CN1152134C - Process for preparing recombinant deoxyribonuclease I - Google Patents

Abstract

The present invention relates to a method for preparing recombination deoxyribonuclease I with biological activity, which comprises: (1) proper prokaryotic host cells are converted by a nucleic acid construction body containing a nucleotide sequence used for coding the deoxyribonuclease I; (2) the converted host cells are cultivated under the condition suitable for selected prokaryotic cell growth; (3) the host cells are caused to generate the deoxyribonuclease I under the condition suitable for deoxyribonuclease I expression; (4) the deoxyribonuclease is recovered from obtained escherichia coli thallus and/or a culture solution. The deoxyribonuclease I prepared by the method of the present invention can be used for preparing medicines used for treating respiratory diseases, reducing the phlegm viscosity of patients and communicating respiratory tracts.

Description

A kind of method for preparing recombinant deoxyribonuclease I

Technical field

The present invention relates to a kind of recombinant deoxyribonuclease class preparation method, particularly prepare the preparation method of recombinant deoxyribonuclease I (being designated hereinafter simply as DNase I) with prokaryotic host cell.Can be used for preparing treatment respiratory system disease, the viscosity that reduces patient's phlegm, the medicine of unobstructed respiratory tract by the prepared deoxyribonuclease I of the inventive method.

Background technology

Deoxyribonuclease (being designated hereinafter simply as DNase) is the phosphodiesterase of a kind of hydrolyzable thymus nucleic acid (DNA), comprises DNase-I and DNase-II, compares with restriction enzyme enzyme require identification particular sequence, and it can non-degrade specifically DNA.The optimum pH of DNase-I must have divalent cation to participate in hydrolysis reaction near neutral, produces the acid of 5 '-phosphoric acid nucleoside and 5 ' phosphoric acid oligonucleotide.

From the separated purifying of the DNase of many species, ox DNase A, B, C and D are early than the purifying in 1973 the and (Liao etc. that check order, journal of biological chemistry (J.Biol.Chem.) 249:2354,1973), the DNase of pig and sheep is separation and purification and order-checking (Paudel etc. also, journal of biological chemistry, 261:16006,1986) and (Paudel, journal of biological chemistry, 261:16012,1986).

Known DNase has multiple function, and can be used for medical purpose.Its topmost medical applications is that reduction lung viscous secretion is the viscosity of phlegm, and for example in treatment pneumonia process, it can help to clean respiratory tract.The respiratory tract obstruction that is caused by viscous secretion can cause breathing discomfort, even causes death by suffocation.

The DNase that extracts preparation from natural ox pancreas once was that trade(brand)name is sold with Domavae, but be to use DNase to cause that tangible complication of part patient such as lung swell and anaphylaxis (Raskin, U.S.'s respiratory system disease summary (Am.Rev.Resp.Dis.), 98:697,1986), finally from market, recall.The reason that produces this complication is because contain the impurity proteolytic enzyme that causes above-mentioned discomfort in the product of this natural origin.In addition, many diseases produce the symptom of heavy-gravity pulmonary secretions normally chronic with recurrent, it is also improper that life-time service ox pancreas DNase treats this type of disease.For addressing this problem, can utilize engineered method, prepare recombinant human or ox DNase with protokaryon or eukaryotic cell, thereby avoid the pollution of proteolytic enzyme.

Although the full length sequence of first Mammals DNase just obtained in 1973, the report that after many years, relevant clone has just been arranged and expressed this zymoid work.The specific effect of U.S. Genetic company in 1994 comes out in recombinant human deoxyribonuclease (rhDNase) expelling phlegm drugs of macromole DNA, the length (shortening to 0.3-0.5kbp) that is used for reducing the macromole dna content of sputum and shortens the extracellular dna molecular from 0.6-2.6kbp, thereby obviously reduce the viscosity of sputum, improve the pulmonary function (FEV1 and FVC all improve more than 10%) of lung diseases such as obstructive, improve patient's quality of life, prolonged patient's lifetime.Thereby rhDNase can be used for preparing the active drug of treatment chronic bronchitis, pulmonary emphysema, pulmonary heart disease, bronchial asthma and other respiratory tract disease.

Because DNase is toxic to host cell, the recombinant expressed especially examples of many successful of expression DNase and few in prokaryotic cell prokaryocyte.For recombinant expressed specific objective protein in intestinal bacteria particularly in prokaryotic cell prokaryocyte, at present general method is that nucleotide sequence with the coding target protein inserts suitable expression plasmid, transform the host bacterium with described recombinant expression plasmid then, obtain recombinant bacterial strain, under suitable culture condition, cultivate the gained recombinant bacterial strain, the target protein that separation and purification is expressed.But, for such as DNase such for the virose target protein matter of host cell, use common described recombinant expression method, the expression of very small amount of DNase will be killed host cell, thereby can't obtain the target product of requirement.Therefore, need to adopt a kind of special recombinant expressed strategy, by utilizing rigorous inducible promoter controlled target protein expression, that is: express recombinant gene not in the recombinant host cell process of growth, when recombinant host cell grows into appropriate concentration, add suitable inductor and induce the expression of target protein.The inducible promoter that this area is known as the lac promotor, can start recombinant protein expression in prokaryotic host cell after adding inductor ITPG.But the lac promotor is not a kind of inducible promoter of strictness, still has a spot of background and express under the situation that does not have ITPG to add.Because it all is fatal concerning the recombinant expressed host of gained that a spot of background of DNase I of the present invention is expressed, so need a kind of very strict abduction delivering system of special design.Utilize general expression strategy, not only be difficult to obtain product when expressing, and will run into very big difficulty in the process that obtains recombinant bacterial strain, for example owing to the degeneration rapidly of bacterial classification, work such as the screening of bacterial classification, preservation and property analysis all will be difficult to carry out.People such as Sheild utilize thermoinducible method at escherichia coli cloning ox DNase-I gene fragment, and identified that expressing fusion protein clone has DNase biologic activity and immunologic competence (Biochem.Soc.Trans. 16:195,1988).Yet because the toxicity that DNase has also is difficult to extract plasmid from the clone even be in holddown at cold-starting.This has greatly limited the application that DNase expresses in prokaryotic cell prokaryocyte.So present only report of secreting, expressing people DNase (Genentech, INC.EP 0 449 968 B1,02/24/1999) in zooblast.The DNase productive rate is low, cost is high, has limited the application of DNase as medicine greatly but animal cell culture is produced.People DNase is recombinant expressed never successful report in prokaryotic cell prokaryocyte.

In order to solve the problem of above-mentioned recombinant expressed deoxyribonuclease, the present invention has designed unique expression strategy, utilize a kind of system of rigorous abduction delivering, that is: the T7-RNA polymerase promoter expression system (this expression system only could be expressed DNase after the phage induction that contains the T7-RNA pol gene) of phage induction, successfully in T7-RNA polysaccharase defective escherichia coli, expressed the DNase I of people and Niu, realized reorganization DNase I production, for road has been paved in the extensive medicinal application of DNase.This expression method has not only solved the toxicity problem of DNase pair cell effectively, and since the characteristic of DNase itself be difficult in bacterial cell, forming inclusion body, solved exogenous protein in intestinal bacteria in the high expression level process common inclusion body form and inclusion body protein matter folds problems such as renaturation complex process, renaturation yield are low.

Summary of the invention

One aspect of the present invention relates to the method for the recombinant deoxyribonuclease I (DNase I) that a kind of preparation has biologic activity, and it comprises that (1) transforms suitable prokaryotic host cell with the recombinant expression vector of the nucleotide sequence that contains coding DNA enzyme I; (2) be suitable for cultivating under the selected prokaryotic cell prokaryocyte growth conditions through transformed host cells; (3) under the condition that is suitable for the deoxyribonuclease I expression, induce host cell to produce deoxyribonuclease I; (4) from gained through transforming prokaryotic host cell and/or nutrient solution reclaim deoxyribonuclease I.

The present invention relates to a kind of zymin that contains the deoxyribonuclease I of useful aforesaid method preparation on the other hand.

Another aspect of the present invention relates to the purposes that is used to prepare the medicine for the treatment of respiratory system disease, the viscosity that reduces patient's phlegm, unobstructed respiratory tract with the deoxyribonuclease I of aforesaid method preparation.

Description of drawings

The collection of illustrative plates of the expression vector pBGI-2 that Fig. 1, the present invention adopt.

The collection of illustrative plates of Fig. 2, the recombinant expression plasmid pBGI-2-hDNase that contains people DNase I encoding gene that makes up with pBGI-2.

The collection of illustrative plates of Fig. 3, the recombinant expression plasmid pBGI-2-bDNase that contains ox DNase I encoding gene that makes up with pBGI-2.

One aspect of the present invention relates to a kind of recombinant DNA with biology activity for preparing The method of enzyme I, it comprises that (1) usefulness contains the nucleotide sequence of coding DNA enzyme I Nucleic acid construct transform suitable prokaryotic host cell; (2) be suitable for selected prokaryotic life Cultivate the host's cell through transforming under the elongate member; (3) be suitable for the deoxyribonuclease I expression Condition under induce host's cell to produce deoxyribonuclease I; (4) from gained through transforming Reclaim deoxyribonuclease I in prokaryotic host cell and/or the nutrient solution.

Term used herein " activity " refers to have non-specific hydrolysis DNA (DNA) phosphoric acid diester linkage causes the property of 3 ' phosphoric acid base deoxidation of sugar-phosphoric acid DNA skeleton Matter.

For realizing rigorous type abduction delivering of the present invention, selected expression system generally comprises band Suitable rigorous expression vector of inducing the type promoter is arranged, can effectively induce luring of described promoter Lead thing, and with selected expression vector and the suitable expressive host of inducing phase to adapt to. Can be used for Expression vector of the present invention comprises chromosome, achromosomal and synthetic dna sequence dna, example As, bacterium plasmid, bacteriophage DNA, yeast plasmid, be derived from plasmid and bacteriophage DNA knot The carrier that closes, viral DNA. But, as long as can in the host, copy and survive, other carrier Also can utilize. Dna sequence dna in the expression vector is controlled with a suitable expression effectively Sequence (promoter) connects together, thereby instructs synthesizing of mRNA. This area is known, can With multiple suitable method, such as temperature transition, chemicals induce, the side such as bacteriophage is induced Method is induced the selected type promoter of inducing, the present invention can with induce the type promoter to comprise but do not limit In, for example: the P of colibacillary lac, trp or tac promoter, bacteriophage_LPromoter, T5, Other promoter of gene expression in T7 promoter and the known control prokaryotic. Express and carry Body also comprises the needed ribosome bind site of translation initiation and transcribes termination. Carrier also can Strengthen the proper sequence of expressing to have. In addition, obtain for the ease of screening recombinant expressed year Body, it preferably comprises one or more selectable marker genes that phenotypic characteristic can be provided, so that The screening of transformed host cell, for example Escherichia coli can use tetracycline, ampicillin, chlorine mould Element, neomycin or kalamycin resistance.

Also should contain suitable restriction enzyme site on the described carrier of the present invention, be beneficial to The insertion of destination protein matter coded sequence. In one embodiment of the invention, preferably make Use the expression vector with the T7-RNA polymerase promoter, for example the T7 that contains of the present invention's structure opens The recombinant expression plasmid pBGI-2 (as shown in Figure 1) of mover. In case the DNase gene with people or ox Be inserted in the position that can be subjected to the adjusting of T7 promoter, only have and in system, contain the T7-RNA polymerization During enzyme, the genes of interest that regulated by described promoter could be transcribed and be shown in selected host Reach.

In order to realize rigorous type abduction delivering, selected host cell should be the bacterial strain that there is or produces the inductor that can induce selected promoter expression in non-natural.Known as this area, can select multiple suitable expressive host for use, as long as the expression of target protein matter can not independently be induced in this host's growth.In a preferred embodiment of the present invention, select a kind of T7-RNA polysaccharase defective escherichia coli for use, particularly to containing the T7 rna polymerase gene and containing the bacterial strain of the phage-sensitive of corresponding promotor, as T7-RNA polysaccharase defective escherichia coli JM109.Can induce selected inducible promoter to express required inductor in order to realize rigorous type abduction delivering of the present invention, can to adopt multiple mode to introduce.Those skilled in the art should know, and the expressive host of selected inducible promoter and employing is depended in the introducing of selected inductor, as long as can realize rigorous type abduction delivering, any method all can be used.In a preferred embodiment of the present invention, adopt the selected host strain of λ CE6 phage-infect.In one embodiment of the invention, recombinant expression plasmid with the nucleotide sequence that contains encoding D Nase I transforms T7-RNA polysaccharase defective escherichia coli JM109, cultivate the gained recombinant bacterial strain under proper condition, during host's bacteria growing, owing to there is not endogenous T7 polysaccharase, the target DNase I gene that regulated by the T7 promotor can not be expressed.The density of recombination engineering bacteria reaches certain expected value in fermented liquid, and wherein contained plasmid copy number adds λ CE6 phage, abduction delivering DNase I when also reaching desired amt.Be not limited to specific theory, the inventor unexpectedly finds, under described condition of the present invention, although the generation of DNase I has toxic action to thalline, can cause bacteriolysis to take place, but when bacteriolysis took place, recombinant expressed host cell of the present invention had produced a large amount of DNase I.Because the expression of DNase I causes the cracking of host cell, a large amount of target proteins are present in the nutrient solution, be different from the routine operation that utilizes the escherichia coli high-level expression target protein, need not the aftertreatment that multisteps such as sex change, refolding waste time and energy, just can directly from the nutrient solution of cultivating recombinant host cell of the present invention, reclaim deoxyribonuclease with biologic activity.

Deoxyribonuclease I of the present invention can be any source, includes but not limited to people, ox, pig, mouse, rat etc.In one embodiment of the invention, preferred end user or ox deoxyribonuclease I.

In order to obtain DNase I, can pass through known pcr amplification technology and the recombinant DNA technology of those skilled in the art.Design corresponding PCR primer according to disclosed DNase I encoding sequence, respectively the DNase of above-mentioned people of amplification or ox from the pancreas cDNA library of people or ox.Gained PCR product is imported suitable expression vector by the known method of those skilled in the art, make up the recombinant DNA construction body.Transform proper host cell with the gained DNA construct, under the condition that is suitable for the target protein expression, cultivate transformed host cells like this, and reclaim and purification of target protein by the known technology in this area.

Suitable dna sequence dna can be inserted in the carrier by many methods.Usually, dna sequence dna is inserted on the suitable restriction endonuclease sites with method as known in the art.These methods are believed in the ken that is in those skilled in the art.

Comprise above-mentioned suitable dna sequence dna, suitable promotor or the carrier of control sequence and can be used to transform appropriate host, allow this albumen of host expresses.

The method that construct is imported host cell has: calcium chloride conversion, magnesium chloride conversion, electroporation or particle gun method, TSS conversion method, protoplast transformation method, Lithium Acetate conversion method etc.

Usually, recombinant expression vector comprises replication orgin and is used for the selective marker of transformed host cell, for example colibacillary kalamycin resistance gene and cereuisiae fermentum TRP1 gene also comprise from the promotor that efficiently expresses gene, thereby mediate transcribing of downstream configurations sequence.Allogenic structure sequence is assembled together with suitable orientation and translation initiation and terminator sequence and other preferred sequence.Selectable situation is, the dna sequence dna fusion rotein of can encoding, and this albumen contains a N-terminal or C-terminal is confirmed peptide, shows the feature of expection, the stabilization of for example expressed recombinant products and simplify purification step.Peptide that is merged or albumen can be that Thiadiazolidine isomerase GST, maltose binding protein MBP, staphylococcus aureus protein A, Trx Trx A, paraxin acyltransferase CAT, beta-galactosidase enzymes, Flag peptide, Intein label, poly are histidine-tagged, poly arginine label and other polyamino acid label etc.Because 6 * and histidine-taggedly making target protein be easy to, can not change proteinic structure, activity and antigenicity and needn't remove etc. by the metal chelate chromatography purifying, the present invention preferably adds 6 at the N-terminal of DNase I or C-terminal * and histidine-tagged.The effective expression carrier that bacterium is suitable for can make up like this: the structural DNA sequence of the target protein of will encoding is inserted in the steerable reading frame that has function in company with suitable translation initiation and termination signal.Carrier should comprise one or more Phenotypic Selection marks and replication orgin, and replication orgin has guaranteed that carrier can remain in the host, and better is to make carrier obtain amplification.The prokaryotic hosts that is suitable for transforming has the various bacteriums of intestinal bacteria, subtilis, Salmonella typhimurium and Rhodopseudomonas, streptomyces and Staphylococcus, and other host also can be selected.

In a preferred embodiment of the invention, according to known person and ox DNase-I gene order, the synthetic upstream and downstream primer of design, with the amplification of PCR method, from human pancreas cDNA storehouse, obtain ripe DNase gene order, from ox pancreas cDNA storehouse, obtain full-length gene order, recombinate among the expression plasmid pBGI-2, and transformed into escherichia coli JM109, after for some time cultivates, adopt λ CE6 phage induction to give expression to target protein people or ox DNase-I under the certain condition.

In one embodiment of the invention, from pancreas, extract total RNA and purified mRNA, 5 ' end at each cDNA adds a specific connexon (adaptor), obtain people's pancreas or ox pancreas, have specific connexon, can be used for the people's pancreas or the ox pancreas cDNA storehouse of the terminal method of rapid amplifying cDNA (RACE-PCR).According to the DNase-I sequence of having announced, design contains the specific 5 ' primer and the 3 ' primer of DNase I gene.With design synthetic 5 ' primer and 3 ' primer and connexon primer, adopt the RACE-PCR amplification gene then, obtain 3 ' RACE fragment and the 5 ' RACE fragment of DNase I respectively.Be connected with cloning vector pGEM-T respectively.Transform and choose anti-Amp ⁺Positive colony.Extraction contains DNase I5 ' fragment and 3 ' segmental two recombinant cloning vectors respectively, and enzyme is cut the back and connected two fragments with dna ligase, obtains full length sequence DNase I gene.DNase I gene fragment is connected with cloning vector pGEM-T, obtains carrying the recombinant clone of complete DNase I gene.According to the gene order of DNase I with need the restriction endonuclease sequences Design primer that adds, pcr amplification goes out to have the DNase I gene of restriction endonuclease sequence, and enzyme is cut the back and is connected with the expression vector pBGI-2 that enzyme is on the same group cut, and obtains recombinant expression vector.Transform and choose anti-Kan ⁺Positive colony.And gained contained insert segmental recombinant plasmid order-checking, expression product is carried out sds polyacrylamide gel electrophoresis (SDS-PAGE) and activity identification.The pBGI-2 expression vector contains f1 replication orgin, ribosome bind site, kalamycin resistance gene (Kan except that containing T7 rna polymerase promoter and T7 terminator ⁺), and contain the encoding sequence of His label.

Adopt the growing state that detects fermented liquid optical density value method check host strain, determine the joining day of phage.The time that adds phage in the time of can adopting intermittently culture method to be cultured to O.D.600 to be 0.6-2 for the recombinant host bacterium.Those of ordinary skills are known to cultivate the recombinant host bacterium by methods such as feeding culture, high-density culture, and the nutrient solution O.D.600 when it adds phage should corresponding increasing.In one embodiment of the invention, when the recombinant host bacterium grows to O.D.600=0.4-0.8 in nutrient solution, add λ CE6 phage, induce DNaseI to express.

Usually can after adding described inductor, continue to cultivate host strain about 0.5-20 hour, preferably about 1-10 hour, more preferably about 1.5-5 hour, thalline O.D. value beginning in nutrient solution obviously descends, collect nutrient solution then, adopt that the known method in this area is as described below to reclaim DNase I from nutrient solution and in the reorganization bacterium thalline.

Common method harvested cell with centrifugal or membrane filtration, the fragmentation that is used for the microorganism cells of marking protein can be with any method easily, usually with physics, chemistry or biological method smudge cells, comprise freeze-thaw cycle, ultrasonic disruption, Mechanical Crushing such as high-pressure homogenization, high speed pearl mill, perhaps use chemosmosis method, the molten method of enzyme, these methods all are well-known to those having ordinary skill in the art.Remove cell debris and other impurity of getting in the cytoclasis liquid by methods such as known centrifugal, the filtrations of those skilled in the art again, obtain broken supernatant liquor.As contain DNase I in the bacteria culture fluid of recombinating, then cytoclasis liquid and nutrient solution can be merged DNase I crude extract, get single step purification ready.After adjusting SOLUTION PROPERTIES such as ionic strength and pH value, available following purification process: the precipitator method such as sulfuric acid amine, ethanol, polyethylene glycol precipitation, affinity precipitation; Chromatography method such as ion exchange chromatography, hydrophobic interaction chromatography, gel permeation chromatography, hydroxyapatite chromatography, reversed phase chromatography, chromatofocusing, affinity chromatography such as DNase I antibody chromatography directly reclaim DNase I.In a preferred embodiment of the invention, for the DNase I crude extract that transforms with recombinant expression plasmid pBGI-2-DNase, the DNase I that contains the His label that adopts metal chelating and chromatography purification to express adopts competitive type of elution as using the imidazoles wash-out, purifying DNase I.Can also adopt membrane filter method such as micro-filtration, ultrafiltration, affinity membrane and aqueous two-phase extraction method, reverse micelle method etc. that DNase is carried out purifying.Micro-Ca in whole purge process ²⁺Existence will help the active maintenance of Dnase I.DNaseI after purified does not contain proteolytic enzyme.

Another aspect of the present invention relates to a kind of zymin that contains the deoxyribonuclease I that utilizes the method for the invention preparation.Described zymin randomly also comprises the required cofactor of maintenance DNase I biologic activity.Zymin of the present invention randomly comprises and keeps the compatible stablizer of DNaseI biologic activity.Zymin of the present invention can be liquid, solid, aerosol or lyophilize product form.In one embodiment of the invention, zymin of the present invention is an aerosol.When being aerosol form, contain 50-300mM NaCl and 0.01-10.0mM Ca in the solution D Nase goods ²⁺To contain 150mM NaCl and 1.0mMCa ²⁺For good.The scope of pH is 4-10.The lyophilize product are the lyophilize product of solution D Nase goods.Can also contain a spot of protein stabilizing agent such as human serum albumin, monose such as glucose, polyoxyethylene glycol etc. in the DNase goods; Can contain other composition such as proteolytic enzyme.

DNase I in the zymin of the present invention randomly also can modify through macromole, and its activity of DNaSe after modified remains unchanged, and stability is improved, the transformation period prolongs, antigenicity is covered.The macromole that is used to modify DNase can be polyoxyethylene glycol, polypropylene glycol etc. linear or branch polyalcohols compound, also can be polysaccharide compounds such as dextran.

The DNase I that the present invention relates to the preparation of method as described herein on the other hand be used to prepare treatment and/alleviate the purposes of the pulmonary disorder patient's who suffers from the concentrating secreted thing of following unusual, viscosity or purulence medicine.For example acute or chronic pulmonary branches tracheal disease (infective pneumonia, bronchitis or tracheobronchitis, bronchiectasis, pulmonary cyst fibrosis pathology (belonging to heredity pancreas disease), pulmonary emphysema, asthma, pulmonary tuberculosis or fungus-caused obstructive pulmonary disease) or since in the complication that atelectasis that tracheobronchial influence causes or bronchotomy cause especially effectively.DNA in the sticking phlegm of described DNase I degraded, thus the viscosity of patient's phlegm, the obturator of removing respiratory tract reduced.Through the DNase I of the present invention preparation for abscess or the auxiliary therapy that seriously closes with the infection (for example empyema, meningitis, abscess, peritonitis, sinusitis, otitis, periodontitis, pericarditis, pancreatitis, chololithiasis, endocarditis and infective sacroiliitis) of property also be effectively, the topical therapeutic for multiple abscess such as for example skin infections wound or mucositis, trauma wounds and festering property wound or infected wound also is very effective equally.People's DNase I is playing good effect aspect the flow velocity of the therapeutic medical pipelines such as the drain catheter that comprises surgery, the conduit of urinating, abdominal cavity film dialysis port and endotracheal oxygen cathete that keep being connected with body cavity.The DNA enzyme may improve the efficient of antiseptic-germicide in the treatment course of infection.

Except being used for medical treatment, solution goods or freeze-dried products can also be used for biological chemistry, molecular biological research and need remove the removal of the process such as the medication preparation process amplifying nucleic acid of thymus nucleic acid as the DNase I of preparation as described in the inventive method.DNase I can free form (in solution) in this type of is used or is played the effect of degradation of dna with immobilized form (being fixed on the solid-phase media).Mentioned herein to solid-phase media be meant various gel medias, silica gel medium, the organic synthesis macromolecular medium that is used for chromatography, and comprise various mineral membranes, organic membrane, comprise that the various microcarriers that are used for cell culture of animals comprise magnetic microcarrier etc.

Embodiment

Embodiment 1: the clone of human pancreas DNase

Gene order (SEQ ID NO.1 according to the human pancreas DNase that has announced, the coding region is positioned at the 160-1008 position of nucleotide sequence, the coding region of signal peptide is positioned at the 160-225 position of nucleotide sequence, SEQ ID NO.3 is the aminoacid sequence that contains signal peptide) (Shak, S. etc., institute of NAS newspaper, 87 (23): 9188,1990) design primer:

5 ' forward primer:

5′-GCAGCCTTCAACATCCAGACATTTGGGGAG-3′

3 ' reverse primer:

5′-GCTTGTCCACAGGCGGATGGATGACCACTG-3′

Get freezing people's pancreas, adopt RNeasy Midi system (Qiagen, the U.S.) from pancreas, to extract total RNA.Use Oligotex mRNA system (Qiagen, the U.S.) purified mRNA again.In order to obtain being used for people's pancreas cDNA storehouse of RACE-PCR clone technology, also must add a specific connexon at 5 ' end of each cDNA, application Marathon cDNA amplification kit (Clontech, the U.S.) has obtained the cDNA storehouse that has specific connexon of people's pancreas.Then with designing synthetic 5 ' forward primer and 3 ' reverse primer and connexon primer (Marathon cDNA amplification kit as mentioned above, Clontech), adopt terminal method (RACE-PCR) amplification gene of rapid amplifying cDNA, the PCR reaction conditions is: 95 ℃ of sex change 1 minute, annealed 1 minute for 55 ℃, 72 ℃ prolong 3 minutes.Carry out 35 circulations.72 ℃ kept 7 minutes then.4 ℃ of preservations.Obtain DNase I 3 ' RACE fragment and 5 ' RACE fragment respectively.After 1% agarose gel electrophoresis confirms segmental existence and size, connect with pGEM-T Easy Vector (Promega, the U.S.) respectively.Use CaCl ₂Conversion method transformed clone carrier bacillus coli DH 5 alpha.Choose anti-Amp ⁺Positive colony, the cloning vector that extracts reorganization also carries out enzyme and cuts evaluation.And gained contained insert segmental recombinant plasmid order-checking, the existence of conclusive evidence target gene fragment in the PCR process and do not morph or go wrong.Extraction contains DNaseI5 ' fragment and 3 ' segmental two reorganization T carriers respectively, with 3 ' fragment of Nco I and Eco47 III cutting DNase I, uses EcoR I and Eco47 III to cut 5 ' fragment of DNase I.Two fragments using Qiaquick Gel Extraction (Qiagen) purifying enzyme to cut then connect two fragments with dna ligase, obtain full length sequence DNase I gene.Use T then ₄Dna ligase connects from the total length DNase gene fragment of agarose gel electrophoresis recovery and purifying with the pGEM-T Easy Vector (Promega, the U.S.) that Nco I and EcoR I cut, and obtains recombinant cloning vector pT-DNase-h1.Use CaCl ₂Conversion method transformed clone carrier bacillus coli DH 5 alpha.Choose anti-Amp ⁺Positive colony, the cloning vector that extracts reorganization also carries out enzyme and cuts evaluation.And gained contained insert segmental recombinant plasmid order-checking, conclusive evidence target gene fragment in the PCR process does not morph or goes wrong.The used method that checks order is two deoxidation cessation method.Confirm that the gene fragment length of being inserted is 849bp, be human pancreas DNase I gene (comprising signal peptide sequence).

Embodiment 2: the clone of ox pancreas DNase

Gene order (SEQ ID N0.2 according to the ox pancreas DNase that has announced, the coding region is positioned at the 27-812 position of nucleotide sequence, SEQ ID NO.4 is an aminoacid sequence) (Worrall and Connolly, journal of biological chemistry (J.Biol.Chem.), 265:21889,1990) design ox DNase I primer:

5 ' forward primer:

5′-GCAGCCTTCAACATCCGCACCTTTGGGGAG-3′

3 ' reverse primer:

5′-GCTCGTACGCAGGCGGATGGATGACCACTG-3′

Get freezing ox pancreas, adopt RNeasy Midi system (Qiagen, the U.S.) from pancreas, to extract total RNA.Use Oligotex mRNA system (Qiagen, the U.S.) purified mRNA again.In order to obtain being used for the people's pancreas or the ox pancreas cDNA storehouse of RACE-PCR clone technology, also must add a specific connexon at 5 ' end of each cDNA, application Marathon cDNA amplification kit (Clontech, the U.S.) has obtained the cDNA storehouse that has specific connexon of people's pancreas or ox pancreas.Then with design synthetic 5 ' forward primer and 3 ' reverse primer and connexon primer (Marathon cDNA amplification kit, Clontech), adopt terminal method (RACE-PCR) amplification gene of rapid amplifying cDNA, the PCR reaction conditions is: 95 ℃ of sex change 1 minute, annealed 1 minute for 55 ℃, 72 ℃ prolong 3 minutes.Carry out 35 circulations.72 ℃ kept 7 minutes then.4 ℃ of preservations.Obtain DNase I3 ' RACE fragment and 5 ' RACE fragment respectively.After 1% agarose gel electrophoresis confirms segmental existence and size, connect with pGEM-T EasyVector (Promega, the U.S.) respectively.Use CaCl ₂Conversion method transformed clone carrier bacillus coli DH 5 alpha.Choose anti-Amp ⁺Positive colony, the cloning vector that extracts reorganization also carries out enzyme and cuts evaluation.And gained contained insert segmental recombinant plasmid order-checking, the existence of conclusive evidence target gene fragment in the PCR process and do not morph or go wrong.Extraction contains DNase I5 ' fragment and 3 ' segmental two reorganization T carriers respectively, with 3 ' fragment of Nco I and Eco47 III cutting DNase I, uses EcoR I and Eco47 III to cut 5 ' fragment of DNase I.Two fragments using QiaquickGel Extraction (Qiagen) purifying enzyme to cut then connect two fragments with dna ligase, obtain full length sequence DNase I gene.Use T then ₄Dna ligase connects from the total length DNase gene fragment of agarose gel electrophoresis recovery and purifying with the pGEM-T Easy Vector (Promega, the U.S.) that Nco I and EcoR I cut, and obtains recombinant cloning vector pT-DNase-b1.Use CaCl ₂Conversion method transformed clone carrier bacillus coli DH 5 alpha.Choose anti-Amp ⁺Positive colony, the cloning vector that extracts reorganization also carries out enzyme and cuts evaluation.And gained contained insert segmental recombinant plasmid order-checking, conclusive evidence target gene fragment in the PCR process does not morph or goes wrong.The used method that checks order is two deoxidation cessation method.Confirm that the gene fragment length of being inserted is 786bp, be ox pancreas DNase I gene.

Embodiment 3: the structure that contains the recombinant expression plasmid of people DNase I encoding gene

Design primer according to the gene order of people DNase I and the restriction endonuclease sequence Nde I (5 ' primer) and the Xho I (3 ' primer) of adding:

5 ' forward primer:

5′-CATATGACTGCAGGTGCCGTGTCC-3′

3 ' reverse primer:

5′-CTCGAGTCACTTCAGCATCACCTCCACTGG-3′

From pT-DNase-b1, use PCR method amplification DNase I gene, reclaim purifying DNase I gene fragment with 1% agarose gel electrophoresis, then DNase I gene fragment and pGEM-TEasy Vector (Promega, the U.S.) are connected, obtain recombinant plasmid pT-DNase-h2.Use CaCl ₂Conversion method transformed clone carrier bacillus coli DH 5 alpha.Extract pT-DNase-h2, cut, reclaim purifying DNase I gene fragment with 1% agarose gel electrophoresis again with restriction endonuclease Nde I and Xho I enzyme.With the T4 dna ligase people DNase I gene fragment and Nde I are connected with expression vector pBGI-2 after Xho cuts then.Described expression vector pBGI-2 is by utilizing SphI and HpaI (Promega, the U.S.) condition of recommending with manufacturer is with pET28 expression vector (Novagen, the U.S.) reconnect acquisition in behind the 598-1629 position nucleotide excision, destroyed lac I gene wherein thus.The pBGI-2 expression vector also contains f1 replication orgin, kalamycin resistance gene (Kan except that containing T7 rna polymerase promoter and T7 terminator ⁺) and the encoding sequence of His label.With DNase gene and carrier T ₄Dna ligase obtains recombinant expression vector pBGI-2-hDNase after connecting.HDNase I represents people DNase I gene.CaCl ₂Method transforms the host bacterium such as the e. coli jm109 of T7 rna polymerase gene defective.Through Kan ⁺Resistance screening obtains positive colony.Expression product is carried out sds polyacrylamide gel electrophoresis (SDS-PAGE) and activity identification.

Embodiment 4: the structure that contains the recombinant expression plasmid of ox DNase I encoding gene

Design primer according to the gene order of ox DNase I and the restriction endonuclease sequence Nde I (5 ' primer) and the Xho I (3 ' primer) of adding:

5 ' forward primer:

5′-CATATGAGGGGCACCAGGCTGATG-3′

3 ' reverse primer:

5′-CTCGAGTTATGTCAGCGTCACCTCCACCGG-3′

From pT-DNase-b1, use PCR method amplification DNase I gene, reclaim purifying DNase I gene fragment with 1% agarose gel electrophoresis, then DNase I gene fragment and pGEM-TEasy Vector (Promega, the U.S.) are connected, obtain recombinant plasmid pT-DNase-b2.Use CaCl ₂Conversion method transformed clone carrier bacillus coli DH 5 alpha.Extract pT-DNase-b2, cut, reclaim purifying DNase I gene fragment with 1% agarose gel electrophoresis again with restriction endonuclease Nde I and Xho I enzyme.With the T4 dna ligase ox DNase I gene fragment and Nde I are connected with expression vector pBGI-2 after Xho cuts then.Described expression vector pBGI-2 is by utilizing SphI and HpaI (Promega, the U.S.) condition of recommending with manufacturer is with pET28 expression vector (Novagen, the U.S.) reconnect acquisition in behind the 598-1629 position nucleotide excision, destroyed lac I gene wherein thus.The pBGI-2 expression vector also contains f1 replication orgin, kalamycin resistance gene (Kan except that containing T7 rna polymerase promoter and T7 terminator ⁺) and the encoding sequence of His label.With DNase gene and carrier T ₄Dna ligase obtains recombinant expression vector pBGI-2-bDNase after connecting.BDNase I represents ox DNase I gene.CaCl ₂Method transforms the host bacterium such as the e. coli jm109 of T7 rna polymerase gene defective.Through Kan ⁺Resistance screening obtains positive colony.Expression product is carried out sds polyacrylamide gel electrophoresis (SDS-PAGE) and activity identification.

Abduction delivering and the preliminary purification of embodiment 5:DNase I

Picking is the reorganization bacterium colony of DNase I high expression level through sds polyacrylamide gel electrophoresis (SDS-PAGE) and DNase I activity identification, inserts to contain in the sterilization LB substratum of 10-50 μ g/ml kantlex 37 ℃ of following 280 rev/mins of overnight incubation.Inoculum size with 1% inserts and contains in the sterilization LB substratum of kantlex 37 ℃ of cultivations.When the O.D.600 of reorganization bacteria culture fluid is 0.6, add the 1M MgSO of 1ml ₄, 1ml 20% maltose (during preparation need with 0.45 μ m filter membrane bacteriological filtration) and the ready λ CE6 of 2ml phage bacterium liquid induce, 37 ° of C continue to cultivate 5 hours, until there being bacteriolysis (O.D. value is decline obviously) to occur.

With 4 ℃ of the inoculums after inducing, 5, centrifugal 10 minutes collecting cells of 000g transfer pH standby to identical with the chromatography level pad or very close back with ionic strength supernatant liquor.Add the level pad of step chromatography down, the following step is 10ml sample-loading buffer (5mM imidazoles, 500mM NaCl, the 20mM Tris-HCl that metal chelating and chromatography are then used 4 ℃ of precoolings, pH=7.9) suspension cell, ice-bath ultrasonic fragmentation (JY92-II type ultrasonic cell disruptor, Ningbo Xin Zhike device institute), the condition of ultrasonication is: 400W, 8 seconds working hours, 10 seconds intermittent times, 80 circulations.4 ℃, 10, centrifugal 20 minutes of 000g collects supernatant liquor, and merges with the cell culture supernatant of transferring ionic strength and pH value, can prepare to use chromatography purification.Use the Kunitz method to measure the DNase I activity of amalgamation liquid as described in embodiment 9, the expression amount of people DNase I is 2.06 * 10 ⁵Every liter of fermented liquid of Kunitz Unit/, the expression amount of ox DNase I is 2.6 * 10 ⁵Every liter of fermented liquid of Kunitz Unit/.

The preparation method of λ CE6 phage bacterium liquid is as follows: with LE392 (a kind of hdsR514 (r that has _k ^-Mk ⁺) genotypic coli strain) be inoculated in and contain 0.2% maltose and 10mMMRSO ₄The LB substratum in, 37 ℃, 280 rev/mins until O.D.=1.0.Get 5ml bacterium liquid and add 2 * 10 ⁸λ CE6 phage (available from Novagen company) and mix.37 ℃ are incubated 15 minutes (can not shake) down.Above host/phage mixed solution is added 500ml contain 10mMMaSO ₄The LB substratum in, 37 ℃, 250 rev/mins take place until bacteriolysis.Add the 5ml chloroform and shook again 10 minutes.4 ℃, 10, centrifugal 10 minutes of 000g carefully shifts out supernatant liquor, adds DMSO to final concentration 7%.

Embodiment 6: metal chelate affinity chromatography is to the purifying of crude product DNase I

The DNase I that separation and purification has the His label can adopt the metal chelate affinity chromatography method.Chromatography media is the Chelating Sepharose of Sweden peace Pharmacia biotech company ^TMFastFlow, tomography devices is AKTA Purifier (Pharmacia Biotech AB, a Sweden).

With the chromatography column that the sterilized water pre-equilibration of 3 times of column volumes is loaded, use Charging Buffer (the 50mM NiSO of five times of column volumes again ₄) the balance chromatography column, use Binding Buffer (5mM imidazoles, 0.5MNaCl, 20mM Tris-HCl, pH7.9) the balance chromatography column of 3 times of column volumes then.Cleer and peaceful on the centrifugal after the cell ultrasonication among the embodiment 2 to transfer the cell culture supernatant of ionic strength and pH value be last all product, flow velocity 30cm/ hour.Behind the last sample, with the Binding Buffer drip washing of 10 times of column volumes, (20mM imidazoles, 0.5MNaCl, 20mM Tris-HCl pH7.9) remove in conjunction with weak foreign protein to use the WashingBuffer of 6 times of column volumes then.(0.5M imidazoles, 0.5MNaCl, 20mM Tris-HCl pH7.9), collect elution peak, promptly get expressing protein DNase I with the Elute Buffer of 6 times of column volumes.(10mM EDTA, 0.5MNaCl, 20mM Tris-HCl pH7.9) slough Ni to use 6 times of column volume Strip Buffer at last ²⁺, with 4 ℃ of preservations after the 20% ethanol balance.Obvious attenuating of flow velocity or chromatography media are through filling Ni when chromatography ²⁺After when but no longer showing blue-greenish colour, pillar need be regenerated.Through the purity of the DNase of metal chelate affinity chromatography purifying I greater than 85%.The rate of recovery is greater than 87%.

Embodiment 7: gel permeation chromatography is refining to DNase I's

Gel permeation chromatography post Supredex 75 HR 10/30 (Sweden peace Pharmacia biotech company) are used enzymes soln (150mM NaCl and 10mM Ca ²⁺, pH7.0) fully carry out balance, will be through the people of metal chelate chromatography purifying or ox DNase I by last sample less than chromatography column column volume 5%, flow velocity is 30cm/ hour, continues to use the enzymes soln wash-out behind the last sample, collection DNase activity peak.This step, the chromatography rate of recovery was greater than 95%.Living through the ratio of the recombinant human DNase I of metal chelate affinity chromatography and gel permeation chromatography purifying is 3200u/mg, and the ratio work of the reorganization ox DNase I of purifying is 4000 u/mg, and purity is all greater than 95%.

The PEG of embodiment 8:DNase I modifies

The dissolving 5 the gram methoxy poly (ethylene glycol)s (mPEG) or at polyoxyethylene glycol (PEG) in 25 milliliters of anhydrous dioxanes.Stirring adding 6mmol is dissolved in N-succinimide chloro-formic ester or the N in the 10ml anhydrous propanone, N-N-two succinimdyl carbonates.Add 6mmol again and be dissolved in 4-dimethylaminopyridine in the anhydrous propanone.Continue stirring reaction 2 or 6 hours.Cross the filtering precipitation, collect supernatant.Add diethyl ethyl phosphonate, obtain the polyoxyethylene glycol (SC-PEG) of succinimdyl carbonateization.Purified reorganization ox DNase I or people DNase I are dissolved in 0.1M, the phosphate buffered saline buffer of pH7.5, concentration is 1-10mg/ml.Add SC-PEG in the stirring, reaction overnight promptly obtains the DNase I of PEGization.Select to have the PEG-DNase I of certain modification degree through gel permeation chromatography Superdex 75 HR.

The activity identification of embodiment 9:DNase I (Kunitz, M, 1950. general physiology magazine (J.Gen.Physiol.) 33:349-362).

With 2 times of reaction buffer (8mM CaCl of 500 μ l ₂, 8mM MgCl ₂, 20mM Tris-HCl, pH8.0), 10 μ l substrates (the calf thymus DNA sodium salt of 50 μ g/ml (SigmaD3664, the U.S.)) and 440 μ l distilled waters add in the cuvette mixing under the room temperature.Add 50 μ l testing samples and quick mixing, insert the absorbance value of surveying 260nm in the spectrophotometer rapidly.With A ₂₆₀To the time mapping, obtain activity curve.The unit of activity of DNase is defined as and utilizes reaction buffer and substrate as mentioned above, and per minute makes the A of reactant under light path 1cm and 260nm condition ₂₆₀The amount of the DNase of increase by 0.001 is defined as 1Kunitz unit.

Embodiment 10:DNase reduces the experiment of patient's phlegm viscosity

By the fluidity testing of sample, the people DNase I to reorganization has carried out vitro test with the effect that reorganization ox DNaseI treats the phlegm of patient's generation of suffering from pulmonary cyst fibrosis pathology respectively.100 microlitre sputum sample product in the Eppendorf testing tube respectively with 10 microlitre 0.01mg/ml, than living to the recombinant human DNase I of 3200u/ml and 10 microlitre 0.01mg/ml, having carried out incubation for the recombinant human DNase I of 4000u/ml than living.Under 37 ℃,, put upside down test tube, in the measurement numerical range of 5 minutes (flowing smoothly), phlegm is not assessed along the ability that the test tube wall flows down along tube wall 0 minute (not flowing) through the incubation in different time cycle.The phlegm that adds recombinant human DNase I produced the result that 3-4 divides at 10 minutes behind the incubation.The phlegm that adds reorganization ox DNase I produced the result that 4-5 divides at 10 minutes behind the incubation.The phlegm that does not add DNase I does not demonstrate mobile.Experimental results show that can degrade the rapidly phlegm of purulence of the pure reorganization DNase I that does not contain proteolytic enzyme.Therefore, pure DNA enzyme is being very effective aspect the viscosity that reduces phlegm.

Sequence table

SEQ?ID?NO.1

1 tcctgcacag?gcagtgcctt?gaagtgcttc?ttcagagacc?tttcttcata?gactactttt

61 ttttctttaa?gcagcaaaag?gagaaaattg?tcatcaaagg?atattccaga?ttcttgacag

121 cattctcgtc?atctctgagg?acatcaccat?catctcagga?tgaggggcat?gaagctgctg

181 ggggcgctgc?tggcactggc?ggccctactg?cagggggccg?tgtccctgaa?gatcgcagcc

241 ttcaacatcc?agacatttgg?ggagaccaag?atgtccaatg?ccaccctcgt?cagctacatt

301 gtgcagatcc?tgagccgcta?tgacatcgcc?ctggtccagg?aggtcagaga?cagccacctg

361 actgccgtgg?ggaagctgct?ggacaacctc?aatcaggatg?caccagacac?ctatcactac

421 gtggtcagtg?agccactggg?acggaacagc?tataaggagc?gctacctgtt?cgtgtacagg

481 cctgaccagg?tgtctgcggt?ggacagctac?tactacgatg?atggctgcga?gccctgcggg

541 aacgacacct?tcaaccgaga?gccagccatt?gtcaggttct?tctcccggtt?cacagaggtc

601 agggagtttg?ccattgttcc?cctgcatgcg?gccccggggg?acgcagtagc?cgagatcgac

661 gctctctatg?acgtctacct?ggatgtccaa?gagaaatggg?gcttggagga?cgtcatgttg

721 atgggcgact?tcaatgcggg?ctgcagctat?gtgagaccct?cccagtggtc?atccatccgc

781 ctgtggacaa?gccccacctt?ccagtggctg?atccccgaca?gcgctgacac?cacagctaca

841 cccacgcact?gtgcctatga?caggatcgtg?gttgcaggga?tgctgctccg?aggcgccgtt

901 gttcccgact?cggctcttcc?ctttaacttc?caggctgcct?atggcctgag?tgaccaactg

961 gcccaagcca?tcagtgacca?ctatccagtg?gaggtgatgc?tgaagtgagc?agcccctccc

1021?cacaccagtt?gaactgcag

SEQ?ID?NO.2

1 aattcgattc?ctaggaggtg?agctctatgc?ttaagatcgc?tgctttcaac?atacgtacct

61 tcggtgaatc?taaaatgtct?aacgctacgc?tagcatctta?catcgtacgc?atcgtacgcc

121 gttacgatat?cgttctgatc?caggaagttc?gcgactctca?cctggttgca?gttggtaaac

181 ttctagacta?cctgaaccag?gacgacccga?acacctacca?ctacgttgtt?tctgaacccc

241 tcgggcgtaa?ctcttacaaa?gaacggtacc?tgttcctgtt?ccgtccgaac?aaagtttcag

301 tactggatac?ctaccagtac?gacgacggat?gcgaatcttg?cggtaacgac?tctttctccc

361 gggaaccggc?tgttgttaaa?ttctcgagcc?actctaccaa?ggttaaagag?ttcgctatcg

421 ttgctctgca?cagcgcgccg?tctgacgctg?ttgctgaaat?caactctctg?tacgacgttt

481 acctggacgt?tcagcagaaa?tggcacctga?acgacgtcat?gctgatgggt?gacttcaacg

541 ctgactgctc?ttatgtaacc?tcttctcagt?ggtcatcgat?tcgtctgcgc?acctcgtcga

601 ccttccagtg?gctgatcccg?gactccgctg?acaccaccgc?tactagtacc?aactgcgctt

661 acgaccgtat?cgttgttgct?ggatccctgc?tgcagtcttc?tgttgtaccg?ggtagcgcgg

721 ccccgttcga?cttccaggct?gcatatggtc?tttcgaacga?aatggcgctg?gccatctctg

781 atcactaccc?ggttgaggta?accctgacct?aataga

SEQ?ID?NO.3

1?MRGMKLLGAL?LALAALLQGA?VSLKIAAFNI?QTFGETKMSN?ATLVSYIVQI?LSRYDIALVQ

61 EVRDSHLTAV?GKLLDNLNQD?APDTYHYWS EPLGRNSYKE?RYLFVYRPDQ?VSAVDSYYYD

121?DGCEPCGNDT?FNREPAIVRF?FSRFTEVREF?AIVPLHAAPG?DAVAEIDALY?DVYLDVQEKW

181?GLEDVMLMGD?FNAGCSYVRP?SQWSSIRLWT?SPTFQWLIPD?SADTTATPTH?CAYDRIVVAG

241?MLLRGAVVPD?SALPFNFQAA?YGLSDQLAQA?ISDHYPVEVM?LK

SEQ?ID?NO.4

1 MLKIAAFNIR?TFGESKMSNA?TLASYIVRIV?RRYDIVLIQE?VRDSHLVAVG?KLLDYLNQDD

61 PNTYHYVVSE?PLGRNSYKER?YLFLFRPNKV?SVLDTYQYDD?GCESCGNDSF?SREPAVVKFS

121?SHSTKVKEFA?IVALHSAPSD?AVAEINSLYD?VYLDVQQKWH?LNDVMLMGDF?NADCSYVTSS

181?QWSSIRLRTS?STFQWLIPDS?ADTTATSTNC?AYDRIVVAGS?LLQSSVVPGS?AAPFDFQAAY

241?GLSNEMALAI?SDHYPVEVTL?T

Claims (10)

1. one kind prepares the method for recombinant deoxyribonuclease I active polypeptide by rigorous type abduction delivering strategy, and it comprises that (1) transforms suitable prokaryotic host cell with the rigorous type inducible expression carrier of the reorganization of the nucleotide sequence that contains coding DNA enzyme I; (2) be suitable for cultivating under the selected prokaryotic cell prokaryocyte growth conditions through transformed host cells; (3) under the condition that is suitable for the deoxyribonuclease I expression, induce host cell to produce deoxyribonuclease I; (4) from gained through transforming prokaryotic host cell and/or nutrient solution reclaim deoxyribonuclease I.

2. the process of claim 1 wherein that the nucleotide sequence of described coding DNA enzyme I is shown in nucleotide sequence 27-812 position among nucleotide sequence 226-1008 position among the SEQ ID NO:1 or the SEQ ID NO:2.

3. the process of claim 1 wherein that described prokaryotic host cell is a T7 RNA polymerase defective escherichia coli.

4. the method for claim 3 is wherein from reclaiming deoxyribonuclease through the coli somatic of abduction delivering deoxyribonuclease I and/or nutrient solution.

5. the process of claim 1 wherein and also contain the T7 promotor in the described recombinant expression vector.

6. the process of claim 1 wherein that described the expression of deoxyribonuclease I need be by containing the T7 rna polymerase gene and having the phage induction of the promotor of the independent T7 of expression rna polymerase gene in described host cell.

7. the method for claim 6, wherein said phage is a λ CE6 phage.

8. zymin that contains the deoxyribonuclease I of method as claimed in claim 1 preparation.

9. the zymin of claim 8, wherein said deoxyribonuclease I is water miscible monomer, PEGization modifier or other derivative.

10. the deoxyribonuclease I of method preparation as claimed in claim 1 is used to prepare the purposes of the medicine for the treatment of respiratory system disease, the viscosity that reduces patient's phlegm, unobstructed respiratory tract.

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