CN1016796B - Process of producing useful substances - Google Patents

Abstract

The present invention discloses a method by which useful substances are generated by that the nucleopolyhedrosis viruses (BmNPV) of Chinese silkworms are proliferated and recombined in a cultural cell or a parasitifer body. The recombined BmNPVDNA generated by the recombination is double-stranded DNA which comprises a 5' upper stream BmNPVDNA segment originally existing in the upper stream of a polyhedrin structural gene, a promotor area of the structural gene, a translation initiation codon, and a gene for generating useful substances with or without a down stream BmNPVDNA segment originally existing in the down stream of the polyhedrin structural gene.

Description

Process of producing useful substances

The invention relates to the method for utilizing gene engineering to produce the material of available medicine, particularly method, especially relate to the method for utilizing viral DNA in external and body, to produce this class material about more effectively producing these materials than ordinary method.On the other hand, the present invention relates to utilize nucleopolyhedrosis virus, the method for in silkworm (fortunatus (Bombyx mori)) body, producing various useful matteies effectively.The present invention also relates to be used to implement the carrier and the recombinant virus DNA of aforesaid method, and produce the method for same material with it.

Once reported many superiority by recombinant DNA technology in the past, use carrier etc. are in intestinal bacteria, Bacillus subtilus.Produce the method for useful matter in the microbies such as cereuisiae fermentum.

There is one piece of report to address to attempt to utilize the viral DNA (Aut-ographa californica nucleopolyhedrosis virus DNA) after a kind of its structure gene is replaced, in cultured cells (a kind of Spodoptera frugiperda clone of foundation), produce beta-interferon and beta-galactosidase enzymes (Molecular and Cellular Biolgy, 3(12), 2156-2165(1983); Ibid., 4(3), 399-406(1984)).People such as people such as Iddekinge and Miller had once reported Autog-rapha Californica and had examined polyhedrosis polyhedrosis gene (Virology, 131,561-565(1983) and Genetic Engineering in Eukaryotes, 89-97(1982), Plenum Publishubg Corp, New York).But it is can be at harmful strain of external world's existence that problem is to be used for to finish the A.Calif-ornioa virus of this method.Therefore producing useful matter with said method is can not be gratifying.About the fortunatus polyhedrosis gene, once there was one piece of report to address progress (the Rohrmann et al of preservation fortunatus polyhedron N-terminal aminoacid sequence, J.Nol-ecular Evolution, 17,329-333(1981)), but it does not propose to prepare various materials with recombinant DNA technology.The inventor has carried out some and has attempted to provide a kind of research of better method, to produce useful matter, has now finished the present invention.

The invention provides a kind of DNA that utilizes fortunatus nucleopolyhedrosis virus (below be abbreviated as BmNPV), method with gene engineering production such as useful matteies such as protein or glycoprotein, particularly provide the gene recombination of a kind of functional advantage of utilizing promoter region among the said BmNPV DNA and useful matter and recombinant DNA that obtains and the recombinant vectors that is used for this reorganization, to produce the method for useful matter effectively.The present invention also provides a kind of method that influences transfection with BmNPV recombinant transfer vector combination of utilizing, the transfer vector of this reorganization contain one its be present in the upstream of polyhedron protein structure gene originally, and still comprise 5 ' upstream BmNPV dna fragmentation of the promoter region of said structure gene, a translation initiation codon and a gene of producing useful thing, have or do not have the 3 ' downstream BmNPV dna fragmentation that originally was present in said polyhedrin structural gene downstream.The present invention further provides a kind of method of producing useful matter, it comprises with the silkworm body of the cell of the mixture inoculation culture of recombinant transfer vector and BmNPV DNA or work so that form recombinant chou BmNPV, thereby at said cell or in vivo produce said reorganization BmNPV, be by preparing a DNA who is connected in kind perhaps by BmNPV DNA and escherichia coli plasmid such as PBR322 with the cell of reorganization BmNPV inoculation culture or silkworm-this reorganization BmNPV that lives, further replace polyhedron protein structure gene with other proper method again and make up with the gene of producing useful matter, and at said cell or in vivo breed said recombinant chou BmNPV.A special aspects of the present invention is, the method of the manufacturing useful matter that it provided is included in the fortunatus nucleopolyhedrosis virus (BmNPV) of cultured cells or host's (particularly clone or silkworm body alive that is obtained by fortunatus) internal breeding reorganization, this recombinant chou is to contain by reorganization originally to be present in the polyhedrin structural gene upstream and still to comprise said structure gene promoter region, translation initiation codon and the 5 ' upstream BmNPV dna fragmentation that produces the gene of useful matter produce, and it has or originally be not present in 3 ' downstream BmNPV dna fragmentation in said polyhedrin structural gene downstream.On the other hand, the invention provides a kind of carrier, it contains 5 ' upstream BmNPV dna fragmentation of the promotor that is present in said polyhedrin structural gene upstream at first and still comprises said structure gene, and 3 ' downstream BmNPV dna fragmentation that is present in said structure gene downstream at first, in addition, the 5 ' upstream dna fragmentation in this carrier of just having mentioned is to follow after the gene of translation initiation codon and generation useful matter.Another aspect, the invention provides a kind of method of producing useful matter, it comprises by cutting polyhedrin structural gene on the BmNPV DNA, together with having from 5 ' upstream portion said structure gene and promoter region that comprise said structure gene, and from the 3 ' downstream part in said structure gene downstream, the structure gene part of removing to replace resulting dna fragmentation with the gene that produces useful matter, substitution product is inserted in the carrier, the BmNPVDNA that so produces and BmNPVDNA united transfer among cell or the host, so that transfection and breed resulting reorganization BmNPV DNA; Reorganization BmNPV DNA through producing with the gene recombination that produces useful matter is a heterologous gene; This reorganization BmNPV DNA that has just mentioned, its reorganization is by at the polyhedron gene regional replacement, or other zone is inserted and realized at some; A kind of method with gene engineering production useful matter comprises utilizes BmNPV DNA to make carrier; And with a kind of method that gene engineering is produced useful matter comprise utilization be present in the upstream of polyhedrin structural gene originally and still comprise said structure gene promoter region 5 ' upstream BmNPV DNA part and be present in 3 ' downstream BmNPV DNA part of swimming under the said structure gene originally.In the appended Fig. 1 of this paper, the establishment figure of the reorganization BmNPV DNA that drawn.

Fig. 2 represents EcoR I-segmental restriction map of EcoR I of pBmE36.

Fig. 3 is the base sequence of Hpa I-Hind III fragment part.

Fig. 4 is near the base sequence of ATG of polyhedron gene.

Fig. 5 represents connexon building-up reactions outline.

Fig. 6 illustrates the method for preparing pBMO30.

Fig. 7 is the restriction map of pBMO30, illustrates the splitting position of the base sequence of multi-link subregion and restriction enzyme wherein.

Fig. 8 illustrates the preparation method that pIFN2BN is a plasmid.

Fig. 9 illustrates the preparation method of pBMO34.

Description by following Implementation Modes to the present invention will make the present invention more clear, obvious. BmNPV is a kind of insect viruses that belong to baculoviral. It has height host specificity and energy infected silkworm (Bombyx mori), and it can assemble polyhedrin in a large number in bombyx mori cell. This kind virus has about 140 kilobase of bifilar length to the cyclic DNA genome of (Kbp), and this DNA can pass through conventional method. As Protease Treatment, dodecyl sodium sulfate (S-DS) process and the method such as extraction by obtaining in the virion. This bifilar cyclic DNA (following real viral DNA or the BmNPV DNA of claiming) is a kind of sizable cyclic DNA. Therefore preferably choice for use is processed the littler dna fragmentation that contains polyhedrin structural gene that dna fragmentation produces and said gene front or the part (5 ' upstream and 3 ' downstream part) of back by restriction enzyme. Various restriction enzymes all are known and can have bought from the market, therefore be easily that above-mentioned purpose is selected one or more suitable enzymes and according to employed such as T.Maniatis, E.F.Fritsch, J.Sambroo-k, Molecular Cloning(is called for short later on Mol.Clon.); Cold Spring Harbor Lab, 1982, pp97148, one or more enzymes of narrating are selected its suitable service condition. Be included in synthetic (chemical synthesis, with one or more restriction enzyme enzymatic lysises), separation and evaluation, dna sequence analysis and the colibacillary processing (conversion, cultivation, plasmid recovery etc.) of dna fragmentation in the present invention's the technology contents, all be to use such as A.D.Riggs and K.Itakura, Am.J.Hum.Genet.31,531-538(1979) and Mol.; The described known genetic engineering technology of Clon is finished.

When screening contained the dna fragmentation of structure gene part in the many dna fragmentations that made by BmNPV DNA, one of them useful method was to use the probe of ordinary method preparation to carry out Southern hybridization (Mol.Coln; The 382-389 page or leaf).Because require selecteed fragment to contain the structure gene part, be very important so said fragment has one section quite long DNA chain in addition in 5 sides, certainly in some cases, such DNA chain also can be present in 3 ' side (downstream side).The length of every side chain is crucial for the desired useful matter of effective generation.As what will mention hereinafter, though operation also is an important factor easily, whether a given chain length enough can be from experimentally determining actually.The suitable length of DNA chain is measured by following method.With the dna fragmentation of different lengths, some contain the recombinant virus that produces the useful matter gene, and for example α-IFN presses the method structure of example 1.Then, infect culturing cell, relatively the productivity of useful matter with these recombinant virus.Although want expensive work, but the description below relying on, particularly by each embodiment, the personnel with ordinary skill level in this area can not run into too big difficulty in finishing these experiments.Because through obtaining a fragment that is applicable to about 10.6Kbp of the object of the invention with the cracking of EcoR I, so also can obtain various suitable fragments with some other restriction enzyme, for example, Cla I-Cla I, Pst I-Pst I etc., Cla I are that the Pst I is to be produced by Takara Shuzo company limited by U.S. New England biology laboratory product.

Can use effectively in the fragment that contains polyhedron protein structure gene of various technology by the front or rear face DNA chain of ining succession and remove the structure gene part, and take out for suitable length being arranged and contain the DNA chain of promoter region at said structure gene upstream side of use, also may be to have suitable length and DNA chain that be present in said structure gene downstream originally sometimes.

Therefore, must examine and determine dna sequence dna,, thereby can use restriction enzyme enzymatic lysis (as digesting) to remove said part with Bal31 or exonuclease with evaluation structure gene part by the resulting fragment of various restriction enzyme enzymatic lysises.Can prepare needed upstream or downstream DNA chain,, perhaps after the dna sequence dna of measuring separately, use the restriction enzyme enzymatic lysis, perhaps obtain it with chemical synthesis process as can after Bal31 digestion, keeping these parts.

Analyzed and reported the aminoacid sequence of silkworm polyhedrin protein already, and reported it according to (J.Invertebrate Patholoqy.30,442-443(1977)) such as S.B.Serebryani and include 244 amino acid.Whether thereby might check the polyhedron gene part that is obtained by BmNPV conforms to this aminoacid sequence, perhaps by measuring said segmental base sequence, on said base sequence basis, set up an aminoacid sequence according to codon-amino acid rule that conforms to, and, see that whether said fragment is from other parts rather than said gene with the sequence comparison that people such as this aminoacid sequence and Serebryani report.

The inventor proved conclusively already, in order to remove the structure gene part and to utilize the upstream and downstream dna fragmentation, with the available BmNPV dna fragmentation of EcoR I cracking (about 10.6Kbp) is the most suitable, obtains two fragments of being convenient to utilize with Hind III (Takara Shuzo company limited product) the said fragment of cracking.Yet only can not reach purpose of the present invention with a said fragment.By also obtaining some other suitable fragment with various restriction enzyme screenings.In the description of back, use above-mentionedly when being suitable for segmental example when meeting, will at any time enumerate out.Except that special instruction person, general dna sequence etc. all is indicated with ways customary.5 ' terminal in the left side, 3 ' terminal on the right side, the fragment that is obtained by restriction enzyme then indicates together with the title of restriction enzyme.In addition in this case, the sequence on the left side or the right also has the same meaning of as above pointing out.

Above-mentioned EcoR I-EcoR I fragment is summarized as follows.

Also can with methods such as Southern hybridization it be screened afterwards, determine the Gene Partial of polyhedrin by further above-mentioned fragment being cracked into than small segment.Therefore in these cases, can determine that the structure gene of being studied is to be present in Hpa I-Hind III fragment (about 1.8Kb).In for reference, in the end provided in one page the part of said Hpa I-Hind III fragment base sequence (by Hpa I cracking site upstream+201 bases are to Hind III cracking site (from-382 to 1185 1567bp)).In order to utilize the upstream and downstream part of structure gene, be to use two Hind III-Hind III (about 3.9Kb) and Hind III-Hind III (about 3.1Kb) fragment eaily by Hind III Processing of Preparation.The then available agarose electrophoresis commonly used of these two fragments is separated.

Isolating two fragments like this can be separated easily to insert and be contained in the plasmid of artificial connexon (Linkers).The plasmid that is applicable to this purpose is the production of Pharmacia P-L Biochemicals company if any commodity plasmid PUCP and PUC8().

To take from the Hind III-Hind III fragment (about 3.1Kb) in downstream side inserts PUC8 and it is utilized in its Hind III site.And contain the upstream side Hind III-Hind III fragment of polyhedron protein gene, and then insert PUC9 in its Hind III site, cut off in a site with restriction enzyme (as the EcoR I) and insert product, and the dna fragmentation that will so obtain digests through Bal31.Because Bal31 digests base one by one by cutting off the both sides, site, thereby can calibrate the length of DNA chain very expediently.By changing the treatment time, and produce the fragment of several different lengthss, and it is carried out the base sequence analysis, just may find out the fragment that has not now had polyhedron gene.

After removing polyhedron gene with aforesaid method, can obtain a dna fragmentation that has contained from the viral source promoter region of said upstream region of gene.This fragment is used to produce vector plasmid, can swim dna fragmentation by inserting to plasmid under the same polyhedrosis gene that also can in above-mentioned removal polyhedrosis gene, obtain of said fragment, and further insert plasmid by the site between gene to two fragment of inserting a generation useful matter (it is the site before polyhedrosis gene occurs), to produce recombinant plasmid.For example, will insert PUC9 with the upstream portion that obtains after the processing of Hind III and obtain a kind of plasmid; And PUC8 is inserted in the downstream part in Hind III site with Hind III-Hind III fragment (about 3.1Kb) form, then obtain another kind of plasmid.Utilize the feature of restriction endonuclease sites all on two kinds of plasmids afterwards, upstream and downstream partly can be inserted same plasmid, and further make up the another kind of plasmid that contains the upstream and downstream part.This situation, artificial connexon of design between this part is convenient to insert the gene that produces useful matter sometimes very much.

When finding around the translation initiation codon place of polyhedron gene or its, and when around translation stop codon place or its, disappearance being arranged, can by add synthetic DNA or with some other proper method fill.This repairing is to use general recombinant DNA technology commonly used to finish.

In order to produce useful matter, except communicant, expectation is still isolated and synthetic these and other gene in the future.What should readily understand is that all these genes can both be used for practice of the present invention.Said useful matter comprises peptide, protein and glycoprotein, as Interferon, rabbit (interferon-alpha, interferon-, IFN-), Regular Insulin, human growth hormone, somatomedin, interleukin and various lymphokines etc.Generally continue after programstep before, be suitable for translation initiation codon ATG is linked with these genes that produce useful matter.

The gene two ends that will produce useful matter afterwards connect connexon, make recombinant plasmid in inserting carrier or with certain other suitable conventional process.As a result, the recombinant plasmid that so obtains just contains the fragment from virus, promptly contains the dna fragmentation of promoter region and the downstream DNA fragment that is begun by translation initiation codon, and it is respectively from the upstream and downstream of the gene that produces useful matter.In above-mentioned programstep, the base sequence reversing when inserting, dna fragmentation may take place.Therefore before carrying out next procedure, must confirm that further its grafting is accurate, must confirm that promptly base sequence possesses correct directivity.

The recombinant plasmid itself that obtains with aforesaid method can be used for transformed into escherichia coli and propagation therein.

5 ' upstream fragment that might not require to contain promoter region is possessed the base sequence that originally comes across among the BmNPV DNA.Even when said fragment has more or less change, also be expected to obtain similar expression rate.

The typical TATA sequence frame (TATAbox) that is present in the promoter region generally can not identify with limited method, and that part of BmNPV DNA has been the upstream of ATG on polyhedral angle protein structure gene.But as Embodiment C-2 and continue after example and table 1 in as shown in, as some disappearance is arranged in the upstream portion of ATG, promptly disappearance-7 to-1bp ,-18 to-1 and 119 to-1bp, can't really have influence on available excellent results when not having these disappearances.When disappearance-29 to-1bp, also can produce satisfied result.But then, as disappearance-82 to-1bp, then can cause the trend of obvious reduction effect.This shows and as if near-80 bases, comprised an important promoter region.5 ' upstream DNA comprises that said promoter region and one are used for the upstream DNA of viral DNA reorganization, and its length can have bigger difference, and for example, the ATG front can have that to reach several kilobase right.More specifically be, will mention as this paper back, segmental that part of Hpa I-Hind III of the about 1.8Kb in ATG upstream is taken from use, or uses segmental that part of Hind III-Hind III or its part of taking from the about 3.9Kb in ATG upstream, can obtain satisfied result.5 ' upstream DNA contains by the upstream more parts of said about 3.9Kb fragment, is not an indispensable condition probably.As already mentioned, said promoter region is crucial for the fabulous albumen productive rate of obtaining BmNPV DNA.

Will address as the back, be positioned at the dna sequence dna of said promoter region upstream and 3 ' downstream DNA sequence in polyhedrin structural gene downstream, it prepares recombinant virus as key component for the gene (having translation initiation codon) that inserts a generation useful matter in BmNPV DNA is vital.Like this, that be used for shifting with BmNPV DNA and one, as to contain the dna fragmentation that produces the useful matter gene and said gene upstream and downstream, its dna sequence dna carries out polyinfection with coming from the recombinant DNA (as plasmid) that BmNPV DNA respectively pays this for oneself, because said key component causes producing exchange and the transfer of the gene and the BmNPV DNA of useful matter for the homology that produces the BmNPV DNA that recombinates.

With reference to above-mentioned method above and hereinafter and among the embodiment carry out the upstream of polyhedrin structural gene and each dna sequence dna of downstream part mensuration, repair and become and utilize, for those of skill in the art in this area, be not have what difficulty.Those skilled in the art can get a thorough understanding of and put into practice relevant technology, if but without any the invention step of technical standpoint, just may be difficult to accomplish.What it should be noted that is that any specific pattern that can put into practice with its method all falls in the present invention's the category.

Use above-mentioned recombinant plasmid, the reorganization BNPV DNA of the gene that obtains containing useful matter of can ining all sorts of ways, for example-and be to carry out external, then be in the silkworm body, to carry out in addition.In the practice model in a dark step, it is not to use such recombinant plasmid, and be to use the recombinant chou that between BmNPV DNA and PBR322 or other plasmid, produces, and polyhedrosis gene is replaced by a kind of gene of useful matter in said recombinant chou, to obtain a recombinant virus dna.

In external use BmNPV DNA and the recombinant plasmid polyinfection cultured cells that has the gene that produces useful matter.For example the clone of being set up by the fortunatus cell (Bm cell) can cause the exchange and the transfer of required gene (producing the gene of useful matter) and viral DNA, and obtains recombinant virus dna (reorganization BNPV DNA).

In the embodiment that has, this reorganization is to realize in the mode with needed gene substitution polyhedrin structural gene zone, and in other embodiments, then be to insert one or more modes that are used to produce the gene of useful matter in certain or some zones in addition to viral DNA to realize.Cause the propagation of recombinant virus dna in this polyinfection of external enforcement, and cause desired useful matter to gather then.May contain not reorganization and virus reorganization in the substratum, but available general method is separated recombinant virus, for example use H.A.Wood, J.Invertebr, Pathol.29,304-307(1977) and S.M-aeda, J.Seric.Sci.Jpn.53,547-548(1984) described dilution method or plaque method.

Above-mentioned polyinfection also can be finished in the silkworm body.

By to be grown in virus in the Bm cell (mixture of reorganization or non-recombinant virus or by wherein isolated recombinant chou) in Infection in Vitro Bm cell, or viral percutaneous injection in the silkworm body or in its body cavity, can be produced needed useful matter effectively.In addition, also can be from the mouthfeel of silkworm the poison of catching an illness.Available afterwards a kind of suitable currently known methods for example, separation and these useful matteies of purifying such as affinity chromatography, ion exchange chromatography, molecular sieve.

Be dependent on the present invention, thus might safety, economical and produce useful peptide, protein and glycoprotein in large quantities.

Be dependent on the present invention, a kind of peptide or protein produce in eukaryotic cell.Therefore when using the gene that obtains by eukaryotic cell, peptide that is produced or protein can be subjected to being repaiied change as what it stood in the eukaryotic cell body, connect as signal peptide deletion and sugar chain and to add, to expect that these products are than with the product that uses the bacterium acquisition in the common gene engineering bigger effectiveness being arranged.In addition, because these products are to be secreted into extracellularly, so also be easy to utilize for example affinity chromatography, ion exchange chromatography, molecular sieve etc. are purifying in addition.

Moreover the mankind have the sericulture experience in many years and have accumulated a large amount of achievements in research, thereby are easy to realize a large amount of breed.Nowadays also may lean on artificial breeding breed silkworms (example: " Vita-Silk ", Japanese Nagoya Vita-Silk company limited product).So, use virus vector on the live organism level, to produce useful matter and probably more be suitable for the suitability for industrialized production purpose, and more more economical than on cell levels, producing these materials.

Down routine embodiment be address produce a kind of protein-interferon-alpha as medicine (α's-IFN), used α-IFN gene as the gene that produces useful matter in the example, be intended to further elaborate Implementation Modes of the present invention.But it should be noted that these embodiment never constitute the restriction to the present invention's scope.

Embodiment 1

A. with plaque technology clone and propagation BmNPV

Be in the 3rd silkworm (fortunatus Bombyx mori) that makes the phase with the BmNPV peroral infection, collect body fluid after several days, with TC-10 substratum (the J.Invertebrate Patho Iogy that contains 1% tire intestines serum, 25,363-370(1975)) dilution and be used to infect the Bm cell of cultivating with form of single sheet (a kind of generally acknowledged fortunatus clone be by Dr.L.E.Volkman, the University of California, Barkeley, ATCCNo.CRL-8851 provides, Appl.Environ, Microbiol.44，227-233（1982）〕。,, cultivated several days after the culture medium solidifying at the TC-10 substratum upper berth layer that contains 0.75% agarose and 5% tire intestines serum with the cell that infects in 27 ℃.With the plaque that forms on the pasteur pipet separating plate.This plaque technical program is repeated twice, obtain the hereditary virus that goes up homogeneous and peel off.With the typical strain BmNPV T3 of these viral coniviums be used to continue after experiment.

Propagation strain T3 in the Bm cell is used for through the bark graft kind afterwards to infect the 5th silkworm that makes the phase.Formed after 6 days and contained polyhedrosis infected tissue liquid, to thin up wherein it, centrifugal, be suspended in throw out in the distilled water and grind it.Classified centrifugal (3,000rpm, 30 minutes) and discontinuous density gradient centrifugal (45% and 55%(W/W) sucrose solution, 20,000rpm, 30 minutes) the purifying polyhedron.Fractional centrifugation polyhedron suspension (3,000rpm, 10 minutes) removing sucrose solution, is suspended in the polyhedron of purifying in 0.1M yellow soda ash (pH11)-0.05M sodium-chlor, and handles 30 minutes in 25 ℃, causes virion to discharge.This viral suspension is through 10-40%(W/W) sucrose density gradient centrifugation (18,000rpm, 30 minutes) is with purifying.Remove sucrose through dialysis or fractional centrifugation (4,0000rpm, 30 minutes), obtain the virion of purifying.

B, preparation viral DNA are identified polyhedrosis gene, and cloning

The extraction of B-1 viral DNA

Add the SDS(sodium laurylsulfonate to containing in the viral solution of so obtaining, 1W/W%) and Proteinase K (Merck, 1mg/ml).37 ℃ of incubations added above-mentioned solution with equal-volume phenol solution (saturated solution that contains 10mM Tris-HCl damping fluid (pH7.6)-1mM EDTA) after 2 hours, this mixture is shaken about 5 minutes gently after, with 12, centrifugal 5 minutes of 000rpm.Collect water layer, and repeat phenol and handle twice.In this contains the water layer of DNA, add the equal-volume chloroform, shake about 5 minutes afterwards gently, and with 12, centrifugal 2 minutes of 000rpm.Collect water layer, repeat chloroform and handle twice, and water layer was dialysed about 2 days to 10mM Tris-HCl damping fluid (pH7.6)-1mM EDTA.The viral DNA that so obtains (ATCCNo.40188) be used to continue after gene cloning and Bm cell transfecting.

The cloning of B-2 polyhedrosis gene

Probe preparation: the 5th makes phase silkworm percutaneous injection strain Bm NPVT3, after several days with guanidine hydrochloride method by the total RNA of fatty body tissue extraction and use Oligo(dT) the cellulose column purifying, obtain containing Poly(A) mRNA.In external use rabbit reticulocyte (Wei-ssbach, Hand Ochoa, S; Ann.Rev.Biochem.45,191 page 1976) translation system checks this mRNA.Find the polyhedrosis mRNA of coding account for total mRNA 90% or more than.Use Mol.Clon, the said mRNA of PP211-246 makes template, with Oligo(dT) make primer, with reverse transcription enzymic synthesis cDNA.Be to be substrate with the dCTP that contains 32P at this moment, so target cDNA is as the probe of screening polyhedrosis gene.

Contain the segmental cloning of polyhedrosis gene Eco R I: with sublimed DNA among the Eco R I digestion Bm-NPVT3.In power on arteries and veins separating digesting fragment and transferring on the nitrocellulose filter paper of 0.7% sepharose, afterwards with above-mentioned probe hybridization.The dna fragmentation of about 10.6kb of specific hybrid is inserted the EcoR I cracking site of pBR322.So far with EcoR I digestion viral DNA, make same phenol processing and chloroform by method described in the top B-1 more afterwards and handle.Separate water layer,, sedimentary DNA be dissolved in Tris damping fluid (10mM Tris-HCl(pH7.6)-1mM EDTA in a small amount to the cold ethanol of 4M sodium-chlor that wherein adds 1/20 volume and 2 volumes) in.With EcoR I digestion pBR322, handle with above-mentioned same method afterwards in addition, further use BAP (bacterial alkaline phosphatase (Bethesda Research Laboratories)) to handle, remake that phenol is handled and the chloroform processing, and add ethanol sedimentation it.Precipitation is dissolved in a small amount of T-ris damping fluid.

With the EcoR I digestion product that the T4 ligase enzyme is obtained by viral DNA and PBR322 in 5 ℃ of processing, handle 10 hours to make it connection.To connect product and introduce e. coli k12 JM83, make probe with the CDNA of above-mentioned mark, by the tetracyclin resistance transformed bacteria of colony hybridization method screening formation.Thereby promptly obtain carrying a coli strain that contains the plasmid of the 10.6Kb EcoR I fragment (wherein containing polyhedron gene) of having an appointment.Cultivate this bacterial strain and with this plasmid DNA of cesium chloride method purifying, name and be pBmE36.The bacterial strain that contains plasmid pBmE36 is called e. coli k12 JM83 DGB-0036 and deposits in fermentation research association, industrial technology agency, Japanese FERM BP-813.Fig. 2 has supplied with the cleavage map of EcoR I-EcoR I insertion portion of pBm E36.Further make the DNA part that probe inserts with the calibrating of Southern hybrid method with above-mentioned cDNA.Said cDNA is only hybridized with Hpa I-Hind III fragment (about 1.8kp).

C, to remove polyhedron structure gene part-p9B be the plasmid structure

C-1 contains the cloning of polyhedrosis gene and downstream part thereof

Hind III-Hind III the fragment (about 3.1kb) that will comprise the segmental Hind III of above-mentioned Hpa I-Hind III-Hind III fragment (about 3.9kb) and comprise the polyhedrosis gene downstream part is inserted among commercialization cloning vector pUC9 and the pUC8 in Hind III site respectively, obtains p9H18 and p8H225.

C-2 removes the polyhedrosis gene part

With EcoR I cracking plasmid p9H18, handle with Bal31 afterwards, the either side of cracking site is cut away.According to the time length difference that Bal31 handles, the segmental length difference that is produced.These fragments are handled with the Hind III and, extracted afterwards, obtain dna fragmentation from the different lengths of virus through the separation of 0.7% sepharose electricity arteries and veins.

Handle pUC9 with Sma I (Takara Shuzo company limited product) and Hind III, be connected with the preceding dna fragmentation that has obtained (a passivity end and a Hind III end are arranged) afterwards, with the plasmid transformation escherichia coli K12 JM83 that is produced and cultivate it, reclaim plasmid, with-primer (primer that is used for 15 base sequences of M13) according to dideoxy method (dideoxy method) (F.Sanger, 214,1205-1210,1981) each viral source dna fragmentation 3 ' side base sequence of mensuration insertion, thereby identifying virus polyhedrosis gene part.In the base sequence of this viral source dna fragmentation, found the base sequence described in people's papers such as Serebryani corresponding to the aminoacid sequence of polyhedrin, and translation initiation codon ATG also is identical, 29 base pairs of the upstream of ATG disappearance in the different plasmids of the different lengths fragment gained that foundation Bal31 handles, and the plasmid of disappearance polyhedrin structural gene (comprising ATG) is named and is p9B241.

Similarly, each lacks 82 base pairs in ATG upstream, 19 base pairs, 18 base pairs and 7 base pairs, and the plasmid of disappearance ATG and subsequently base pair is named respectively and is p9B587, p9B310, p9B276 and p9B585.The base sequence that related this is regional such as Fig. 4.

The structure of the plasmid of D, disappearance polyhedron structure gene part

Handle plasmid p9B241 with EcoR I and Aat II, handle the p8H225(that makes up by the described method of C-1 with EcoR I, Aat II and Sca I again and the fragment that nonessential pUC8 obtains is converted into small segment) with the Sca I.Two reaction mixtures are handled with phenol earlier, handle with chloroform again, use ethanol sedimentation afterwards.Connect two parts of sediment D NA, with resulting plasmid transformation escherichia coli K12 JM83.The plasmid that the upstream of screening viral source and downstream DNA fragment are inserted with correct direction.With various restriction enzyme enzymatic lysises, show that this recombinant plasmid has an amicillin resistance sign, and the upstream portion that comprises a viral DNA, this part rise from polyhedrosis gene initiator codon ATG upstream the 30th base pair, and are upstream extended by said the 30th base pair.The said part of about 3kb(is a untranslated zone that comprises promotor); This recombinant plasmid also comprises one by the terminator codon of said gene the downstream part of about 3.1Kkb (but about 300 base pairs in disappearance terminator codon downstream) downstream, and above-mentioned upstream and downstream respectively are same direction with protovirus.This plasmid can be bred in the intestinal bacteria system, and names and be p89b241.Similarly, make up p89B587, p89B276 and p89B585 with p9B587, p9B310, p9B276 and p9B585 respectively.

The structure of the insertion-pIFN-1-B241 of E α-IFN gene

I) by finding out white corpuscle IFN gene (Lawn et al.cell, 15,1157-1174(1978)) in the human genome lambda particles phage Charon 4A recombinant chou library.Handle the DNA that contains α-IFN-J gene that so obtains with Hind III and EcoR I, separate the fragment that contains α-IFN-J gene with sepharose electricity pulpating method, and by being connected with the pUC9 that handles with Hind III and EcoR I.With resulting plasmid transformation escherichia coli K12 JM83.Contain the dna fragmentation of α-IFN-J gene and a Sma I connexon (Takara Shuzo) is connected to said segmental two ends by reclaiming this plasmid in the transformed bacteria and handle it, separating with Mst II (U.S., New England's biology laboratory are produced) and Pvu II.Handle this connection product with the Sma I, and be connected, to obtain a recombinant plasmid with the Sma I fragment of the plasmid p89B241 that in the D step, obtains.After further confirming that α-IFN really inserts this plasmid with correct direction,, thereby produce a large amount of plasmids promptly with this plasmid transformation escherichia coli k12 JM83 and cultivate it.This plasmid named be PIFN-1-B241.In this plasmid, follow the polyhedrosis gene upstream portion (from approximately-the 3kb base pair is to-30 base pairs) afterwards be a 13bp residue that is making up used connexon the plasmid p89B241, being a untranslated district of 32bp5 ' that derives from chromosomal α-IFN-J gene again, then is the α-IFN-J gene that begins and have correct direction of insertion with ATG after this zone.The downstream part that also has a polyhedrosis gene that links to each other with said α-IFN-J gene in addition (by the base pair of the about 300bp in terminator codon downstream to the about 3.1kbp base pair in said base pair downstream).

II) the above-mentioned plasmid of structure of the insertion of α-IFN gene-pIFN-2B plasmid, the product transformed into escherichia coli k12 JM83 that the Hind III of the promptly above-mentioned α of containing-IFN-J gene-EcoR I fragment is connected with PUC9.By reclaim this plasmid in the transformed bacteria and handle, separate with HPa II (Takara Shuzo company limited products) dna fragmentation that contains α-IFN-J gene and with use 32p-ATP and T ₄The oligomer (CGGGCCATC) of the chemosynthesis of polynucleotide kinase (Takara Shuzo) phosphorylation is connected with another synthetic oligomer (CCGGGATGGCC) annealed product.Remove to separate this connection product with sepharose electricity pulpating method and extract it afterwards, use 32p-ATP and T again ₄Polynucleotide kinase makes it phosphorylation and is connected with Xma I (the Britain New England biotron is produced) fragment of the plasmid p89B241 that obtains among the step D, to obtain a recombinant plasmid.After confirming that α-IFN-J gene inserts this plasmid with correct direction really,, thereby produce this plasmid in a large number promptly with said plasmid transformation escherichia coli K12 JM83.This plasmid is named is pIFN-2-B-241.

In this plasmid, the upstream portion (from about 3kb upstream base pair to-30 base pairs) that is connected on polyhedrosis gene afterwards be the 13bp residue that makes up the used connexon of plasmid p89B241, be to begin and, next be the downstream part (about 300bp base pair is to certain base pair of the about 3.1kb in base pair downstream from the terminator codon downstream) of a polyhedrosis gene again with α-IFN-J gene that correct direction is inserted with ATG thereafter.

According to making parent material with p89B587, p89B310, p89B276 and p89B585 respectively, make up pIFN-2-B587, pIFN-2-B310, pIFN-2-B276 and pIFN-2-B585 with quadrat method.

III) connexon is synthetic

The chemosynthesis of oligodeoxyribonucleotide:

(Nucleic Acids Research, method 101755(1982) use polystyrene resin with the synthetic oligomer of solid phase method according to people such as H.Ito report.Warp is at TERTIARY BUTYL AMINE-pyridine (1: 9V/V) handle in the solution; that protected difunctional dimer (I) (about 100mg) is converted into is corresponding 3 '-phosphodiester (II); sym-trimethylbenzene sulphonyl-nitrotrimethylolmethane hygron (the MSNT that uses; 100mg) be condensing agent, make 3 ' phosphodiester (II) and 5 ' free nucleosides resin (III) condensation.(1: 9V/V) solution makes 5 ' acylated hydroxy of unreacted resin (III) with acetic anhydride-pyridine.Continue after in 2% Tricholroacetic Acid (TCA)-methylene dichloride, handle, obtain a kind of detritylation product (IV).Repeat above-mentioned reaction train, until the sequence of synthetic special requirement.In the ion exchange resin that combines the oligomer that so obtains (about 20mg), add 0.5ml 0.5M tetramethyl guanidine-pyridine-2-ethylidenehydroxylamine (being dissolved in pyridine-water (15: 4 volumes))., with strong aqua this reaction mixture was handled 8 hours after 8 hours in 37 ℃ of reactions in 55 ℃.(2.5 * 60Cm) filter this solution, obtain a kind of DMTr-oligomer with the meticulous post of sephadex G-50.Use high pressure liquid chromatography (HPLC) method (HPLC) to be further purified this oligomer afterwards again, use a reversed-phase column (SSCODS-272 in the chromatography, 0.6 * 20Cm) and solution A (0.2M triethyl ammonium acetate (TEAA), pH7.0) and solution B (be dissolved in 50% acetonitrile of 0.02M TEAA, pH7.0), to produce a linear concentration gradient.The DMTr-oligomer of purifying like this was handled 20 minutes with 80% acetic acid under room temperature, so that the trityl removal effect is more complete, afterwards by the HPLC once more of method as mentioned above.Elutriant is to 10mM Tris-Hcl(pH7.5)-1mM EDTA(pH8.0) dialysis, obtain the oligomer that needs.The complete de-protected oligodeoxyribonucleotide that so obtains is made reversed phase column chromatography a peak only occurs, using (r in addition ^-32P)-ATP and T ₄Behind polynucleotide kinase mark 5 ' end,, a band only occurs, thereby confirm the homogeneity of this synthetic product with 15% polyacrylamide gel-7M urea system electricity arteries and veins.

IV) insertion of α-IFN gene-pBM034 structure

As shown in Figure 6, the pBM030 plasmid is made by the p9B312 plasmid.Usually, except three alkali to (3bp), all can occur promoter region among the BmNPV of this plasmid and stop the zone, simultaneously, between these zones, have a multi-link son to replace as shown in Figure 7 polyhedrosis gene.Among Fig. 6, the method that is prepared p9M312 by p9B312 is called external mutagenesis (mutagenesis of oligonucleotide); Nucleic acids research 9(15) 3647-3656(1981), can be used for some other effect equally.

By method shown in Figure 9,, make pBM034 with α-IFN gene insertion vector (pBM030).

The formation of the transfection of F, Bm cell-recombinant chou BmNPV.BmNPVT strain viral DNA (ATCCNo.40188) and pIFN-1-B241, its ratio is 1: 100, mixes with the solution I and the II that have following component respectively:

I. distilled water 2.1ml

Carrier DNA (salmon spermary, 1mg/ml) 50 μ l

BmNPV DNA 10μl

pIFN-1-B241 DNA 50μg

2M calcium chloride 300 μ g

II. contain the 50mMHEPES damping fluid (pH7.1) of 0.28M sodium-chlor

2.5ml

Phosphate buffered saline buffer (35mM Na ₂HPO ₄-35mMNaH ₂PO ₄)

50μl

The 1ml suspension is added to 4ml cultivates in the substratum of Bm cell, so that above-mentioned DNA is introduced in the fortunatus cell.Change fresh culture after 20 hours.Continue to cultivate 5 days, reclaim substratum and centrifugal afterwards.Get that clear liquid carries out α-IFN activation analysis on the clarification.With 1, centrifugal 5 minutes of 000rpm reclaims the supernatant liquor that is obtained by substratum, dilutes and does the plaque analysis.In the test under microscope plaque, collect no polyhedron plaque after four days.This program is repeated 3 times, obtain single strain.With the Bm of this virus strain infection cell, and after cultivating 5 days, reclaim substratum.

G. α-IFN is in the intravital expression of fortunatus

The 5th makes and cultivates the substratum that reclaims after 5 days above the silkworm percutaneous injection of first day phase described in the F; Perhaps give the single virus strain of injection with 0.5ml/ only (10 ⁷Pfu) dosage infects the back and cultivates the substratum that reclaimed in 5 days, raises 5 days with mulberry leaf in 25 ℃ afterwards.Collection is needled into the abdominal cavity annex and with its humor collecting in the ice-cooled EPPendorffShi pipe.In body fluid, add isopyknic TC-10 substratum, centrifugal (10,000rpm, 5 minutes) and the α of clear liquid-IFN activity analytically.

H. the bioassay of α-IFN

(ⅰ) determination of activity

Method (Method in Enzymoloqy according to people such as C.Philip report, 78,387-394(1981)) use derives from human amniotic membrane FL cell and vesicular stomatitis virus (VSV), suppress (50%CPE inhibition) analytical method with the cell pathology effect and carry out α-IFN determination of activity on 96 hole titer plate, the while compares with α-IFN standard model that U.S. sanitary research association is provided.Table 1 and table 2 display analysis result (respectively with different single virus stains).

The mensuration of the N-end of (II) α-IFN

After a kind of antibody column purifying, can record the N end of the α-IFN that uses pB034 gained silkworm.11 terminal amino acid conform to the amino acid of the α of standard.

This means that bombyx mori cell is correctly debated the signal sequence of others' body protein and with program accurately with in the signal transfered cell.

J. contain β-IFN gene recombinant chou BmNPV generation with and external and intravital expression.

Filter out β-IFN gene in the phage Charon 4A recombinant chou routine library by human genome.The DNA that contains β-IFN gene of gained, with the cracking of Hind II, and the fragment that contains β-IFN gene is separated by sepharose electricity arteries and veins.This fragment is linked the position of the Hind II of pUC9.The plasmid of gained is handled with Hind II and Bg/ II.The fragment that contains β-IFN gene adopts the method for above-mentioned E to separate and be connected with the pBM010 that handled with EcoRV.This plasmid is named and is pBM211.With the method same, the virus of recombinant chou is separated with above-mentioned F.

Utilize the recombinant chou BmNPV of aforesaid method gained, can handle expression formula in the silkworm by the method identical with above-mentioned G.

When with the method for H, with standard β-when the IFN sample was measured the activity of β-IFN, discovery recombinant chou BmNPV produced about 8 * 10 respectively in hemolymph and Bm cell ⁵U/ml and 8 * 10 ⁴β-IFN of U/ml.

By the IFN that recombinant virus produces, be neutralized into the β-IFN of human body with a kind of unicolor antibody.

The present invention at length is illustrated, and simultaneously according to wherein particular embodiment, the technician of this area can understand wherein various variations surely and can be improved and do not violate spirit of the present invention and standard.

Claims (4)

1, a kind of this method of method of producing exogenous genes products comprises:

From the structure gene of NPV DNA excision (a) coding polyhedrin together with 5 of said structure gene upstream '-upstream portion and comprise the promoter region of said structure gene and (b) said structure gene downstream 3 '-downstream part,

With the structure gene part of an encoding exogenous in the resulting dna fragmentation of gene substitution of said virogene product,

In the metathetical gene insertion vector,

The recombinant DNA carrier that so produces is introduced among the host being used for transfection with NPV,

At the resulting recombinant chou NPV of host internal breeding,

It is characterized in that said polyhedron disease virus be by fortunatus (Bombyx mori) produce and said recombinant chou NPV is to breed in a kind of established cell line of fortunatus or in the silkworm in a kind of work.

2, according to the method for the production exogenous genes products of claim 1, wherein said recombinant chou NPV is to breed in a kind of silkworm body of work.

3, a kind of method of producing the recombinant chou NPV, this method comprises:

From the structure gene of NPV DNA excision (a) coding polyhedrin together with 5 of said structure gene upstream '-upstream portion and comprise the promoter region of said structure gene and (b) said structure gene the downstream 3 '-downstream part

With the structure gene part of an encoding exogenous in the resulting dna fragmentation of gene substitution of the gene product of said virus,

In the metathetical gene insertion vector,

The recombinant DNA carrier that so produces is introduced among the host being used for transfection with NPV,

It is characterized in that described NPV is produced by fortunatus.

4, a kind of method of producing the recombinant DNA carrier, this method comprises:

From the polyhedrosis protein structure gene of NPV DNA excision (a) coding together with 5 of said structure gene upstream '-upstream portion and comprise the promoter region of said structure gene and (b) the 3-downstream part in the downstream of said structure gene

With the structure gene part of an encoding exogenous in the resulting dna fragmentation of gene substitution of the gene product of said virus,

In the metathetical gene insertion vector,

It is characterized in that said NPV is produced by fortunatus.

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