CN102277327A - Colon bacillus for over-expressing RimL and application on preparing N-extrasin alpha acetylate - Google Patents

Abstract

The invention discloses an Escherichia coli overexpressing RimL and its application in preparing N-acetylated thymosin α. The invention provides a recombinant bacterium, which is obtained by introducing the coding gene of RimL and the coding gene of thymosin α into the host bacterium. Experiments of the present invention prove that the present invention constructs a recombinant bacterium obtained by co-expressing the coding genes of N-acetyltransferase RimL and thymosin α, ferments the recombinant bacterium, and obtains thymosin α mostly as N-acetylated thymosin α, has obvious application prospects.

Description

Cross intestinal bacteria and the application in preparation N-acetylated thymosin alpha thereof of expressing RimL

Technical field

The present invention relates to biological technical field, relate in particular to intestinal bacteria and the application in preparation N-acetylated thymosin alpha thereof that a kind of mistake is expressed RimL.

Background technology

The terminated acetylated modification of the N-of albumen and polypeptide is ubiquitous a kind of modification in the eukaryotic cell, and the eukaryotic cell plasmosin more than 50% all has this modification.Preliminary functional study shows the terminated acetylated electric charge that passes through change N-end of N-, and characteristic generation material impacts such as degraded are discerned, resisted to modes such as sealing N end to space structure, the part of many albumen and polypeptide.Terminated acetylated modification has promoted the identification of itself and membrane receptor Sys1p/hSys1 as the proteic N-of small molecules GTPaes-Arl3P, and it is navigated on the film.Terminated acetylated its tetrameric depolymerization ability that can make of the N-of foetal haemoglobin improves 30 times.The N-of thymosin (thymosin α 1, T α 1) is terminated acetylated to have vital role to its stability in blood plasma.Except T α 1; also just like melanocyte-stimulating hormone (Melanocyte-Stimulating Hormones; α-MSH), the extrasin beta 4 a collection of polypeptide that has important application clinically such as (Thymosin β 4, T β 4) also need the terminated acetylated modification of N-.

Visibly different with the ubiquity phenomenon in the eukaryote is that the terminated acetylated modification of procaryotic N-is very rare.The terminated acetylated modification of known generation N-has only several albumen few in number such as ribosomal subunit L7, S5, S18, elongation factor EF-Tu and chaperone SecB in the intestinal bacteria intrinsic protein.The acetyltransferase of the participation albumen that has been found that in the intestinal bacteria and the terminated acetylated modification of N-of polypeptide has only the RimI of responsible ribosomal subunit S5 acetylation modification; the RimL of the RimJ of ribosomal subunit S18 acetylation modification and ribosomal subunit L7 acetylation modification.These acetyltransferases have very strong Substratspezifitaet.Except that the specific substrate of above-mentioned each enzyme correspondence, unknown its can be modified colibacillary other albumen or polypeptide.

Extrasin alpha is the immunoloregulation polypeptide that gang has same or analogous N-terminal sequence, and they comprise prophymosin-alpha, thymosin, thymosin 1 and various fusion roteins thereof etc.Thymosin and thymosin 1 are the products of prophymosin-alpha degradation in vivo, and they have comprised 28 and 35 amino acid of N-end of prophymosin-alpha respectively.The extrasin alpha of different plant species has very high conservative property.There is polymorphism in some site on the aminoacid sequence of extrasin alpha, holds the 13rd amino acids residue as its N-, has plenty of Threonine, has plenty of Isoleucine, and they have same or analogous function.

Zadaxin is the immunological reagent that comprises various extrasin alphas and other thymus gland source peptide class that extracts in the class animal thymus, has been widely used in the treatment of viral hepatitis, tumour etc.But there is complicated component in the Zadaxin that animal is extracted, and purity is low, wherein is mixed with the pollutent of animal-origin, easily produces problems such as anaphylaxis.The N-acetylated thymosin alpha 1 of chemosynthesis, it is a kind of N-acetylated thymosin alpha 1 that adopts the preparation of chemiluminescent polypeptide synthetic method, the T α 1 purity height that this kind method obtains, active high, do not have a tangible side reaction, as " Zadaxin " of SciClone company, the treatment that a plurality of state approvals that comprised China are used for viral hepatitis obtains as immunoregulation druge; But such 28 amino acid whose polypeptide of chemosynthesis N-acetylated thymosin alpha 1, synthetic and purifying process complexity, yield is lower, so goods price height, has limited the widespread use of this medicine.

Summary of the invention

An object of the present invention is to provide a kind of reorganization bacterium.

Reorganization bacterium provided by the invention is following 1) or 2):

1) encoding gene of RimL and the encoding gene of extrasin alpha are imported the reorganization bacterium that obtains in the host bacterium; Described host bacterium is the host bacterium that contains the RimL encoding gene in the genome;

2) encoding gene with extrasin alpha imports the reorganization bacterium that obtains in the reorganization bacterium as described below;

Described RimL be following (a) and (b) or (c) shown in albumen:

(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 2;

(b) with the aminoacid sequence shown in the sequence in the sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have the albumen of identical function;

(c) and a) or b) dna sequence dna that limits has 90% homology at least and has the albumen of identical function;

Described extrasin alpha is a peptide species or albumen, in 28 amino-acid residues of its N-terminal and the sequence table in 28 amino-acid residues of identical or its N-terminal of 28 amino acid of the N-terminal of sequence 3 and the sequence table 28 amino acid of the N-terminal of sequence 3 have 90% homology at least.

The replacement of described one or several amino-acid residue and/or disappearance and/or be added to replacement and/or the disappearance and/or the interpolation of 10 amino-acid residues.

1) in the reorganization bacterium shown in, described encoding gene and the encoding gene of the extrasin alpha method that imports the host bacterium with RimL comprises the steps:

A) encoding gene with described RimL imports described host bacterium by recombinant vectors 2, and bacterium A obtains recombinating;

B) encoding gene with described extrasin alpha imports the described reorganization bacterium A that step a) obtains by recombinant vectors 1, obtains the bacterium of recombinating;

Described recombinant vectors 1 is that described extrasin alpha encoding gene inserts in the expression vector 1, obtains expressing the recombinant vectors 1 of extrasin alpha; Described expression vector 1 is preferably pBV220; Described recombinant vectors 1 is specially described extrasin alpha encoding gene is inserted between the BamHI and EcoRI restriction enzyme site of pBV220, obtains expressing the recombinant vectors 1 of extrasin alpha;

Described reorganization bacterium A be encoding gene with described RimL by in the recombinant vectors 2 importing host bacterium, the reorganization bacterium A that obtains; Described expression vector 2 is preferably pOKBV; During described recombinant vectors 2 is specially and inserts the encoding gene of described RimL between EcoR I in the pOKBV carrier and Sal I restriction enzyme site, obtain expressing the recombinant vectors 2 of RimL.

1) in the reorganization bacterium shown in, described host bacterium is intestinal bacteria;

Described RimL encoding gene is following 1) or 2) dna molecular shown in arbitrary:

1) dna molecular shown in the sequence 1 in the sequence table;

2) with 1) dna sequence dna that limits has the proteic dna molecular that 90% homology and coding have identical function at least;

Described extrasin alpha encoding gene is a kind of polynucleotide, the sequence 4 in its nucleotide sequence and the sequence table 5 ' terminal rise 84 Nucleotide identical or with sequence table in 5 ' terminally the playing 84 nucleotide homologies and have 90% dna molecular at least of sequence 4.

Another object of the present invention provides a kind of reorganization bacterium A.

Reorganization bacterium A provided by the invention is for the encoding gene with described RimL imports in the host bacterium reorganization bacterium A that obtains by recombinant vectors;

Described recombinant vectors is that the encoding gene of described RimL is inserted in the expression vector, obtains expressing the recombinant vectors of RimL, and described expression vector is to express by the encoding gene of inducible promoter control RimL.

Described inducible promoter is lactose promotor, pectinose promotor, alkaline phosphatase promotor, trp promoter, phage t7, P _L, P _RPromotor or contain hybrid promoter Tac, the P of these promotor subelements _LP _R, described expression vector is preferably pOKBV.

The 3rd purpose of the present invention provides a kind of reorganization bacterium.

Reorganization bacterium provided by the invention, the upstream for the external source inducible promoter is inserted the acetyltransferase encoding gene of host bacterium is used to start the expression of described acetyltransferase encoding gene, the reorganization bacterium that obtains.

Described inducible promoter is lactose promotor, pectinose promotor, alkaline phosphatase promotor, trp promoter, phage t7, P _L, P _RPromotor or contain hybrid promoter Tac, the P of these promotor subelements _LP _R

Described host bacterium is intestinal bacteria;

Described inducible promoter is the phage t7 promotor, its nucleotides sequence classify as sequence 5 in the sequence table from 5 ' terminal 1119-1135 position Nucleotide;

Described acetyltransferase is RimL,

Described RimL be following (a) and (b) or (c) shown in albumen:

(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 2;

(b) with the aminoacid sequence shown in the sequence in the sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have the albumen of identical function;

(c) and a) or b) amino acid that limits has 90% homology at least and has the albumen of identical function.

Above-mentioned reorganization bacterium is prepared as follows:

1) the pKD46 plasmid is imported in the host bacterium, bacterium 1 obtains recombinating;

2) dna molecular that will contain the T7 promotor imports in the reorganization bacterium 1 that step 1) obtains, and bacterium 2 obtains recombinating;

3) with step 2) the reorganization bacterium 2 that obtains is respectively at the substratum that contains paraxin with contain in the substratum of penbritin and cultivate, if can grow containing on the substratum of paraxin, but is not the reorganization bacterium in the substratum growth that contains penbritin;

The nucleotides sequence of the dna molecular of the described T7 of containing promotor is classified the 1119-1135 position Nucleotide of the sequence 5 in the sequence table as.

The application of described reorganization bacterium in preparation N-acetylated thymosin alpha also is the scope of protection of the invention.

The 4th purpose of the present invention provides a kind of method of the N-of preparation acetylated thymosin alpha.

Method provided by the invention comprises the steps:

The described reorganization bacterium of fermenting is collected tunning, promptly obtains the N-acetylated thymosin alpha;

The temperature of described fermentation is 42 ℃, and described fermentation time is 12h.

Of the present invention experimental results show that; the encoding gene that the present invention has made up N-acetyltransferase RimL and extrasin alpha carries out the reorganization bacterium that coexpression obtains; this reorganization bacterium of fermenting; obtain extrasin alpha and all mostly be the N-acetylated thymosin alpha greatly; not only can improve the productive rate of N-acetylated thymosin alpha; and can make things convenient for the separation of N-acetylated thymosin alpha by reducing the ratio of non-acetylated thymosin alpha, have tangible application prospect.

Description of drawings

Fig. 1 was that the SDS-PAGE that expresses RimL analyzes

Fig. 2 expresses the influence (X-coordinate represent elution time (min), ordinate zou represent ultraviolet absorption value (mAU)) of RimL to ProT α acetylize level for crossing in the intestinal bacteria

Fig. 3 is a red homologous dna schematic arrangement

Fig. 4 for RimL in the T7 abduction delivering genome in the intestinal bacteria to the influence (X-coordinate is represented elution time (min), and ordinate zou is represented ultraviolet absorption value (mAU)) of ProT α acetylize level

Embodiment

Employed experimental technique is ordinary method if no special instructions among the following embodiment.

Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.

But following sequence is synthetic all.

The RimL of embodiment 1, expression extrasin alpha crosses the colibacillary structure of expression

One, N-acetyltransferase RimL crosses the colibacillary structure of expression

1, the structure of pOKBV plasmid

Because of wanting coexpression RimL and extrasin alpha, consider plasmid incompatibility, then pBV220 (is dodged brilliant molecular biosciences Science and Technology Ltd. available from Shanghai.) replicon and ampicillin resistance gene replace with plasmid pOK12 (Jeffrey Vieira and Joachim Messing.New pUC-derived cloning vectors with different selectable markers and DNAreplication origins.Gene, 1991,100:189-194, can from the information that the document provides, make up, construction of carrier is open (as J. Sa nurse Brooker etc. at many documents, " molecular cloning experiment guide " second edition, Science Press, 1995; The pOK12 carrier is for Genbank number AF223639, according to disclosed sequence library, finds as in the gene pool (Genbank) of NIH (NIH) etc., therefore can utilize the complete synthesis mode of gene to obtain.) p15A replicon and kalamycin resistance gene, specific as follows:

With plasmid pBV220 is template, uses primer 2 20-5 ' SphI

(AAAA GCATGCAGAGCGCCCTTATCTTTCCCTTTAT) and 220-3 ' SacI

(AAAA GAGCTCATCAGGGTTATTGTCTCATGAGCGG) increase, obtain the PCR product of 1.8k, be the temperature sensitive operon of pBV220.

Cut the temperature sensitive operon of the above-mentioned pBV220 that obtains with SphI and SacI enzyme, obtain enzyme and cut product, enzyme is cut product to be obtained plasmid pOK12 carrier segments and is connected with cutting through same enzyme, the connection product that obtains changes DH5 α competent cell over to, the picking mono-clonal, extract plasmid and send to order-checking, the result inserts the plasmid that obtains between the SphI of plasmid pOK12 and SacI restriction enzyme site for this plasmid for the temperature sensitive operon with pBV220, with this plasmid called after pOKBV, will contain the bacterium called after DH5 α/pOKBV of this plasmid.

2, the clone of RimL gene

Extract bacillus coli DH 5 alpha (available from the Beijing Quanshijin Biotechnology Co., Ltd, catalog number (Cat.No.) CD201) genomic dna is that (method is seen J. Sa nurse Brooker etc., " molecular cloning experiment guide " second edition, Science Press to template, 1995), use primer L5 '-EcoR I

(AAAA GAATTCATGACTGAAACGATAAAAGTAAGCGAA) and L3 '-Sal I

(AAAA GTCGACTTATTGTGAATCGATAATACGCGCGTA) carry out pcr amplification, obtain the PCR product of 540bp, through order-checking, this PCR product has the Nucleotide shown in the sequence 1 in the sequence table, and its aminoacid sequence is a sequence 2, is N-acetyltransferase RimL through comparison.

Table 1 is the online comparison partial results of RimL aminoacid sequence

3, cross the colibacillary structure of expressing RimL

1), crosses the colibacillary structure of expressing RimL

The above-mentioned 2 PCR products that obtain (the RimL gene of amplification) are cut with EcoR I and Sal I enzyme, the enzyme that obtains is cut product and is connected with the plasmid pOKBV that is obtained by step 1 that cuts through same enzyme, the connection product that obtains changes DH5 α competent cell over to, the picking mono-clonal, extract plasmid and send to order-checking, the result is the plasmid of this plasmid for obtaining between the EcoR I that sequence in the sequence table 1 inserted plasmid pOKBV and Sal I restriction enzyme site, with this plasmid called after pOKBV-RimL, will contain the bacterium called after DH5 α/pOKBV-RimL of this plasmid.

2), DH5 α/pOKBV-RimL crosses expression RimL

DH5 α/pOKBV-RimL and contrast bacterium DH5 α/pOKBV are inoculated the 5mlLB substratum that contains 100 μ g/ml kantlex respectively, 30 ℃, 200 rev/mins of shaking culture are spent the night, be seeded in the LB substratum that 100ml contains 100 μ g/ml kantlex 30 ℃, 200 rev/mins shaking culture 3-5 hour, to the nectar degree be 0.4-0.6A600, rising culture temperature to 42 ℃, 200 rev/mins shaking culture 3-6 hour again, centrifugal collection supernatant, analyze with 15%SDS-PAGE, coomassie brilliant blue staining, the result as shown in Figure 1, wherein (1 is contrast bacterium DH5 α/pOKBV, 2 is DH5 α/pOKBV-RimL), obtain the target protein about 18KD, consistent with the expection size, expression amount is about 200 μ g/mL, therefore explanation, DH5 α/pOKBV-RimL has tangible RimL to cross the expression band.

Two, the RimL that expresses extrasin alpha crosses the e. coli bl21 (DE3) of expression

The structure of (pOKBV-RimL/pBV220-proT α)

PBV220-proT α (Thr ¹³) be prepared as follows: with ProT α (Thr ¹³) gene (a kind of extrasin alpha, nucleotides sequence are classified sequence 4 as, and its aminoacid sequence is a sequence 3) inserts the BamHI and the EcoRI site of pBV220 carrier, the carrier that obtains, proT α gene is by the P on the pBV220 carrier _LP _RPromotor control.

Plasmid pBV220-proT α (Thr with above-mentioned acquisition ¹³) shocking by electricity is transformed among the above-mentioned one DH5 α/pOKBV-RimL that obtains, and obtains transformant, extracts plasmid, through order-checking, is pBV220-proT α (Thr ¹³) and pOKBV-RimL, will contain the bacterium called after DH5 α (pOKBV-RimL/proT α (Thr of these two kinds of plasmids ¹³)).

Use same quadrat method, with pBV220-proT α (Thr ¹³) shocking by electricity transforms among DH5 α/pOKBV, obtains transformant, extracts plasmid, the result is pBV220-proT α (Thr ¹³) and pOKBV, will contain the bacterium called after DH5 α (pOKBV/proT α (Thr of these two kinds of plasmids ¹³)).

Extract DH5 α (pOKBV-RimL/proT α (Thr ¹³)) and DH5 α (pOKBV/proT α (Thr ¹³)) plasmid, change over to respectively among the BL21 (DE3), obtain recon 1 and recon 2 respectively, extract the plasmid of recon 1 and recon 2, through order-checking, be respectively pOKBV-RimL/proT α (Thr ¹³) and pOKBV/proT α (Thr ¹³), prove that therefore recon 1 is BL21 (DE3) (pOKBV-RimL/proT α (Thr ¹³)), recon 2 is BL21 (DE3) (pOKBV/proT α (Thr ¹³)).

Embodiment 2, reorganization bacterium BL21 (DE3) (pOKBV-RimL/proT α (Thr ¹³)) former (Thr of expression N-acetylated thymosin alpha ¹³)

1, induces the reorganization bacterium

With BL21 (DE3) (pOKBV-RimL/proT α (Thr ¹³)) and contrast bacterium BL21 (DE3) (pOKBV/proT α (Thr ¹³)) be seeded to I substratum (50mM Sodium phosphate dibasic/phosphate sodium dihydrogen buffer solution that 5mL contains 50 μ g/ml kantlex and 100 μ g/ml penbritins respectively, the 20g/L yeast extract, the 10g/L Tryptones, 2g/L glucose) in, 30 ℃ of overnight incubation, be forwarded to the 500ml that contains 150mL I substratum according to 1% ratio with the bacterium liquid of incubated overnight and shake in the bottle next day, treats A _600nmBe about at 0.6 o'clock and be warming up to 42 ℃ of abduction deliverings, induce that centrifugal after 12 hours (the described centrifugal time is that 20 minutes, centrifugal temperature are 4 ℃, and centrifugal force is 10,000g; ) gather in the crops thalline respectively.

2, the former (Thr of preparation N-acetylated thymosin alpha ¹³)

1) the saturated phenol extracting of Tris

The wet thallus that per 1 gram above-mentioned steps 1 obtains adds 10mL deionized water, carrying out ultrasonic bacteria breaking, 10, centrifugal 20 minutes of 000 * g gets supernatant, adds the 2M pH4.5HAc-NaAc damping fluid of 1% volume in broken bacterium supernatant, the heavy foreign protein of acid, centrifugal 20 minutes of 10,000 * g gets supernatant, and the saturated phenol of the Tris that adds 1/4 volume, mixed centrifugal 20 minutes of 8000 * g, water intaking phase supernatant liquor 10 minutes.

2) HiTrap Q Sepharose Fast Flow chromatography

With supernatant liquor HiTrap Q Sepharose Fast Flow chromatography column (available from U.S. GE company) purifying, instrument is that AKTA UPC 214nm detects (available from U.S. GE company), flow velocity is 1mL/min, earlier with A liquid (A liquid: balance Q anion chromatography post 20mM pH4.5 acetate-sodium acetate buffer), use 20%B liquid (B liquid: 20mM pH4.5 acetate-sodium acetate buffer behind the last sample, 1M NaCl) B liquid consumption is 1 column volume flush away foreign protein, uses 30%B liquid wash-out again, collects elutriant.

3) high performance liquid chromatography

Elutriant is carried out chromatography on HP 1090 type high-performance liquid chromatographs, actual conditions is for using Spherigel 300AC18 reversed-phase column ((4.6mm * 250mm is available from Shen, big Liaanjiang county separation science technology company), mobile phase A is the pure water that contains 0.1%TFA, Mobile phase B is for containing the trifluoroacetic acid aqueous solution of 0.1%TFA (trifluoroacetic acid] trifluoracetic acid), use earlier the 100%A balance, applied sample amount 25 μ L, 0%-100% Mobile phase B gradient elution in 28 minutes, detect with 214nm and 550nm dual wavelength, flow velocity is 1mL/min, collect the elutriant that retention time is 9.9min respectively, obtain A peak sample, collecting retention time is the elutriant of 10.1min, obtains B peak sample.

Mapping is schemed a left side as shown in Figure 2: contrast bacterium BL21 (DE3) (pOKBV/proT α (Thr ¹³)) preparation prophymosin-alpha (Thr ¹³); Figure is right: BL21 (DE3)/(pOKBV-RimL/proT α (Thr ¹³)) preparation prophymosin-alpha (Thr ¹³).

The A peak sample and the B peak sample that will contrast the collection of bacterium and experimental bacteria are respectively used mass spectrometric detection respectively, method Wu J, Chang S, Gong X, Liu D, Ma .Q.Identification of N-terminal acetylation of recombinant human prothymosin alpha in Escherichia coli.Biochim Biophys Acta.2006; 1760 (8): 1241-7., molecular weight differs 42Da between two components of mass spectroscopy A, B, the molecular weight ratio A component of B component big by 42 (molecular weight of ethanoyl).Quality peptide spectrum determines that the increase of this molecular weight occurs in N-end peptide section; with tandem mass spectrum this peptide section is checked order and to find that this peptide section is the prophymosin-alpha N-end peptide section of N-end acetylation modification, finds that acetylation modification occurs in N and holds first amino acid---on the serine residue.

The above results shows that peak A sample is the prophymosin-alpha of non-acetylation modification, and peak B sample is the prophymosin-alpha of acetylation modification.

As can be seen, A peak sample is the former (Thr of non-acetylated thymosin alpha ¹³); B peak sample is the former (Thr of N-acetylated thymosin alpha ¹³),, from figure, can find the prophymosin-alpha (Thr that the contrast bacterium is expressed in conjunction with shown in Figure 2 ¹³) in have 60% approximately (instrument area integral and learn) be the former (Thr of N-acetylated thymosin alpha ¹³), and reorganization bacterium BL21 (DE3)/(pOKBV-RimL/proT α (Thr ¹³)) prophymosin-alpha (Thr that expresses ¹³) in, 90% is the former (Thr of N-acetylated thymosin alpha ¹³), the A peak has disappeared substantially.

Above presentation of results, the reorganization bacterium BL21 (DE3) of structure/(pOKBV-RimL/proT α (Thr ¹³)) can efficiently express the former (Thr of N-acetylated thymosin alpha ¹³).

The T7 promotor structure that embodiment 3, utilization are inserted in the genome contains the former (Thr of expression N-acetylated thymosin alpha ¹³) the reorganization bacterium

Adopt the Red recombinant technology; the T7 promotor that will have lactose operon (derives from the PET22b plasmid; this plasmid is available from Novagen company) be inserted into the upstream of the N-acetyltransferase RimL gene open reading frame (ORF) of e. coli bl21 (DE3), the t7 rna polymerase gene that is subjected to the control of lactose promotor that has in this bacterium genome.In substratum, add lactose or its analogue; during as IPTG; the t7 rna polymerase genetic expression of lactose promotor control; the synthetic t7 rna polymerase is that the T7 promotor that is inserted into the upstream of RimL gene open reading frame (ORF) combines with the people; the lactose operon in T7 promotor downstream derepresses simultaneously; efficiently transcribing of control N-acetyltransferase RimL gene, thus crossing of realization N-acetyltransferase RimL expressed.

Concrete grammar comprises:

One, two ends have the amplification of the chloramphenicol resistance gene in RimLORF upstream homology arm and ORF initiator homology arm and FRT site

1, the pcr amplification of chloramphenicol resistance gene.

Synthetic primer:

5Cm AgCgATTgTgTAggCTggAg

3Cm TAATTAACggCTgACATgggAATTAg

Extract BW25141/pKD3 (available from the CGSC of Yale University, CGSC#7631) plasmid pKD3 is a template, is that primer carries out pcr amplification with 5Cm and 3Cm, the 1055bp PCR product that obtains, through the order-checking for sequence 5 from 5 ' terminal 41-1095 position Nucleotide, with this PCR product called after Cm.

2, the pulsating pcr amplification of T7 promoter DNA that has lactose operon

Synthetic primer:

3Cm5T7?cTAATTcccATGTcAGccGTTAATTAtcgagatctcgatcccgcga

3T7 catatgtatatctccttcttaaag

Wherein the 3Cm5T7 primer has 5 ' sequence of chloramphenicol resistance gene 3 ' sequence and T7 promotor.

With the PET22b plasmid is template, is that template is carried out pcr amplification with 3T7 and 3Cm5T7, the 140bp PCR product that obtains, through order-checking T7 promotor be sequence 5 from 5 ' terminal 1119-1135 position Nucleotide, with this PCR product called after T7.

3, have the pcr amplification of the Red reorganization of RimLORF upstream homology arm and ORF initiator homology arm with DNA.

Synthetic primer:

5RimLCm AGCCAGGCGGCTTTTTTAACAACTGCATGGATTGACTGGAAgCgATTgTgTAggCTggA

RimLORF5pt7 GTAATTCAAGTGATTCGCTTACTTTTATCGTTTCAGTCATatgtatatctccttctta

Wherein 5RimLCm contains RimLORF upstream homology arm (line part) and chloramphenicol resistance gene 5 ' sequence, and RimLORF5pt7 is the reverse complementary sequence of RimLORF initiator homology arm (line part) and T7 promotor 5 ' sequence.

Dna fragmentation Cm and each 1 μ l of dna fragmentation T7 with the preparation of above-mentioned acquisition are template, are primer with 5RimLCm and RimLORF5pt7, carry out pcr amplification, obtain the 1246bpPCR product, through order-checking, nucleotides sequence is classified sequence 5 as, called after Red recombinant DNA, structure iron as shown in Figure 3.

Plasmid pKD3 may be mixed with in the dna fragmentation of gene-amplification, the false positive phenomenon may be caused, thus the dna fragmentation that reclaims is handled 1h with 37 ℃ of waters bath with thermostatic control of Dpn I, to eliminate plasmid pKD3.

4, the acquisition of homologous recombination positive colony BL21 (DE3)/T7RimL

1) preparation of e. coli bl21 (DE3)/pKD46

With pKD46 plasmid (genbank AY048746, its construction process is seen Datsenko KA, PNAS USA2000,97 (12): 6640-5, also can obtain from various preservations center, as the CGSC of Yale University, the preserving number of the intestinal bacteria BW25141/pKD46 that has the pKD46 plasmid at this center is 7634.Anti-penbritin.This plasmid contains the RED recombinase.) transform BL21 (DE3) (available from vast Tyke, Beijing biological gene technology company limited, catalog number CD601) coating contains the LB flat board of penbritin, 30 ℃ of incubated overnight, next day the picking mono-clonal, extract plasmid, send to order-checking and be the pKD46 plasmid, will contain reorganization bacterium called after BL21 (the DE3)/pKD46 of this plasmid.

2) the two ends electricity of dna fragmentation that has a chloramphenicol resistance gene in RimLORF upstream homology arm and ORF initiator homology arm and FRT site transforms

BL21 (the DE3)/pKD46 of above-mentioned acquisition is inoculated in the LB substratum 30 ℃ is cultured to OD _600nmBe about 0.2, add final concentration and be 0.2% L-arabinose (L-ara) and induce about 1h (OD _600nmLess than 0.6), cell is put in the ice cooling 10min rapidly, with 4000rpm in 4 ℃ of centrifugal 10min, cell is given a baby a bath on the third day after its birth inferior with 10% ice-cold glycerine, at last cell is managed packing with 1/200, the 100 μ l/ that 10% ice-cold glycerine is concentrated into the original bacteria liquid volume ,-70 ℃ of preservations are standby.Per 100 μ L competent cells add the Red recombinant DNA of the above-mentioned preparation of 0.2-1 μ g, go to behind the mixing in the 0.1cm electric shock cup, make electric shock with Bio-lab electric shock instrument and transform.Add 600 μ l LB substratum after the electric shock immediately, 150rpm, cultivate 1h for 30 ℃, coating contains the LB flat board of paraxin (Cm), 37 ℃ of incubated overnight, and pKD46 is unstable in 37 ℃ of culturing process, can spontaneously lose, next day the picking mono-clonal, and the single bacterium colony of streak culture separation on the LB flat board that contains paraxin (Cm) again is the mono-clonal that obtains the Red recombinant DNA.

The mono-clonal that obtains the Red recombinant DNA is inoculated LB flat board that contains paraxin (Cm) and the LB flat board that contains penbritin (Amp) respectively, and 37 ℃ of incubated overnight can be grown on the Cm flat board, but can not be the clone who loses the pKD46 plasmid that is of the dull and stereotyped growth of Amp.

The clone's of pKD46 plasmid plasmid is lost in extraction, and carry out PCR with synthetic primers designed 5RimLPout and 3RimLpout and identify,

5RimLPout GCGTCAAAGAGGTGTAAACT

3RimLpout GATAAAGAGGTTTGACGTGA

With the PCR product through 1% agarose electrophoretic analysis, the T7 promotor for having the Cm gene that obtains 1Kb is inserted the positive plasmid of intestinal bacteria RimL gene ORF upstream, the negative clone who obtains 100bp does not contain the T7 promotor of Cm gene, will contain clone's called after BL21 (the DE3)/T7RimL of positive plasmid.

5, reorganization bacterium BL21 (DE3)/T7RimL expresses RimL

With BL21 (DE3)/T7RimL concentration is the IPTG (sec.-propyl-D-thiogalactoside of 1mM, available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd) induce (behind 37 ℃ of cultivation 2h, induce 6h), collect supernatant, carry out the SDS-PAGE electrophoresis, obtain the fragment of 20KD, illustrate to give expression to RimL.

Two, utilize the T7 promotor of inserting in the genome to make up and contain the former (Thr of expression N-acetylated thymosin alpha ¹³) the reorganization bacterium

PBV220-proT α (Thr ¹³) be prepared as follows: with ProT α (Thr ¹³) gene (nucleotides sequence is classified sequence 4 as, and its aminoacid sequence is a sequence 3) inserts the BamHI and the EcoRI site of pBV220 carrier, the carrier that obtains, proT α gene is by the P on the pBV220 carrier _LP _RPromotor control.

Plasmid pBV220-proT α (Thr with above-mentioned acquisition ¹³) shocking by electricity is transformed among above-mentioned one BL21 that obtains (DE3)/T7RimL, obtains transformant, extracts plasmid, through order-checking, pBV220-proT α (Thr ¹³), will contain bacterium called after BL21 (the DE3) (T7RimL/proT α (Thr of this plasmid ¹³)).

Use same quadrat method, with pBV220-proT α (Thr ¹³) shocking by electricity transforms among the BL21 (DE3), obtains transformant, extracts plasmid, the result is pBV220-proT α (Thr ¹³), will contain bacterium called after BL21 (the DE3) (proT α (Thr of this plasmid ¹³)).

Embodiment 4, BL21 (DE3) (T7RimL/proT α (Thr ¹³)) preparation N-acetylated thymosin alpha is former

1, protein crude extract

With BL21 (DE3) (T7RimL/proT α (Thr ¹³)) and contrast bacterium BL21 (DE3) (proT α (Thr ¹³)) be inoculated in respectively in the 50ml LB liquid nutrient medium, 37 ℃ of shaking table overnight incubation, be forwarded to then and contain 500ml substratum (yeast extract 10g/L, Tryptones 10g/L, SODIUM PHOSPHATE, MONOBASIC 20mM, Sodium phosphate dibasic 30mM) 3L shakes in the bottle, behind 37 ℃ of cultivation 2h, the IPTG (0.5mM) that adds 1.25ml 0.2mol/L induces, and continues to cultivate 6h, fermented liquid is centrifugal, collects thalline.

With the thalline water of collecting resuspended (every gram bacterium adds 10ml water), supersonic wave wall breaking, centrifugal collection supernatant adds glacial acetic acid and regulates pH to 4.5 in supernatant liquor, left standstill 30 minutes, and centrifugal collection supernatant is protein crude extract.

2, purifying

1) SP FF Φ 1.6 * 20cm column purification (cation-exchange chromatography)

The supernatant liquor of above-mentioned acquisition is carried out purifying with SP FF Φ 1.6 * 20cm post (medium is available from U.S. GE company, and void column is available from magnificent laboratory apparatus factory).Actual conditions is: the A liquid (damping fluid that 20mM sodium acetate and acetate are formed, pH4.5), B liquid (A liquid+1M NaCl), SP FF post is earlier with A liquid balance, then from A channel with sample on the above-mentioned crude extract that contains the N-acetylated thymosin alpha to SP FF post, use the unconjugated albumen of A liquid flush away again, at last at 0 → 30 minute: A is from 100% → 0%; B is from 0% → 100% linear gradient elution, the Fractional Collections elutriant.

SDS-PAGE analysis and HPLC analysis are done in the at different levels parts of elutriant samplings of collecting, merged the elutriant (in the 30%-60%B elutriant) that contains prophymosin-alpha, obtain the former raw product of N-acetylated thymosin alpha.

The RP-HPLC chromatography is carried out in sampling, adopts the HP1090 high pressure liquid chromatograph, and (A liquid is for containing the pure water of 0.1%TFA (volumn concentration) for 4.6 * 250mm, Dalian chemical physics institute of the Chinese Academy of Sciences for the C18 post; B liquid is the trifluoroacetic acid aqueous solution that contains 0.1%TFA (volumn concentration), gradient elution: 0min, and A 100%, and B 0%; 5min, A 82%, B18%; 25min A 78%, B 22%; 28min A0%, B 100%; 30min A0%, B 100%; 31min A 100%, B 0%, and 214nm ultraviolet detection, flow velocity are 1mL/min, collect the elutriant that retention time is 17.0min respectively, obtain A peak sample, and collecting retention time is the elutriant of 20.0min, obtains B peak sample.

Map as shown in Figure 4, I is contrast bacterium BL21 (DE3) (proT α (Thr ¹³)) preparation prophymosin-alpha (Thr ¹³); II is BL21 (DE3) (T7RimL/proT α (Thr ¹³)) preparation prophymosin-alpha (Thr ¹³).

The A peak sample and the B peak sample that will contrast the collection of bacterium and experimental bacteria are respectively used mass spectrometric detection respectively, and method is the same, and the result shows that peak A sample is the prophymosin-alpha of non-acetylation modification, and peak B sample is the prophymosin-alpha of acetylation modification.

As can be seen, A peak sample is the former (Thr of acetylated thymosin alpha not ¹³); B peak sample is the former (Thr of N-acetylated thymosin alpha ¹³),, from figure, can find contrast bacterium BL21 (DE3) (proT α (Thr in conjunction with shown in Figure 4 ¹³)) the N-acetylated thymosin alpha was 41% (B peak) originally in the extrasin alpha of preparation, and BL21 (DE3) (T7RimL/proT α (Thr ¹³)) the N-acetylated thymosin alpha was 82% (B peak) originally in the prophymosin-alpha of preparation, the raising of this N-acetylation modification rate provides the foundation for improving productive rate, therefore has a good application prospect.

Embodiment 5, prepare various N-acetylated thymosin alphas with crossing the intestinal bacteria express N-acetyltransferase RimL

Adopt the method for embodiment 1 to prepare the reorganization bacterium of expressing the various extrasin alphas (aminoacid sequence of various extrasin alphas is as shown in table 2 below) in the following table 1 respectively, the method for employing embodiment 2 is fermented and is detected, and the result is as shown in table 2 below:

The acetylize rate of the various N-acetylated thymosin alphas of table 2 preparation

As can be seen from the above table, the reorganization bacterium according to the preparation of the method for embodiment 1 all can obtain the high N-acetylated thymosin alpha of content by fermentation.

Claims (10)

1. a reorganization bacterium is following 1) or 2):

1) encoding gene of RimL and the encoding gene of extrasin alpha are imported the reorganization bacterium that obtains in the host bacterium; Described host bacterium is the host bacterium that contains the RimL encoding gene in the genome;

2) encoding gene with extrasin alpha imports the reorganization bacterium that obtains in arbitrary described reorganization bacterium among the following claim 6-8;

Described RimL be following (a) and (b) or (c) shown in albumen:

(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 2;

(b) with the aminoacid sequence shown in the sequence in the sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have the albumen of identical function;

(c) and a) or b) dna sequence dna that limits has 90% homology at least and has the albumen of identical function;

Described extrasin alpha is a peptide species or albumen, in 28 amino-acid residues of its N-terminal and the sequence table in 28 amino-acid residues of identical or its N-terminal of 28 amino acid of the N-terminal of sequence 3 and the sequence table 28 amino acid of the N-terminal of sequence 3 have 90% homology at least.

2. reorganization bacterium according to claim 1 is characterized in that:

1) in the reorganization bacterium shown in, described encoding gene and the encoding gene of the described extrasin alpha method that imports the host bacterium with RimL comprises the steps:

A) encoding gene with described RimL imports described host bacterium by recombinant vectors 2, and bacterium A obtains recombinating;

B) encoding gene with described extrasin alpha imports the described reorganization bacterium A that step a) obtains by recombinant vectors 1, obtains the bacterium of recombinating;

Described recombinant vectors 1 is that described extrasin alpha encoding gene inserts in the expression vector 1, obtains expressing the recombinant vectors 1 of extrasin alpha; Described expression vector 1 is preferably pBV220;

Described recombinant vectors 2 is that the encoding gene of described RimL is inserted in the expression vector 2, obtains expressing the recombinant vectors 2 of RimL, and described expression vector 2 is preferably pOKBV.

3. reorganization bacterium according to claim 1 and 2 is characterized in that:

1) in the reorganization bacterium shown in, described host bacterium is intestinal bacteria;

Described RimL encoding gene is following 1) or 2) shown in dna molecular:

1) dna molecular shown in the sequence 1 in the sequence table;

2) with 1) dna sequence dna that limits has the proteic dna molecular that 90% homology and coding have identical function at least;

Described extrasin alpha encoding gene is a kind of polynucleotide, the sequence 4 in its nucleotide sequence and the sequence table 5 ' terminal rise 84 Nucleotide identical or with sequence table in 5 ' terminally the playing 84 nucleotide homologies and have 90% dna molecular at least of sequence 4.

4. reorganization bacterium A is for the encoding gene with described RimL imports in the host bacterium reorganization bacterium A that obtains by recombinant vectors;

Described recombinant vectors is that the encoding gene of described RimL is inserted in the expression vector, obtains expressing the recombinant vectors of RimL, and described expression vector is to express by the encoding gene of inducible promoter control RimL.

5. recombinant vectors according to claim 4 is characterized in that: described inducible promoter is lactose promotor, pectinose promotor, alkaline phosphatase promotor, trp promoter, phage t7, P _L, P _RPromotor or contain hybrid promoter Tac, the P of these promotor subelements _LP _R, described expression vector is preferably pOKBV.

6. a reorganization bacterium for the external source inducible promoter being inserted the upstream of the acetyltransferase encoding gene of host bacterium, is used to start the expression of described acetyltransferase encoding gene, the reorganization bacterium that obtains.

7. reorganization bacterium according to claim 6 is characterized in that: described inducible promoter is lactose promotor, pectinose promotor, alkaline phosphatase promotor, trp promoter, phage t7, P _L, P _RPromotor or contain hybrid promoter Tac, the P of these promotor subelements _LP _R

8. according to claim 6 or 7 described reorganization bacterium, it is characterized in that:

Described host bacterium is intestinal bacteria;

Described inducible promoter is the phage t7 promotor, its nucleotides sequence classify as sequence 5 in the sequence table from 5 ' terminal 1119-1135 position Nucleotide;

Described acetyltransferase is RimL,

Described RimL be following (a) and (b) or (c) shown in albumen:

(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 2;

(b) with the aminoacid sequence shown in the sequence in the sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have the albumen of identical function;

(c) and a) or b) amino acid that limits has 90% homology at least and has the albumen of identical function.

9. the application of arbitrary described reorganization bacterium in preparation N-acetylated thymosin alpha among the claim 1-8.

10. a method for preparing the N-acetylated thymosin comprises the steps:

Arbitrary described reorganization bacterium among the fermentation claim 1-3 is collected tunning, promptly obtains the N-acetylated thymosin alpha;

The temperature of described fermentation is 42 ℃, and described fermentation time is 12h.

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