogen free among 172 (2) P481, be resuspended in the A damping fluid, the consisting of of this damping fluid: 140mM NaCl, 5mM KCl, 2.5mMNa ₂PO ₄, 10mM HEPES, 2mM MgSO ₄, 6mM glucose, 2mM CaCl ₂, 0.1% gelatin (wt/vol), pH is 7.4.The pulmonary macrophage final concentration is 10 in the suspension ⁷Cell/ml.

(2) preparation of SP-A: the SP-A1 of natural pig SP-A and reorganization (yeast saccharomyces cerevisiae P1 and P.pastoris P2) is dissolved in 5mM HEPES, and pH is 7.4, break into portions be stored in-70 ℃ standby.

(3) microbial culture: E.coli J5 is incubated at respectively in the LB substratum, and 37 ℃ of shaking culture are spent the night, and are centrifugal under the normal temperature, 3000g * 2min collects bacterium, and is with A damping fluid washing 3 times, centrifugal, 2500g * 10min, the A damping fluid is resuspended, and it is 5 * 10 that fluorescent spectrophotometer assay makes its final concentration ⁸Cfu/ml.

(4) bacterium killing experiments: when having or not having SP-A and/or pulmonary macrophage and exist, test it and engulf killing activity.Adopting bacterium/scavenger cell ratio is 1: 1, guarantees that bacterium is absorbed effectively, and various dose SP-A is added in the mixed solution, and final volume is 200 μ l, slightly rocks 37 ℃ of hatching 30min, the ddH of adding precooling ₂O ends to engulf and lethal effect, with being coated with agar plate after isopyknic LB dilution, 37 ℃ of overnight incubation, counting clone.Three, result

1.pPIC9K/SP-A1 the evaluation of recombinant expression vector: find to cut out the purpose fragment with SnaB I and EcoR I restricted enzyme cutting analysis, as shown in Figure 1,1 among Fig. 1 is DNAMarkers, and 2,3 is pPIC9K, and 4,5 is pPIC9K/SP-A1.Recombinant plasmid and parental plasmid obtain the fragment of 936bp and 188bp respectively through the alpha factor primer amplification, and as shown in Figure 2,1～3 among Fig. 2 is pPIC9K/SP-A1, and 4 is Markers, and 5～6 is pPIC9K.Be cloned into T carrier T7 and SP6 primer sequencing result, sequence and frame are entirely true, and as shown in Figure 3, explanation successfully makes up the pPIC9K/SP-A1 expression vector thus.

2. the GS115 that transforms with Sal I linearization plasmid of yeast conversion result, its phenotype is that HIS4Mut+ has 167 bacterium colonies, phenotype is HIS4 ⁺Mut ^s28 bacterium colonies are arranged, about 6: 1 of the ratio of the two, the visible transformant overwhelming majority that obtains is to utilize the quick growth form of AOX1 promotor.Transform SMD1168 and obtain similar result.The host was that the transformant of GS115, SMD1168 and KM71 has 23,18,21 respectively when G418 concentration was 3.0mg/ml.

3.SP-A1 the abduction delivering in P.pastoris

Recombinant bacterial strain is expressed when methanol concentration is 0.1～1.0% (g/ml), and abduction delivering best results during methanol concentration 0.5% is cultivated 72h, and protein reaches maximum expression amount, i.e. 150mg/L, and its expression amount scope of different strains is 0mg/L～150mg/L.The expression 12%SDS-PAGE of SP-A1 in Saccharomyces cerevisiaeS-78 and Pichia postoris GS115 as shown in Figure 4, wherein, 1 is Protein Markers, 2 is SP-A1 (S-78), 3 is SP-A1 (GS115).

4. the purifying of product: expression product obtains the target protein matter that size is about 32kD through Sephadex G-25, Sepharose 4B and SephadexG-75.

5.HPLC obtain the purpose peak behind the purifying as shown in Figure 5.

6. the antibody with anti-SP-A carries out Western hybridization detection

Utilize the ELISA method to detect the immunogenicity of natural SP-A and gene expression product respectively, as seen the feature similarity of two response curves, it all is typical S type curve, the antigenic characteristic that shows SP-A1 is identical with natural SP-A's, Figure 6 shows that the Western hybridization that is expressed in the SP-A1 among the Pichia pastoris, wherein, 1 is pPIC9K/GS115, and 2 is pPIC9K-SP-A1/GS115.

7.SP-A opsonization

37 ℃ of hatching 30min, observation is the bacterium surviving rate when having or not having pulmonary macrophage and SP-A and exist, the independent kill rate of pulmonary macrophage is 5.6% ± 2%, killing-efficiency obviously increases when adding SP-A, and SP-A itself does not have influence to bacterium, and SP-A conditioning pulmonary macrophage to the kill rate of intestinal bacteria J5 as shown in Figure 7.

Claims (5)

1. the expression of human SP-A 1 in Pichia pastoris, it is characterized in that: with the human SP-A 1 gene clone to expression vector, change P.pastoris over to by the electric shock transformation technology, and from transform bacterial strain, filter out the multiple copied transformant, with methanol oxidase 1 gene (AOX1) promotor of high expression level, utilize methyl alcohol to induce the P.pastoris that recombinates is expressed as sole carbon source.

2. the expression of human SP-A 1 according to claim 1 in Pichia pastoris is characterized in that: in the structure of described human SP-A 1 expression vector, designed amplimer is:

(+)5’-GATACGTAATGTGGCTGTGCCCTCTG-3’

(-)5’-GCGGAATTCGAACTCACAGATGGTCAG-3’。

3. the expression of human SP-A 1 according to claim 1 and 2 in Pichia pastoris is characterized in that: it is 1500V, 25 μ F, 400 Ω that described electric shock transforms parameter.

4. the expression of human SP-A 1 according to claim 1 in Pichia pastoris is characterized in that: described methanol concentration is 0.1～1.0% (g/ml).

5. the expression of human SP-A 1 according to claim 4 in Pichia pastoris is characterized in that: described methanol concentration optimum value is 0.5% (g/ml), and incubation time is 72h.

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