CN1131308C - Artificial synthetic human stem cell factor code gene - Google Patents

Abstract

The present invention discloses a human stem cell factor encoding gene artificially synthesized. The present invention selects a preferred codon of colibacillus to remove the existing sites of EcoRI, XmaI, SmaI, PstI, etc. in natural genes by replacement of the codon on the premise that amino acid sequences are not changed, sites of EcoRI and SalI are introduced on both ends, and sites of HincII and PvuII are introduced in the middle. Because the site of PvuII is positioned in the position near the connecting position of two structure domains in a high-grade structure predicted by hSCF proteins, the present invention provides convenience for the research of the relation of the structure and the functions of hSCF and the design of protein molecules with hSCF as material.

Description

The human stem cell factor code gene of synthetic

Technical field

The present invention relates to the dna sequence dna and the expression method thereof of synthetic.

Background technology

(Stem Cell Factor is to derive from the early stage hematopoiesis regulatory factor of a multiple histiocytic class SCF), in mouse, by 10 to STEM CELL FACTOR ^#Steel seat coding on the karyomit(e) is the outer part of born of the same parents of proto-oncogene c-kit product.Because the characteristic of its stimulate cell growth, bind receptor, be known as mast cell growth factor (Mast Cell Growth Factor again, MGF), the Steel factor (Steel Factor/Steel Locus Fact, SF/SLF), KIT part (Kit Ligand, (Williams such as KL), DE et al, 1990, Cell63 (2): 167-174; Zsebo, KM et al, 1990, Cell 63 (2): 195-201; Martin, FM et al, 1990, Cell, 63 (2): 203-211; Zsebo, KM et al, 1990, Cell, 63 (2): 213-224; Huang, E etal, 1990, Cell, 63 (2): 225-233; Anderson, DM et al, 1990, Cell, 63 (2): 235-243; Williams, DE et al, 1991, Pro Growth Factor Res, 3 (3): 235-242).

From nineteen ninety with Williams DE, three different experiments groups headed by the Zesbo KM, Huang EW report respectively and have found SCF and delivered since the sequence of its cDNA, the research of all respects of SCF has all obtained remarkable progress.The hSCF gene is positioned at chromosomal 12q ^22-24The district, full transmembrane protein of genes encoding, difference splicing by mRNA and proteolytic enzyme are at Ala ¹⁶⁴/ Ala ¹⁶⁵The enzymatic degradation of position also can produce soluble activity form (Martin, FM et al, 1990, Cell, 63 (2): 203-211; Zsebo, KM et al, 1990, Cell, 63 (2): 213-224; Andsern, DM et a1,1991, Cell Growth ﹠amp; Differentiation, 2 (4): 373-378) the soluble form here is meant a kind of activity form of being come through the proteolytic enzyme enzymatic degradation by film mating type, rather than refers to the existence of expression product in intestinal bacteria.The SCF of membranous type, soluble form has biological action important and that have nothing in common with each other, and membranous type SCF is mainly the working in early days of fetal development, and the SCF of soluble form then has effect more widely.

In vivo, the activity form of SCF is non-covalent dimer, and is high glycosylation, but glycosylation to the influence of its biological action little (Zsebo, KM et al, 1990, Cell 63 (2): 195-201; Zsebo, KM etal, 1990, Cell 63 (2): 195-201).Natural gene is recombinant expressed in E.coli, product after renaturation, biologically active.And structure and function relationship aspect, disclose by deletion mutantion, carboxyl terminal part amino acid, to its bioactive keeping not is necessary, but if disappearance has been destroyed the integrity of two disulfide linkage, loss of activity then, this explanation: disulfide linkage is vital for keeping correct senior conformation and biologic activity.Itself and carrier are carried out amalgamation and expression then to be found: merge the uncorrelated small peptide at aminoterminal, carboxyl terminal, to its activity influence little (Nishikawa, M et al, 1992, Biochem Biophys Res Commun, 188 (1): 292-297).

People have also carried out extensive studies to the biological action of STEM CELL FACTOR, find that it has the various biological effect, participate in various physiological processes.To hemopoietic system, SCF be one multispectral be regulatory factor, both can act on early stage hemopoietic stem cell, progenitor cell, can act on late period again than mature blood cell; Both can act on separately, and can work in coordination with other factor again and play the amplification effect; Both can work, cause the mature cell gathering, can be used as a kind of serum factor again, influence the growth of progenitor cell in the part.In addition, research also finds, SCF also plays an important role in the migration of fetal development early archaeocyte, melanocyte and location, the generation of clinical numerous disease all with it or its part defective relevant (Aye, MT et al, 1992, Exp Hematol, 20 (4): 523-527).Because SCF has the various biological function, clinically, SCF has application promise in clinical practice in many aspects.Preliminary animal, human experimentation show its treatment and prevention at some diseases such as multiple anaemia, acquired immune deficiency syndrome (AIDS) (AIDS) and relative disease, the protection of radiotherapy, chemotherapy, aspects such as external hematopoiesis and gene therapy all have good effect (Zsebo, KM etal, 1992, Proc Natl Acad Sci USA, 89 (23): 9464-9468; Luskey, BD et al, 1992, Blood, 80 (2): 396-402).

Under physiological status, SCF content is atomic, and it is very difficult only depending on biochemical means isolation and purification from cell cultures, in-vivo tissue to come for scientific research, clinical application.Though can express at E.coli by the natural hSCF gene that reverse transcription PCR obtains, expression level is not very high, generally accounts for about 13% (Martin, FM et al, 1990, Cell, 63 (2): 203-211 of synthetic proteins total amount; Langley, KE et al, 1992, ArchBiochem Biophys, 295 (1): 21-28), want in E.coli, to efficiently express, certain difficulty is arranged.

Summary of the invention

The purpose of this invention is to provide a kind ofly can in intestinal bacteria, efficiently express, the human stem cell factor code gene of synthetic.

The artificial synthetic human stem cell factor code gene of the present invention, its nucleotide sequence is as follows:

5′GGAATTCAATGG?AAGGTATCTG?CCGTAACCGT?GTTACTAACA?ATGTTAAAGA?CGTTACTAAA 62

3′GTTACC?TTCCATAGAC?GGCATTGGCA?CAATGATTGT?TACAATTTCT?GCAATGATTT

CTGGTAGCTA?ACCTGCCGAA?AGACTACATG?ATCACCCTGA?AATACGTTCC?GGGTATGGAC 122

GACCATCGAT?TGGACGGCTT?TCTGATGTAC?TAGTGGGACT?TTATGCAAGG?CCCATACCTG

GTTCTGCCGA?GCCACTGCTG?GATCAGCGAA?ATGGTTGTAC?AGCTGTCTGA?CTCTCTGACT 182

CAAGACGGCT?CGGTGACGAC?CTAGTCGCTT?TACCTTCATG?TCGACAGACT?GAGAGACTGA

GATCTGCTGG?ACAAATTCTC?TAACATCTCT?GAGGGCCTGT?CTAACTACTC?TATCATCGAC 242

CTAGACGACC?TGTTTAAGAG?ATTGTAGAGA?CTCCCGGACA?GATTGATGAG?ATAGTAGCTG

AAACTGGTAA?ACATCGTTGA?CGACCTGGTT?GAATGCGTTA?AAGAAAACTC?TTCTAAAGAC 302

TTTGACCATT?TGTAGCAACT?GCTGGACCAA?CTTACGCAAT?TTCTTTTGAG?AAGATTTCTG

CTGAAAAAAT?CTTTCAAAAG?CCCGGAACCG?CGTCTGTTCA?CTCCGGAAGA?GTTCTTCCGT 362

GTCTTTTTTA?GAAAGTTTTC?GGGCCTTGGC?GCAGACAAGT?GAGGCCTTCT?CAAGAAGGCA

ATCTTCAACC?GTTCTATCGA?CGCTTTCAAG?GACTTCGTAG?TTGCATCTGA?AACTAGCGAC 422

TAGAAGAAGG?CAAGATAGCT?GCGAAAGTTC?CTGAAGCATC?AACGTAGACT?TTGATCGCTG

TGCGTTGTTT?CTTCTACCCT?GTCTCCGGAA?AAAGACTCTC?GTGTTTCTGT?TACCAAACCG 482

ACGCAACAAA?GAAGATGGGA?CAGAGGCCTT?TTTCTGAGAG?CACAAAGACA?ATGGTTTGGC

TTCATGCTGC?CGCCGGTTGC?TTAAG?3′ 512

AAGTACGACG?GCGGCCAACG?AATTCAGCTG?GCA?5’

The present invention the design SCF synthetic gene the time, with reference to Climie, S et a1,1990, Proc Natl Acad SciUSA, 87 (2): 633-637; Barry, A et al, 1987, Proc Natl Acad Sci USA, 84 (22): the work of 8961-8965, adopt computer software to carry out many-sided assistant analysis, and what time followingly consider:

1), the selection of codon

There are significantly relation in the use of codon and the height of expression efficiency, in order to realize hSCF gene efficiently expressing in intestinal bacteria, have selected the codon of E.coli preference.In other host (as, yeast), express after considering, directly obtain possibility of active result, in the selection of codon, selected both common to have a preference for password as far as possible.

2), the selection of oligonucleotide fragment length

The length of oligonucleotide fragment, the number of the synthetic required oligonucleotide fragment of the full gene of SCF of decision.Length is too short, and then required oligonucleotide fragment number can increase, and correspondingly synthetic, the workload of purifying and the difficulty of gene assembling also can increase; Length is oversize, can make the synthetic middle wrong probability increase that occurs again.Should make full use of the synthesis capability of existing dna synthesizer, the mistake that may occur in will synthesizing again, cloning is reduced to minimum, according to external report, 50-70 the better (Climie of Nucleotide, S et al, 1990, Proc Natl Acad Sci USA, 87 (2): 633-637), the present invention has selected the length of 60 oligonucleotide.

3), the selection of corresponding oligonucleotide fragment complementary sequence length

In order all to mix annealing, connect, realize the disposable assembling of gene, between the homologous segment bigger complementarity must be arranged, the present invention has selected 50% complementarity.

4), the interpolation of restriction enzyme site and removal

For clone, the expression of gene and to carry out operation such as gene fusion later on convenient; the at first selection by synonymous code; removed the inner most restriction endonuclease sites commonly used of gene; and in the N-end front that is equivalent to SCF; codon ATG, restriction enzyme EcoR I site and corresponding protection base have been added; in the C-of SCF end back, termination codon TAA and Sal I site have been added.

5), avoid the formation of complicated secondary structure

As the meander structure that in the synthetic gene, has complicated secondary structure such as repeating structure, hairpin structure, big section etc., will certainly increase the difficulty of gene assembling, influence the efficient of genetic expression.The existence of the complicated secondary structure in translation initiation district; the carrying out that also can directly influence translation initiation efficient even stop translation; therefore; with pBV220 is expression vector; prediction by translation initiation region two-stage structure; by the selection of synonymous code, restriction endonuclease sites and protectiveness base, remove in the synthetic gene, especially the complicated secondary structure before and after the initiator codon as far as possible.

According to the synthesis strategy of above design, the present invention is divided into 18 fragments, has synthesized the full gene of SCF, and its sequence as shown in Figure 1 and Figure 2.According to principle of design, under the prerequisite that does not change aminoacid sequence, replacement by codon, hSCF gene to soluble form has carried out bigger change: change about 23% (114/492) on nucleotide level, on the codon level, change approximately 63% (103/164), and removed sites such as the inner former EcoR I that pre-exists of natural gene, Xma I, Sma I, Pst I, introduced EcoR I, Sal I site at two ends, Hinc II, Pvu II site have been introduced in the centre.Because Pvu II site just is arranged near the junction of two structural domains of higher structure of hSCF albumen prediction, be that material carries out the protein molecule design and provides convenience for the research of the structure-function relationship of hSCF and with hSCF.And synthetic hSCF gene inserts after the pBV220, the secondary structure in the translation initiation district of prediction shows: the SD sequence is arranged in a bigger open loop structure, all do not have complicated secondary structure before and after the codon ATG yet, should help synthetic hSCF gene efficiently expressing in intestinal bacteria.

Use human stem cell factor code gene of the present invention, the target protein that obtains in intestinal bacteria accounts for more than the 4O% of bacterial protein, and expression amount is significantly improved.

The invention will be further described below in conjunction with accompanying drawing.

Description of drawings

Fig. 1 is the nucleotide sequence of the hSCF gene of synthetic soluble form

Fig. 2 is comparison synthetic, natural hSCF gene nucleotide series

Fig. 3 is the PCR product analysis

Fig. 4 is bacterium colony in situ hybridization

Fig. 5 is the restricted enzyme cutting analysis of pUC19/SCF

Fig. 6 is the nucleotide sequencing of the hSCF gene of synthetic soluble form

Fig. 7 is the restricted enzyme cutting analysis of pBV220/SCF

Fig. 8 be 42 ℃ induce after, the total protein electrophoretogram of engineering bacteria different times

The control of phase when Fig. 9 is engineering bacterium fermentation

Embodiment

In the following embodiments, involved material is as follows:

1, bacterial classification and plasmid

E.coli?JM109(recA1supE44endA1hsdR17gyrA96relA1thiΔ(lac-proAB)F′[traD36proAB ⁺?lacIqlacZΔM15])

E.coli?DH5α(supE44ΔlacU169(φ801acΔM15)hsdR17recA1endA1gyrA96thi-lrelA1)

Plasmid pUC19

Plasmid pBV220

2, substratum

The LB substratum: in the water of 900ml, add peptone 10g, yeast extract 5g, sodium-chlor 10g stirs until dissolving fully, regulates pH to 7.0 with 5M NaOH, adds water to 1000ml, 15 pounds of autoclavings 20 minutes.

The LB solid medium: above-mentioned LB liquid nutrient medium adds 1.5% (W/V) agar powder.

M9 substratum: in the water of 900ml, add 6g Na ₂HPO ₄, 3g KH ₂PO ₄, 0.5g NaCl, 1g NH ₄Cl stirs until dissolving fully, and last water is supplied cumulative volume to 1000ml, and autoclaving adds 20% glucose solution 20ml of separately sterilization, 1M MgSO before the use ₄2ml, 1M CaCl ₂0.1ml.

M9 CA substratum: the casein hydrolysate that adds 0.1-0.2% in the above-mentioned M9 substratum.

3, toolenzyme and damping fluid thereof

Restriction enzyme EcoR I, Sal I, T ₄The phage DNA ligase enzyme, T ₄Phage polynucleotide kinase, Taq polysaccharase and DNA Marker etc. are Biolab, Promeg, company's product such as magnificent.

Restriction enzyme high-salt buffer (10 *): 500mM Tris-HCl (pH7.5), 1M NaCl, 0.1MMgCl ₂, 10mM DTT.

T ₄Phage DNA ligase enzyme damping fluid (10 *): 200mM Tris-HCl (pH7.6), 50mM MgCl ₂, 50mM DTT, 0.5mg/ml bSA (BSA)

T ₄Phage polynucleotide kinase damping fluid (10 *): 0.5M Tris-HCl (pH7.6), 50mM DTT, 0.1M MgCl ₂, 1mM hydrochloric acid spermidine, 1mM EDTA (pH8.0)

PCR damping fluid (10 *): 500mM KCl, 100mM Tris-HCl (pH8.3), 15mM MgCl ₂

4, used solution in the experiment

4.1, SDS-PAGE protein isolate agents useful for same

Tris-glycine electrophoretic buffer: 250mM glycine, 25mM Tris-HCl (pH8.3), 0.1%SDS

Separation gel damping fluid: 375mM Tris-HCl (pH8.8), 0.1%SDS

Concentrate the glue damping fluid: 125mM Tris-HCl (pH6.8), 0.1%SDS

30% acrylamide storage liquid: 29% acrylamide, 1% methylene-bisacrylamide

Sample buffer: 50mM Tris-HCl (pH6.8), 2%SDS, 100mM DTT (existing), 0.1% bromjophenol blue, 10% glycerine with now adding

Coomassie brilliant blue R250 staining fluid: 0.25% (W/V) Coomassie brilliant blue R250,45% methyl alcohol, 10% glacial acetic acid

Destainer: 45% methyl alcohol, 10% glacial acetic acid

Albumen Marker:Promega company product

4.2, the used solution of bacterium colony in situ hybridization

SSC liquid (20 *): 3M sodium-chlor, the 0.3M Trisodium Citrate, sodium hydroxide is transferred pH7.0

Denhardt reagent (50 *): 1% ficoll (Ficoll, 400 types; Pharmacia), 1% polyethylene pyrrolidone, 1% bovine serum albumin (component V; Sigma)

Salmon sperm dna: with the water-soluble final concentration to 10mg/ml of salmon sperm dna, the concentration of regulating NaCl is used phenol, phenol to 0.1M: each extracting of chloroform is once reclaimed water, ultrasonic shearing DNA, and ethanol sedimentation reclaims DNA, and solution O D is measured in the suitable quantity of water dissolving ₂₆₀, regulate the DNA final concentration to 10mg/ml, boiled 20 ℃ of preservations 10 minutes.Face with preceding boiling water bath 5 minutes the ice bath quenching.

Sex change liquid: 0.5M NaOH, 1.5M NaCl

Neutralizer: 0.5M Tris-HCl, 1.5M NaCl

Pre-washing lotion: 5 * SSC, 0.5%SDS, 1mM EDTA (pH8.0)

Prehybridization solution: 6 * SSC, 5 * Denhart ' s, 0.1%SDS, 50% methane amide, the salmon sperm dna that 100 μ g/ml are also interrupted through sex change.

Hybridization solution: add the good probe of mark in the prehybridization solution and be hybridization solution

4.3, the electrotransfer damping fluid: 39mM Gly, 48mM Tris alkali, 0.037%SDS, 20% methyl alcohol

4.4, the TE damping fluid: 10mM Tris-HCl (pH8.0), 1mM EDTA

4.5, tbe buffer liquid: 89mM Tris-boric acid, 2mM EDTA (pH8.0)

4.6, the PBS damping fluid: 10mM NaH ₂PO ₄-Na ₂HPO ₄(pH7.0), 0.15M NaCl

5, other material

IPTG, X-gal is a German Boehringer Mannheim company product, yeast extract, peptone, RPMI-1640,3% agar powder is Nihon Pharmaceutical Co., Ltd.'s product, acid hydrolyzed casein (Casamino Acids) is a Difco company product, low melting-point agarose, ultrapure agarose, Coomassie brilliant blue R250 is a Britain Serva company product, polyacrylamide, dithiothreitol (DTT) DTT is a Sigema company import packing product, poly(vinylidene fluoride) (PVDF) film is a Millipore company product, T7 Sequencing Kit is a Parmacia company product, isotropic substance is a Fu Rui company product, penbritin, Streptomycin sulphate, N,O-Diacetylmuramidase is Shanghai No. 3 Pharmaceutical Factory's product, and horse serum is available from Fengtai army horse institute.

In the following embodiments, involved method is as follows:

1, the preparation of plasmid DNA and purifying

The quick extracting of plasmid DNA, a large amount of preparation all are to adopt alkaline denaturation, reference literature Sambrook, and J etal, 1989, Molecular Cloning, 2nd ed., CSHL Press, New York. carries out.Obtaining highly purified plasmid adopts Sephorose 2B column chromatography to remove RNA.Use the TE-NaCl buffer solution elution, A is collected in the monitoring of nucleic acid-protein detector ₂₆₀First peak (concentrating earlier) as the too big available propyl carbinol of volume, add the 3MNaAC (pH5.2) of 1/10 volume and the dehydrated alcohol of two volumes, place the liquid nitrogen gas phase spent the night in 15-20 minute or-20 ℃, 4 ℃ 12, centrifugal 15 minutes of 000g, precipitation washes twice with 70% ethanol, and vacuum is drained, the TE dissolving ,-20 ℃ of preservations are standby.

2, digestion with restriction enzyme

Specific requirement by different restriction enzymes is carried out.Generally get and treat that enzyme cuts DNA 1 μ g, add a restriction enzyme 3-5 unit, reaction volume 15-20 μ l, proper temperature water-bath 1-2 hour, the sample electrophoresis that takes a morsel detected enzyme and cuts effect.

3, the recovery of dna fragmentation

The DNA endonuclease bamhi adopts freeze-thaw method or low melting-point agarose method to reclaim after agarose gel electrophoresis separates.

Freeze-thaw method: under long-wave ultra violet lamp, fragment to be recycled is downcut from sepharose, remove the part that does not contain DNA as far as possible, adhesive tape is placed in the centrifuge tube of 1.5ml, placed the liquid nitrogen gas phase 5 minutes, and made adhesive tape freezing, take out room temperature and melt, 12, centrifugal 5 minutes of 000g gets supernatant, twice repeatedly, supernatant liquor is merged, with the extracting of equal-volume phenol once, get supernatant, use phenol again: chloroform, each extracting of chloroform are once.Get supernatant, reclaim DNA with ethanol sedimentation.

Low melting-point agarose method: after treating the gel cooled and solidified, carry out electrophoresis to guarantee that gel can not melt in 4 ℃.When target DNA band electrophoresis arrives the appropriate location, cutting-out contains the gel of this DNA, the 20mMTrisHCl (pH8.0) that adds 5 times of volumes, the damping fluid of 1mM EDTA, 65 ℃ water-bath 5-10 minute, make low melting-point agarose fusing, after being cooled to room temperature, with the extracting of equal-volume phenol once, get supernatant, use phenol again: chloroform, each extracting of chloroform are once.Get supernatant, reclaim DNA with ethanol sedimentation.

4, the connection of dna fragmentation and conversion

In the sticky end ligation of DNA, the mole ratio of control carrier and target DNA fragment is 1: 1-1: 1.5,12 ℃ connect 12-16 hour, get the about 20-30ng of ligation thing, add 0.2ml competence bacterium, ice bath 40 minutes, insulation is 1-2 minute in 42 ℃ of water-baths, add the 0.8mlLB substratum, 37 ℃ or 30 ℃ leave standstill cultivation 45 minutes.Get the culture 0.1ml of suitable dilution, coat in the flat board that contains penbritin, be inverted overnight incubation, 4 ℃ of preservations for 37 ℃ or 30 ℃.

5, the preparation of ssDNA probe

Use T ₄Phage polynucleotide kinase (T4 PNK) carries out 5 '-end mark.Get the about 10pmol of the good oligonucleotide fragment of purifying, add 3 μ l[γ- ³²P] ATP (10 μ Ci/ μ l), the T that 2 μ l 10 times ₄Phage polynucleotide kinase damping fluid, the T of 8-10 unit ₄PNK and deionized water be to cumulative volume 20 μ l, mixing, and 37 ℃ of reactions 45-60 minute, 68 ℃ of heating 15 minutes are with the deactivation kinases.The probe of this method preparation can be directly used in hybridization, needn't be further purified (Sambrook, J et al, 1989, Molecular Cloning, 2nd ed., CSHLPress, New York.).

6, bacterium colony in situ hybridization

Wait to screen the bacterium colony dibbling on nitrocellulose filter after will transforming with aseptic toothpick, duplicate a cover contrast plate simultaneously, 37 ℃ of overnight incubation are preserved the contrast plates for 4 ℃.Take filter membrane off with blunt-ended forceps, bacterium colony faces up and places with on the 10%SDS impregnated filter paper, with fixing bacterium colony.After 3 minutes, filter membrane is transferred to alkaline denaturation on the saturated filter paper of sex change liquid 7 minutes, and then filter membrane is transferred to on the saturated filter paper of neutralizer, in and 5 minutes, repeat 3 times.Fixed dna was handled 5 minutes in same operation with 2 * SSC liquid.Bacterium colony is faced up, and after at least 30 minutes, 80 ℃ were toasted 1-2 hour in natural drying at room temperature.Take out filter membrane, soaked 5 minutes with 2 * 8SC liquid, move in the pre-washing lotion of large volume and shake gently, 50 ℃ of temperature were bathed 30 minutes, moved on to then in the prehybridization solution, and 55 ℃ are incubated 4-6 hour, again 55 ℃ of incubated overnight in hybridization solution.Take out filter membrane, wash filter membranes 3-4 time, each 15 minutes in 56 ℃ with 2 * SSC/0.1%SDS liquid.After treating the filter membrane seasoning ,-70 ℃ of lower sheetings, radioautograph (Sambrook, J etal, 1989, Molecular Cloning, 2nd ed., CSHL Press, New York.).

7, the mensuration of dna sequence dna

Measure with reference to the terminal cessation method of two deoxidations of Sanger, use Pharmacia company's T 7 SequnencingKit.Directly make template with plasmid, positive anti-primer is measured from both direction.On the U.S. 373A of AppliedBiosystem company type automatic dna sequencer, carry out simultaneously.

Synthetic and the clone of embodiment 1, gene fragment makes up conversion carrier

1, the chemosynthesis of oligonucleotide fragment

On the U.S. 381A of Applied Biosystem company type dna synthesizer, adopt solid phase phosphorous acid amine method synthetic.

2, synthetic segmental aftertreatment and purifying

Strong aqua with 29% gets off the oligonucleotide hydrolysis from solid support in room temperature reaction 3 hours.Hydrolyzed solution is transferred in 60 ℃ of water-baths, and temperature was bathed 8-16 hour, separates with the urea-denatured glue of polyacrylamide/6M of 16% again, and the aqueous solution of gel usefulness 0.5M NaCl that contains the product band is in 37 ℃ of soaked overnight, and leach liquor passes through C ₁₈The reversed phase chromatography post is able to purifying.Purified product is measured OD ₂₆₀, press 10D ₂₆₀=34 μ g single stranded DNA calculating concentrations, vacuum-drying, standby.

3, the phosphorylation of 5 ' of gene fragment-end, annealing connects

With each oligonucleotide fragment deionized water dissolving of purifying, respectively get 100pmol, containing 1 * T ₄PNKbuffer, 10mmol ATP, in the reaction system of the 40 μ l of the PNK of 5 units, 37 ℃ of temperature were bathed 30-45 minute, and 68 ℃ of temperature were bathed 20 minutes, with deactivation PNK, termination reaction.

Get the above-mentioned reactant of 20 μ l, 95 ℃ were boiled 5 minutes, slowly reduced to 58 ℃, kept 30 ℃ after cooling in 1-2 hour, at the T that contains 1mM ATP, 10mM DTT, 1 * ligase enzyme damping fluid, 10 units ₄In the reaction system of dna ligase, spend the night in 12 ℃ of connections.

4, pcr amplification

With the dilution in 1: 10 of ligation thing, get 20 μ l as template, be primer with A1, B1 (as shown in Figure 1), carry out PCR, amplification gene.PCR reaction cumulative volume is 50 μ l, wherein contains each 1 μ M of primer, four kinds of each 0.2mM of deoxynucleotide, 4 units of Taq polysaccharase.Reaction conditions is 95 ℃ of sex change 1 minute, anneals 30 seconds for 58 ℃, and 70 ℃ were extended 45 seconds, repeat 30 circulations, finish the disposable assembling of gene, its PCR result (wherein is with 1 to be dna marker: pBR322+Hinf I as shown in Figure 3, be with 2 to be the pcr amplification result, be with 3 to be pUC19+EcoR I+SalI; Be with 4 for pUC19), obtained the dna fragmentation of 0.5Kb through the amplification conclusive evidence.

5, the clone of synthetic hSCF gene; Make up conversion carrier; Transformed into escherichia coli; Screening transforms bacterium colony

Reclaim the gene fragment of pcr amplification, after EcoR I, Sal I enzyme are cut, be connected with the pUC19 that crosses with same digestion with restriction enzyme, reorganization, transformed into escherichia coli JM109, coat on the flat board that contains X-gal, IPTG and penbritin, overnight incubation, the picking white colony, with synthetic oligonucleotide fragment A3 (as shown in Figure 1) is probe, with the positive contrast of B7, B8 (as shown in Figure 1), carries out bacterium colony in situ hybridization, the result is (bacterial plaque 1 positive contrast, bacterial plaque 2 negative contrasts) as shown in Figure 4.Positive colony of random choose, inoculation culture, extracting DNA carries out the plasmid size, enzyme is cut evaluation.The result shows that the size of recombinant plasmid is 3.2Kb, cuts with EcoR I, SalI enzyme, shows single point of contact; Carry out double digestion, can cut out the fragment of a 0.5Kb size, and as shown in Figure 5 (be with 1 to be dna marker: pBR322+Hinf I, be with 2 to be pUC19/SCF+EcoR I+Sal I; Be with 3 to be pUC19/SCF+EcoR I; Be with 4 to be pUC19/SCF+Sal I; Be with 5 to be pUC19, electrophoresis result is in full accord with design, proves the hSCF gene that contains the total length soluble form really.

6, the complete sequence determination of gene

For further confirming the total correctness of synthetic gene, positive colony has been carried out sequential analysis.With the two strands is template, with positive and negative two kinds of primers, carries out complete sequence determination from both direction simultaneously by the Sanger dideoxy chain termination.In the experiment, a plurality of positive colonies have been measured, same clone's replication twice, the sequence of one of them clone's SCF synthetic gene and design in full accord, this recombinant plasmid called after pUC19/SCF.Again this clone has been carried out full-automatic order-checking, the result as shown in Figure 6, and is in full accord with manual measurement result.

The structure of embodiment 2, engineering bacteria

Cut the pUC-19/SCF plasmid with EcoR I, Sal I while enzyme,, be connected, realize the structure of expression plasmid with the pBV220 that handles with same restriction enzyme with the SCF fragment of the about 0.5kb of low melting point agar method recovery size.The recombinant plasmid transformed bacillus coli DH 5 alpha that connects, coat the flat board that contains penbritin, be inverted overnight incubation for 30 ℃, random choose transformant inoculation culture, extracting plasmid electrophoresis carries out plasmid size and EcoR I, Sal I enzyme and cuts evaluation, as shown in Figure 7, experimental result is consistent with expection, confirms that this is expression plasmid pBV220/SCF, and the bacterial strain that contains this plasmid is the engineering strain that can be used for fermentation expression.

The fermentation culture of embodiment 3, engineering bacteria

Select single bacterium colony from the engineering bacteria LB inclined-plane of the hSCF gene that contains soluble form, be seeded in the LB substratum, 30 ℃ of shaking tables are cultured to logarithm (OD in late period ₆₀₀About 0.65), 5% is inoculated in the M9CA substratum, and the shaking table overnight incubation is inoculated in M9CA substratum, fermentation culture, OD with overnight culture with 2% ratio again ₆₀₀Rise to about 0.5, carry out 42 ℃ of water-bath thermal inductions, continue to cultivate, the appropriate time results.

The shake-flask culture of embodiment 4, engineering bacteria

30 ℃ of cultivations,, forward engineering bacteria to 42 ℃ of cultivations then with the amplification plasmid copy number.Owing to during 30 ℃ of cultivations, accumulated a large amount of plasmid copy numbers, when forwarding 42 ℃ to, make CI repressor inactivation, start the great expression of SCF gene.The electrophoresis behavior of the lysate of engineering bacteria shows, is about the place of 19KD at molecular weight, increased an obvious protein band newly, is equivalent to the proteic size of hSCF of soluble form.Use Pharmacia LKB Ultro ScanXL laser scanner glue is carried out density scan (633nm), this protein band accounts for about 40% of bacterial protein as a result, be higher than domestic and international report (Langley far away, KE et al, 1992, Arch Biochem Biophys, 295 (1): 21-28), the hSCF engineering strain that has obtained to efficiently express soluble form is described.

Also can adopt polyacrylamide gel electrophoresis SDS-PAGE method (Laemmli, UK et al, 1970, Nature 227:680-685) identifies the product that obtains.Its way is that sample is got 10-15 μ l and carried out electrophoresis after boiling 4-5 minute with the sample dissolution damping fluid in advance.During electrophoresis, the concentration that concentrates glue is 5%, and separation gel is 12%-15%, electrophoresis plate 10mm * 7mm * 1mm, 20mA constant current, electrophoresis time 1.5-2 hour.Electrophoresis is finished, with Coomassie brilliant blue R250 dyeing more than 4 hours, the destainer decolouring, to background limpid till.Through the laser intensity scanner scanning, measure the content of target protein then.

The control of phase when embodiment 5, engineering bacterium fermentation

When determining shake-flask culture, what degree engineering bacteria grows at 30 ℃ is induced, and how long 42 ℃ of inducing culture gather in the crops, and can obtain maximum harvest yield, the contriver has at first observed the growth curve of bacterium when cultivating for 30 ℃, and (curve 1 is the growth curve under 30 ℃ as shown in Figure 9; Curve 2 is 42 ℃ of growth curves after inducing; Curve 3 is the synthetic of hSCF under the different periods; Curve 4 is relative crop): 2% inoculation, amount/3000ml shakes in the bottle in the 300ml cultivation, cultivates its OD 4-5 hour ₆₀₀Can reach about 0.5, bacterium is in logarithmic growth mid-term, divides vigorously, therefore selects to carry out this moment 42 ℃ of thermal inductions.42 ℃ induce after, every 1 hour the sampling, measure its OD ₆₀₀Value is also carried out SDS-PAGE and is analyzed, and observes its expression, and (numeral on the gel is the time number after 42 ℃ of processing to the result as shown in Figure 8, numeral under the gel is the per-cent that rhSCF accounts for total protein), 42 ℃ induce after, have one significantly to raise than growth rate, and along with the prolongation of time, its OD ₆₀₀Value rises gradually, continues 6-7 hour, just tends towards stability; The expression amount of SCF also after 6-7 hour, reaches the highest, so its relative harvest yield (OD ₆₀₀Product with expression amount) also the highest, so select 42 ℃ to induce and gathered in the crops in 6-7 hour.

The purifying of embodiment 6, SCF expression product and materialization are measured

1, the preparation of inclusion body

Fermented liquid is collected thalline through 4 ℃ centrifugal (3,000rpm, 15 minutes).Thalline proper volume PBS washed twice, it is resuspended that wet thallus about every then 1g adds 2mg/ml N,O-Diacetylmuramidase/solution W (50mMTris-HClpH8.0/10mM EDTA) of 10ml, the slight vibration of room temperature 30 minutes, and then with the broken bacterium of ultra-sonic generator ice-bath ultrasonic, 4 ℃ centrifugal (14,000g, 20 minutes), abandon supernatant, precipitation is inclusion body.

2, the washing of inclusion body

Precipitation washes twice with 3%Triton X-100/0.2% Sodium desoxycholate/solution W earlier, washes once with 2.0M urea/solution W again.All washing process all carry out in room temperature, and gentle agitation 30-45 minute, 4 ℃ centrifugal (14,000g, 15 minutes).Keep precipitation, the supernatant electrophoresis that takes a morsel is observed washing effect.

3, the dissolving of inclusion body

Precipitation is dissolved with 8M urea/solution S (50mMTris-HCl pH8.0/1mM EDTA/0.03% sodiumazide).The room temperature gentle agitation, to most of resolution of precipitate, 4 ℃ centrifugal (17,000g, 30 minutes) remove insoluble impurity, and supernatant is used for electrophoresis evaluation, folding renaturation and is further purified.In the dissolution process, the total concn of control SCF is about 3-4mg/ml.

4, the renaturation of rhSCF

The Streptomycin sulphate of adding 2% in solubilization of inclusion bodies liquid was placed 20-30 minute for 4 ℃, and 4 ℃ centrifugal (12,000g, 15 minutes) are removed flocks and kept supernatant.Adding DTT again in supernatant is 10mM to final concentration, and 30 ℃ are incubated 30 minutes, dilute with solution S, to the about 0.3mg/ml of SCF final concentration, move in the dialysis tubing, the 4 ℃ of 2M urea with 10-20 times of volume/solution S were dialysed 72 hours, gentle agitation, middle replacing dialyzate once.

4 ℃ of dialyzates centrifugal (17,000g, 30 minutes) keep supernatant, and precipitation is dissolved again, dialysed with a small amount of 8M urea/10mMDTT/ solution S again.Merge dialyzate twice, to the dialysis of large volume solution S, finally remove urea again.This is the good rhSCF crude extract of renaturation.

5, rhSCF's is further purified

(1) Acid precipitation

Crude extract is regulated pH to 4.0 with 10% HAC, with 50mM NaAC-HAC (pH4.0) dialysis 30 minutes, flocks occurs, and 4 ℃ centrifugal (17,000g, 30 minutes) remove precipitation, keep supernatant.

(2) ion exchange chromatography

Get the Acid precipitation supernatant and carry out cationic exchange.

Ion exchange column: Pharmacia C16/20 (2 * 16cm)

Medium: Pharmacia Sp-Sepharose Fast Flow

Balance liquid: 50mM NaAC-HAC (pH4.0)

Elutriant: 0-0.3M NaCl/50mM NaAC-HAC (pH4.0)

Flow velocity: 120ml/hr

Operation:, constant until pH value, electric conductivity value with the abundant balance cylinder of balance liquid.Last sample fully washes with balance liquid, goes up the NaCl gradient then, and up liquid stream wash-out is collected step by step, and each chromatographic peak carries out electrophoresis detection and determination of activity.

(3) gel permeation chromatography

Gel-filtration column: Pharmacia CK 26/100 (5 * 100cm)

Medium: Pharmacia Sephacryl S-200 (HR)

Balance liquid: PBS

Elutriant: PBS

Flow velocity: 60ml/hr

Operation: with after the abundant balance cylinder of balance liquid of at least 2 times of bed volumes, with sample on 5% the column volume, up liquid stream wash-out, substep is collected, and each chromatographic peak carries out electrophoresis detection and determination of activity.

Embodiment 7,-terminal amino acid sequential analysis

After about 10 μ g purification of samples SDS-PAGE further separate, be transferred on the pvdf membrane that soaked 30 minutes in advance with methyl alcohol through 45-60 minute electricity of 10mA constant current, with rinsed with deionized water pvdf membrane 2-3 time, with 0.1% Coomassie brilliant blue, 10% Glacial acetic acid, 50% methyl alcohol film was dyeed 1-2 minute again, with 10% Glacial acetic acid, the decolouring of 50% methyl alcohol, deionized water rinsing is cut the part corresponding to the purpose band, is directly used in the sequential analysis of n terminal amino acid.

The bioactive mensuration of embodiment 8, rhSCF

To the influence that human bone marrow cell's grain, macronucleus assembly fall to forming, determine its biologic activity according to rhSCF.

1, the preparation of normal people's marrow single cell suspension

Take from the thoracic surgery patient's of 301 Hospital rib, aseptic technique is taken out marrow with RPMI-1640 nutrient solution suspension cell, makes cell suspension, makes the individual cells suspension with lymphocyte layering liquid then.

2, CFU-GM is semi-solid cultivates

Add RPMI-1640 successively; 2 * 10 ⁵/ ml cell suspension; The horse serum of final concentration 30%; HMCM (final concentration 10%, 5%); 37 ℃ of SCF (final concentration 10 μ g/ml, 1 μ g/ml, 0.1 μ g/ml) insulation 15 minutes slowly drips 3% agar that boils, and mixing is poured in the 36mm culture dish immediately, and every ware 1ml evenly paves, and places 37 ℃, 5% CO ₂Culture condition was cultivated 10 days, 14 days down, observed colony and formed situation, to count a colony greater than the cell mass of 40 cells.The content of rhSCF is determined by the Coomassie brilliant blue R250 conjugated protein molecular weight standard that dyes.The result shows that rhSCF of the present invention can stimulate human bone marrow cell's propagation, causes grain, macronucleus assembly the fall size of (CFU-GM), the obvious increase of quantity, has the biological activity of natural hSCF.

Claims (1)

1, a kind of human stem cell factor code gene of synthetic, its nucleotide sequence is as follows: 5 ' GGAATTCAATGG AAGGTATCTG CCGTAACCGT GTTACTAACA ATGTTAAAGA CGTTACTAAA 62

3′GTTACC?TTCCATAGAC?GGCATTGGCA?CAATGATTGT?TACAATTTCT?GCAATGATTT

CTGGTAGCTA?ACCTGCCGAA?AGACTACATG?ATCACCCTGA?AATACGTTCC?GGGTATGGAC 122

GACCATCGAT?TGGACGGCTT?TCTGATGTAC?TAGTGGGACT?TTATGCAAGG?CCCATACCTG

GTTCTGCCGA?GCCACTGCTG?GATCAGCGAA?ATGGTTGTAC?AGCTGTCTGA?CTCTCTGACT 182

CAAGACGGCT?CGGTGACGAC?CTAGTCGCTT?TACCTTCATG?TCGACAGACT?GAGAGACTGA

GATCTGCTGG?ACAAATTCTC?TAACATCTCT?GAGGGCCTGT?CTAACTACTC?TATCATCGAC 242

CTAGACGACC?TGTTTAAGAG?ATTGTAGAGA?CTCCCGGACA?GATTGATGAG?ATAGTAGCTG

AAACTGGTAA?ACATCGTTGA?CGACCTGGTT?GAATGCGTTA?AAGAAAACTC?TTCTAAAGAC 302

TTTGACCATT?TGTAGCAACT?GCTGGACCAA?CTTACGCAAT?TTCTTTTGAG?AAGATTTCTG

CTGAAAAAAT?CTTTCAAAAG?CCCGGAACCG?CGTCTGTTCA?CTCCGGAAGA?GTTCTTCCGT 362

GTCTTTTTTA?GAAAGTTTTC?GGGCCTTGGC?GCAGACAAGT?GAGGCCTTCT?CAAGAAGGCA

ATCTTCAACC?GTTCTATCGA?CGCTTTCAAG?GACTTCGTAG?TTGCATCTGA?AACTAGCGAC 422

TAGAAGAAGG?CAAGATAGCT?GCGAAAGTTC?CTGAAGCATC?AACGTAGACT?TTGATCGCTG

TGCGTTGTTT?CTTCTACCCT?GTCTCCGGAA?AAAGACTCTC?GTGTTTCTGT?TACCAAACCG 482

ACGCAACAAA?GAAGATGGGA?CAGAGGCCTT?TTTCTGAGAG?CACAAAGACA?ATGGTTTGGC

TTCATGCTGC?CGCCGGTTGC?TTAAG?3′ 512

AAGTACGACG?GCGGCCAACG?AATTCAGCTG?GCA?5’

Images