JPS63502239A - Lytic virulent phage, host cell containing the phage, and protein production method - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] ``Virulent phage: Main body containing T phage and protein production 1 The present invention relates to lytic viruses such as bacteriophages. host cells infected with viruses such as and heterospecific proteins in the host cells. It also relates to the use of said virus in the production of.

One of the objectives of the present invention is to enable the integration of shrimpsomes into the chromosomes of lytic viruses. and maintain the recombinant nature of the virus throughout its infectious life cycle. The objective is to provide novel biological methods. Here, the present invention is applied to known bacteria. Although the explanation will be made using the phage E.coli-phage T5, the present invention It is not limited to this.

The main purpose of the present invention is to add an alien product to the chromosome. ion gene) inserted therein. This recombination The prey virus is stable throughout its infectious life cycle. The production gene is preferably is the non-essential region of the chromosome of the virus. on).

The virus of the present invention is a bacteriophage such as phage T5. is preferred, the phage is preferably T5+ or an autonomous phage derived therefrom. Select from possible stable deletion mutants, such as T5sto. The production gene The offspring are those introduced into the chromosome of the virus by so-called homologous reciprocal recombination. preferable.

The invention further relates to host cells infected with lytic recombinant viruses such as those described above. Ru. The host cell is preferably a polygon.

Another object of the invention is to produce heterospecific proteins that can be recovered by suitable methods. The goal is to provide a method for

According to this object of the invention, the method for producing a protein by expression in a host cell is It consists of the following steps.

a) introducing a heterogeneous gene into the chromosome of a lytic virus; b) the resulting recombinant introducing a virus into a host cell and infecting the host cell; C) expressing the recombinant protein in the host cell; and d) Recovering the protein thus produced.

Preferably, the production gene of step a) is introduced by homologous reciprocal recombination, This gene is preferably introduced into a non-essential region of the phage chromosome and be under control of the motor.

In this protein production method, the host cells to be infected with recombinant phage are E. Li/l cutilia may be used.

The present invention utilizes three advantageous properties of the T5 system: rapid host protein synthesis; complete arrest, destruction of host chromosomes and high signal strength (signal st A phage-based method that takes advantage of the use of a promoter with developed a production system.

The method of the present invention can be applied to various applications, but the most important are the following two applications. It is.

First, the present invention involves introducing a desired production gene into the virulent phage used. Therefore, recombinant heterospecific proteins can be easily produced. To lytic phage T5 The based system can be used in biotechnology for the expression of foreign genes For example, the regulation of host protein synthesis immediately after infection Complete cessation prevents the infected cells from expressing recombinant protein derived from the phage. Limited to high quality. In addition, since the host chromosome is degraded to the point where it is acid-soluble, recombinant Protein 1. Reduces DNA viscosity problems seen when purifying from blockers ( Fish, N. M. &’Li1ly, M. D.

(1984) Biotechnology 2.623-827), Phage Insertional activation of gene after M change in genome variation), the rule of normal proliferation, and the production of proteins that are fatal to Chikuhi cells. protein can be expressed. Additionally, this expression is similar to that of any other E. coli system. A class of promoters found to have greater signal strength than Based on the promoter (won Gabain, ^, & Bujard, H , (1979) Proc.

Nat, 8cad, sci, Us^　78. 189-193).

The results disclosed here demonstrate that a T5-based expression system is feasible. There is. Various modifications are also possible to facilitate use and optimize the system.

Deletion mutants/plasmid sets designed for insertion can be used to generate inserts of various sizes. and the juxtaposition of the promoter and insert to suit optimal expression. Ru. Also, the plasmid designed for insertion may not be written for other phages. colorimetric plaque test (Messing, J., Ronenborn, B.

Muller-1 (ill, B, & Hofschneider, P, M, (1977) Proc, Natl.

Recombinant phages were generated using Δcad, sci, US^3642-3646). An indicator gene to be identified, for example an indicator gene encoding β-galactosidase It can be equipped with. One further variation of this method is the "early" - "late" switch consists of using a T5 mutant strain that is free of . These phages are sterile and stall at an “early” stage when they infect non-permissive cells. (McCorquodale, DJ).

(1975) CRCCr1tical Reviews in Microb iology 4.213-234). The advantage of a sterile infection system is that the “initial” The protein expression period is long and does not enter the stage of phage DNA replication and cell lysis. There is something wrong with that.

Second, such systems use minicells (Frazer, Δ, C, & Curtiss III, R. (1975) Curr, Top.

Microbiol, Immunol, 69.1-84) or Maxi cells. (Sancar, ^, , Hack, ^, M, & Rupp, D, ( 1979) J, Bacteriol.

137, 692-693) to interfere with cellular protein synthesis, as in It can be used for analysis of recombinant regression products without any This method is related to the CAT gene. Insertion of the gene of interest into phages and short-term release of infected cells, as demonstrated by and analysis of radiolabeled extracts for recombinant proteins. With this invention Expression in these cells is limited to proteins derived from phage.

Hereinafter, the present invention will be explained in more detail by way of non-limiting examples based on the accompanying drawings.

FIG. 1 shows an autoradiogram of DNA plotting.

Figure 2 shows chloramphenicol acetyltransferase in infected cells. The activity is shown in an autoradiogram.

Figure 3 shows a model of the physical structure of the T5 phage.

Figure 4 shows a map of one integrating plasmid used.

Figure 5 shows a map of the different integration plasmids used.

As mentioned above, the present invention will be described herein by way of non-limiting example, with reference to the monophage T5. and explain.

As already pointed out, T5 plays a role in biological research regarding episomal integration into chromosomes. It has a number of unique properties that make it advantageous for research. This phage has 121,284 bases Organized in the form of linear double-stranded DNA molecules exhibiting equal length in pairs (Rhoad es, M.

(1982) J, Virol, 43.566-573), non-essential regions of chromosomes. Stable deletions in the region (McCorquodale, D. J. (1975) CRCCr1ticalReviells in Microbiology 4.213-234) equates the insertion of genetic material to 14,000 base pairs. Can be made up to large sizes.

In addition, approximately 100 restriction enzyme recognition sites are specified along the genome, so Biographical reconstruction is easy to analyze. This phage also probes episomes. unique genes that would be more easily investigated if they could be inserted into the viral genome. It exhibits many functions and characteristics during its life cycle.

During the infection cycle, three sets of genes are involved: r pre-early; "early" and "late" genes are activated in a cascade. These three types of remains Genes are arranged in clusters along chromosomes in the order of “immediate early,” “early,” and “late.” Ru.

First, the r-immediate-early genes are introduced into the host cell, and only after they are expressed are the genes left behind. The new chromosomes actively move within the host. Then the “early” and “late” genes become active. Be sexualized.

Expression of “early” genes regulates phage chromosome replication and phage capsid synthesis. prepare. Both of these are initiated with the onset of "late" genes. dyed Immediately after the first step injection (FST) of the plastid host, protein synthesis is completely inhibited. The host's DNA is degraded to acid-soluble within the first few minutes.

The effects of T5-controlled expression are partially explained by the unusual strength of the promoter be done. The promoter of E.co ratio T5 was found to be by far the strongest among the I made it clear.

The efficiency of phage-controlled biosynthesis during infection is significantly greater, i.e. cells for 45 min with a burst size equal to approximately 200 phage particles per cell. Approximately 2.4·10 7 base pairs are replicated during the infection cycle. This is the host chromosome It is five times that of In these 5-stained cells, the protein synthesis rate also increases 2.5 times (' Chinnadurai, [;, & MacCorquodale, D.J.

(1973) Proc, Nat, ^cad, sci, US^703502- 3505), one reason for the dramatic effect of T5-regulated expression is the abnormal strength of the promoter. Therefore, it is explained (von Gabain, A, & Bujard, H, (1 977) Molec.

gen, Genet, 157.301-31.1 and von Gabain, ^, & Bujard, H.

(1979) Proc, Nat, ^cad, sci, USA76.189- 193).

E Ll Shita pa bizu 11 restriction endonuclease, T4 DN^ ligase, S1 nuclease and Lucari phosphatase was obtained from BoehrinHerMannheim. ; The conditions for use of these enzymes are those specified by the manufacturer, or the conditions for use with ColdS pring Harbor Laboratory, Mo1ecular Cloning, 1982, Maniatis, T.

As described by Frltseh, E. F., and Sambrook, J. We used the following conditions.

Phage and boosmid T5” and T5sto were kindly provided by H. Bujard. offered. Plasmid pACYC177 was kindly donated by S. N. Cohen. It was. ppt, 1 is B, chloramphenicol acetyl from Pumlis ・It is a pPL603 derivative containing the transferase (CAT) gene (Wi lliams, D. M., Duvall, E. J., & Lovett', P. S. (1981) J. BaeLeriol, 146.1162-1165. This was contributed by Rutberg. Regulation derived from the T5 chromosome Limited fragment old nd1110 to pBR322 or 11ACYC177 (7) were inserted into the former ncl111 site of Di-; these constructs were inserted into pBRO and pBRO, respectively. pACYC (named +). Asymmetrically observed in the phage-derived insert EcoR1 site on pBR322 and the next EcoR1 site on pBR322' (Rodriq uez, R, L, F, Bolivar, RJ, Greene, M, C, .

Betlach, H.L., Heynecker, 11. L...Boye r,H,W,, Crosa,,J,H.

& Falkou+, S. (1977) Gene 2.95-113) and the next Xho1 site on pA CY’C177 (Chang, ^, C, Y , & Cohen, S. N. <1978) J. Bacteriol, 134 ．． 1141-1156)) is the insertion direction in the two vectors. (See also Figure 5). into pBR322 vector Remove the 249 base pair EcoRI fragment starting from the phage insert. pBRΔ0 was induced from pBRo by this. fif&, pPLl tends to be The isolated Bglii BamHI fragments are described in the results section below and By inserting into the unique Bam111 site of pPRΔ0, as shown in Figure 5, pPRΔ0CAT? :Configured.

Fuji's and -L T5° and T5sto Bujard, H, & Hend riekson H, E, (1973) Europe, J, Biochem, 33. Propagated as described in 517-528°. Recombinant phages are also The cells were cultured in a medium, solution and solution containing 10mM Mg5O and 1mM spermine. (Labedan, F, T.).

B, & Legault-D6mare, J, (1984) J, Virol , 50.213-219>.

CsCl gradient 33,0 by VTi 50 rotor (Beckman) The test was carried out at 00 rpm and 20° C. for 40 hours. The initial density was adjusted to 1.56 g/ml. 30,000 rpm, 2 Phages were first purified by running a CsCl gradient for 1 hour at 0°C. . Preformed step phages containing equal amounts of these phages at the following densities: layered on the agent: 1.5g/1ml, 1.0g/ml, 0.5g/m l.

of plasmid DNA and phage DNA; Co1d the plasmid DNA Spring Harbor Laboratory, 1982, Mole Maniatis, T., Fr1tsch, E. for cularCloning. According to the experimental procedure described by F., and Sambrook, J. Prepared. Extraction of phage DNA was performed by von Gabain, A., Wayw. ard, G. & Bujard, H.

(1976) Molec4en, Genet, 143.279-290 and Bujard, H. & Hendrickson, 1. E. (1973) Eur. OP, J. Biochem, 33.517-528 as follows: It was performed with a modification: before placing the phage particles on the CsCI step gradient. 37°C with DNasel (10νg/n I, Worthington) This incubation was carried out in 10mM HgSO4. Phage suspension buffer containing (Bujard, H. & Hendrickso n, Il, E. (1973) Europ.

J. Biochem, 33.517-528).

Plaque lift and blowing 4--Co1d the number of plaque forming units Spring l1arbor Laboratory, 1982, Mo- For example, for λ phage, Maniatis ,T.

Described by Fr1tsch, E.F., and Sambrook, J. Measurements were made using the soft agar technique. However, this technique was modified as follows. :Soft agar still contains 2ml of CaCl2, but the recombinant phages are plated. If sprinkled on top, add 10mM MgCl□ and 1mM spermine. I made it. Plaque lift was performed using the procedure described for recombinant λ phage (f oe.

cit, ) on a nitrocellulose filter. This filter's hybrid Dization and washing were performed according to the promoter described above. These frames The filter was finally exposed to X-ray film (Koclak X-OHΔT) for 2-24 hours. Exposed. Plotting of electrophoretically separated RNA or DNA samples as described above. It was carried out according to the procedure (loc, cit,). In any hybridization Plasmid DNA or, in some cases, 14 DNA fragments are Labeled deoxyribonucleotide-triphosphate with radioisotope The nick translation process was performed using the following methods (Ioc, cit,).

Assay of chloramphenicol acetyltransferase CAT on chromosome All cells were simultaneously infected with the recombinant phage containing the integrated CAT gene. (The multiplicity of infection (M, 0.1) was 2); 2 at multiple points in the infection cycle. An aliquot of m1 was removed and Nisson, (:, Be1asco, J. ,G.

Cohen, S. N., & von Gabain, H. (1984) Natu. re　312. CAT enzyme content was determined as described in 75-77.

Target θHLvan Gabain, ^,, Be1asco, J, (:,, 5 chotLel, J.

Chant, ^, C, Y, & Cohen, S, N, (1983) Proc. , Nat, Δcad, sci, USA80, 653-657. RN8 was extracted from T5-infected cells and control cells by the following method. Plot this RNA Berk and 5harp method (Berk, ^, J, & 5ha rp, P, ^, (1977) Cell 12.721-723).

This is the old 1sson, G., Be1a-sco, J.G., Cohen, S. , N. &’ von Gabain, ^, (1984) Nature312 , 75-77 and von Gabain, ^, , Be1asco, J.G., . 　5chottel.

J., Chang, ^, C. Y., & Cohen, S. N. (1983) Pr. oc, Nat, 8 cad.

Sci, tlS88, 653-657 for details.

Husband's blasphemy In order that the experiment may be fully understood, a brief description of the accompanying drawings will now be provided.

Figure 1 shows an autoradiogram of plaque hybridization.

Cells harboring pBROcAT were infected with 75sLo (see “Materials and Methods” section). and table notes). After lysis, 2000 phages were plated on an agar plate. Nell A).

Bracoon using nitrocellulose as described in the “Materials and Methods” section. Plaques were analyzed by lift technique. This filter is labeled with a radioactive label. It hybridizes with ppt and t, and furthermore, Co1d Spring Harbor. LaboratoryM, 1982, Molecular Cloning In Maniatis, T., Frltsch, E.F.

& Sambrook, J.

X-ray film (Kodak X-0M^T) was exposed to this filter for 4 hours, and then Post-processed. Agar plate areas corresponding to positive plaques were punched out.

Co1cl Spring Harbor Laboratory, 1982 , Maniatis, T., Molecular Cloning.

Described by Frltsch, E.F., and Sambrook, J. The phages were isolated using the method described. The eluted phages are displayed in “Results” and “Materials”. and methods, and purified on a CsC1 gradient. . A denser band corresponding to the recombinant phage was recovered. 300 frames The phages were plated and analyzed as described above (Panel B). to panel A In the experiment shown, 1 out of 2000 plaques reacted to the probe. , in the experiment shown in panel B, 98% of the plaques hybridized with the probe. .

Figure 2 shows the autoradiogram of DNA plotting using 5outhern. (Colcl Spring Harbor Laboratory i, 1982, Maniatis, T., Fr1tsch, E.F., & Sa+ebrook, J.

(See Molecular Cloning). cYc to plasmid .

cells with T5sto were infected with T5sto. Collect the phages as described above, and The DNA was extracted and treated with restriction endonucleases HindIII and Ps as described above. As a control, DN isolated from T5sto digested with tl A was decomposed with the two types of enzymes mentioned above, and the DNA from pAcYc177by was converted into Ps. Decomposed with tl (ChaB, ^, C, Y, & Cohen, S, N, (197 8) See J. Bacteriol, 134° 1141-1156). D.N. A fragment to Cold Spring Harbor Laboratory lJ, 1982, Maniatis in Molecular C1onirH ,T.

Described by Frltsch, E.F., and Sambrook, J. Fractionate using 0.8% agarose gel as described above and transfer to nitrocellulose. I ransfered. Hybridization was carried out according to the method of collapsing and drilling.

DNA from plasmid pAcYc177 was used as a probe. fill The samples were exposed to X-ray film (X-OH) for 6 days. (a) cYc1 to p DNA from 77 was digested with Pstl: (b) from E. coli phage. Digest the DNA from Hindlll; (() 1l of DNA from T5sto (d) DNA from T5sLo was digested with Pstl; DNA from T5sto grown on E. coli containing pAcYco and H. Digested with indll (e) and Pstl (f). expressed in kilobases The size is shown in the corresponding position of the signal.

Figure 3 shows chloramphenicol acetyltra in cells infected with recombinant T5. This is an autoradiogram showing the activity of ferase. Various shapes appear in 10 minutes after dyeing Mono- and di-acetylated chloramphenicols have appeared. extraction of cells Aliquot the cells before infection and at 2, 4, 10, and 20 minutes after infection. The cells were removed and destroyed by lysozyme treatment and sonication. child Incubate the cell extract with 14C-labeled chloramphenicol. and analyzed by ascending hole C (Nilsson, G., Be1asco, J.G., Cohen, S.N., & von Gabai n.

^, (1984) Nature 312.75-77).

Figure 4 shows a model of the physical structure of T5st(0) DNA (113kb). It is shown together with ``back pattern''. In addition to the physical model of DNA, various transcribed regions The area and direction of transcription are indicated by arrows. Here is a map of the HindIII restriction site. Shown below the physical map. The plasmid integrates (by homologous recombination) The region has been expanded to include T5, which encodes a stable phage-specific RNA. A physical map of a section of the genome is shown (Ksenzenko, V., N. , , Kamynina, T.P., , Kazantsev, S.I.

5hlyapnikov, M.G., Krykov, V.M., & Baye v, ^, ^-<1982) Bio-chimica et Biophysic a Acta 697, 235-242), the transcription direction of these genes is The possibility of initiating transcription of the recombinant protein is indicated by the arrow below. A promoter has been described (Stueber, D., Delius, H. , & Bujard.

H, (1978) Molec, gen, Genet, 166.141-14 9).

Figure 5 shows the 906bp EcoRi)Iindlll fragment from T5. This is a map of the integrated plasmid. pPL603 (Williams.

D, M., Duvall, E. J., & Lovett, P. S. (1981 ) J, Bacteriol.

CAT-8 obtained from pPLl, a derivative of 146.1162-1165) 6 genes (RuLberg personal communication) have been inserted into the old Bam site. This CAT Genes do not have promoters (Joc, cit,).

The attached table shows how plasmids of different backgrounds are inserted into the viral genome. Frequency is indicated. Each plastic T5sto was dyed 3 times (M, O, 1, is 3? and 5). After lysis, the phage titer was examined and approximately 20,000 phages were found. were plated onto four agar plates and subjected to plaque lift as described above. their respective Homologous region between phage and plasmid ns) size is given in base pairs; plasmid size is given in kilobases.

(length equal to 1200 base pairs) was added to plasmid pBR322 (length equal to 1200 base pairs) as described above. Rodriguez, R.L.F., Bolivar, P.J., Gree ne, H, C,.

Betlach, H.L., Heynecker)1. L. Boyer , H.J.l., Crosa, J.H.

& Falkow, S. (1977) Gene 2.95-113) and p. AcYc177 (Chang.

8, C, Y., & Cohen, S. N., (197,8) J. Bacteriol. , 134.1141-1156) eoli- and its derivatives with T5sto, i.e. 8570 salts in the region that can be deleted next to the region spanning the old ndlll fragment 0. A deletion mutant of the wild-type phage lacking the base pair was infected. Child phage (d (escending phage) on an agar plate to form plaque forming units. I checked the number of The indicator strains did not contain the respective plasmids. nick trance pBR322 and pAcYc177, which had been subjected to lysis treatment, were used as probes. Plates containing 102-104 plaques were subjected to a plaque lift (Ma Niatis, T., Frltsch, E. F., & Sambrook, J. , Molecular Cloning, Co1d Spring Harb or Laboratory, 1982).

Plaques that react positively to the probe have a frequency of 0.5 to 2xlO-' (see attached drawing). The number of plaques that reacted positively Even if 20% of the Hindll fragment 0 is deleted in the (See “Materials and methods used” and attached drawings).

When cells were infected with wild-type phage T5'' or without virus insertion, each protein was No positive plaques were identified when lasmid-containing cells were infected ( (See attached drawing).

We repeated this experiment using the Tsuiho strain, which is rec＾minus as a host. . The results were similar, with the frequency of positive plaques not changing much.

Phage isolated from positive plaques are plated again or onto indicator cells. Reinfection increased the number of plaques reactive to 10-b (Fig. 1). reference). The same results were obtained when these phages were grown in liquid culture. T The ratio of 5sto to recombinant phages is such that the mixture of these phages is Amplification through several passages in host cells without sumids did not change.

b) ″　　phage　, Phage derived from plaques giving a positive signal were cultured in a liquid medium as described above. Proliferated. These phages were purified with a CsCl step gradient (1 t+NΔ was extracted and cut simultaneously with multiple restriction endonucleases. this The DN^ fragments were fractionated on an agarose gel and then transferred to nitrocellulose. Spread it. These filters were added to each plasmid without phage inserts. probed with old ndIII fragment 0. Figure 2 shows E. coli. For example, analysis of recombinant phages containing pAcYco obtained by staining It shows. This DNA was digested with the restriction endonucleases Hincllll and Pst. Cut at I. This phage segment was inserted into the unique old ndIIr site of the vector. As a result of inserting it into the old nd1111, cYcO is cut at 2 places to tp, Luca, P sLI only cleaves a portion of the pAcYcO hector in one place. Hind Ill digests can only be detected with plasmid-specific probes. fragment (4,1 kb), Pst1 degradation revealed that both probes and haphazard Three fragments are hybridized (20kb, 6.8kb and 5.2kb, respectively) kb) was revealed. This result is suitable for multiple insertions of plasmids by homologous recombination. match.

The Hindll fragment corresponds to the excised pAcYco in Figures 2 and 2. 4), the smallest PsLI fragment (Figs. 2 and 4) is pAcYc177 and plasmid and phage fragments. is expected from the calculation of the distance to the double-matching Pst1 site as a result of multiple insertions. (see Figure 4). This result is a recombinant phage. Although it is not possible to determine the exact number of inserted plasmids per Pstl flag The relative signal intensities of the samples indicate an average of 3 inserts per chromosome. pB Amplified on E. coli with another tie 1 plasmid such as RΔ0CAT. Similar results were obtained when chimeric phages were analyzed.

The recombinant phage as described above and the previous phage were purified as described above (CsCI-contaminated). The characteristics of the phages were further investigated by fractionating them on a gradient gradient. Usually 2 Two bands were obtained. For example, use pBRΔCAT (see Figure 5) as a phage genome. When inserted into the nom, the upper band was detected for T5sto1,54 g/cm', and the lower band has a b of 1.56 g/cm3. oyanL density is shown (see Figure 2).

The boyant density in the lower band is greater than the titer measured for wild-type phage. (Hertel, R, Marchi, L, & Mueller, K.

(1963) Virolog)’ 18.576-581), each with two As a result of analyzing the phage obtained from different bands, the upper band contained only 75sto; The lower band reflects 99% of recombinant phage with the given insert. It turned out that. The increment in boyant density is the same for fur whose genome size differs by approximately 8 kb. This b The increment in oyant density will match the insertion of each of the three plasmids.

CsCl-purified recombinant phage was used to reintroduce cells containing the inserted plasmid. Once infected, in addition to the "canonical" positions, there are also gradients that can reflect multiple insertions. Phage located at high density were recovered.

C) Transferring the plasmid to the phage genome Integration leads to the expression of heterospecific genes by reinfection of cells with recombinant phages. mediate. To test this possibility, we selected genes that are easy to assay, namely B, J. Chloramphenicol transferase (CAT) derived from pBR32 2 (see Figure 5 and "Materials and methods used"). This gene was first It was extracted from a plasmid that had been constructed to measure promoter activity. (Williams, D. H., Duvall, E. J.

& LoveLt, P, S, (1981) J, Baceriol, 146. 1162-1165), does not contain any endogenous promoter. Furthermore, this gene In the plasmid, the expression of phage-specific tRN^ is suppressed by homologous recombination. It was oriented to be inserted downstream of the “early” phage promoter that controls the (See Figures 4 and 5).

Cells containing this construct exhibited some basal level of CAT activity. C.A.T. The BglII-BamHI fragment containing the gene is also found upstream of the old ndl ll fragment 0 mo kizue 0 and country vs. ki! does not show any promoter activity in Itame(111illiams.

D, M., Duvall, E. J., & Lovett, P. S. (198 1) J, Bacteriol.

146, 1162-1165 and Ksenzenko, V.N., Kamyn. ina, T, P,.

Kazantsev, S., 1. , 5hlyapnikov, M.G., Kry. kov, V, M,! tBayev, ^, ^, (1982) Biochim ica et Biophysica Acta 697°235-242 and 5tueber, D., Delius, H., & Bujard, H. (1978 ) Holec4en, Genet, I66, 14i-149), expression is It should be derived from the promoter found on pBR322 mentioned above (Chang , A., C., Y., 'Nunberg, J. H., Kaufman, R., Er. 1ieh, H, ^,.

Schi-mke, R.T. & Cohen, S.N. (1982) Na ture 275.617-624 and 5tuber, D. & Bujar d, 11. (1981) Proc, Nat, ^ead.

Sci, LISΔ78.167-171). However, due to homologous recombination, When the plasmid is integrated into the phage genome, the promoter and CAT gene were separated (see Figures 4 and 5). Isolate the recombinant phage and proceed as described above. and (concentrations were increased with a CsCI gradient.

E. coli cells JNaka294 were simultaneously infected with the recombinant phage and treated as described above. Determine CAT activity during the infection cycle by analyzing aliquots of infected cells. monitored (Nilsson, G.

Be1asco, J., Cohen, S. N., & van Gabain, ^, <1984) Nature 312. ’75-77). The results are shown in Figure 3. show. Expression can be detected before the early stages of infection, i.e. approximately 10 minutes after infection. persists until late stages. Demonstrating phage-mediated CAT gene expression An alternative method was performed as follows.

Cells not containing pPRΔCAT were infected with recombinant phages (M, O, 1, was 5). Ten minutes after infection, chloramphenicol was added to the medium (100 g/ml). Infected cells are lysed after the normal infection cycle time (McC orquodale, D. J. (1975) CRCCrltical Rev. ies in Microbiology 4.213-234).

The same experiment was repeated using 75sto for infection. No lysis occurred. recombination Comparing the titers obtained for recombinant phage and 75sto, it was found that when grown in the presence of translation inhibitors, T5sto Amplify the number of phage/ml. The titer obtained for T5sto was 104. E. coliff cells (MM294) containing the mela plasmid pBRΔ0CAT were Deutsche Sammlung van Hi in West Germany G5ttingen Deposited on July 16, 1985 at Kroorganismen. acceptance number The number is DS M 3386.

Ga! and I sweets To perform this experiment, we used a β-galactosidase gene that does not have any promoter. The offspring were infected with the old ndlll fragment of T5 in the same manner as for the CΔT gene described above. (See Figure 5). The insertion direction is the gene is under the control of a T5-specific promoter after integration into the phage genome. It was decided that it would be recombinant. Recombinant fur containing β-galactosidase gene The lesions were identified by plaque lift as previously described.

The phages were further amplified and then used to co-infect Mochichu cells. multiple infections The concentration was 5 phages per bacterial cell.

Aliquots of cells (2,5x 109) were removed from the medium before infection and at designated time points after infection. Determine β-galactosidase content using an enzymatic assay (see table below). (see citation).

Enzyme activity was detectable 10 minutes after infection and steadily increased until cell lysis. In addition, recombination Both phage fractions were not determined for the phage stocks used in this experiment. won. Therefore, the number of units per cell is It cannot be used as a standard for measuring the β-galactosidase profile that may occur in the stem. (For details on the experiment, see ColclSpring Harbor Lab, ed. 1972, Experiments in Mo1 by J. H. Miller (See ecular Biology).

This experiment demonstrates the general applicability of the T5-based expression system and the method can be used in recombinant It was possible to identify the phage as a blue peak in a colorimetric assay.

east Before infection/after infection β-galactosidase units minutes After lysis 275 It is to be understood that the invention is not limited to the specific embodiments described above. subordinate By introducing a gene other than the CAT gene mentioned above into the chromosome of an appropriate lytic virus. , other commercially significant proteins can also be expressed. Such implementation Examples include insulin, interferon, insulin-like growth factors, Examples include antithrombin.

, , , 0 sg+ I! Bam HI BamHJ International survey report

Claims (18)

[Claims] 1. A gene that produces a heterospecific protein has been inserted into the chromosome Lytic virus. 2. Claim 1, wherein the gene is located in a non-essential region of the chromosome of the virus. The virus described. 3. The method according to claim 1 or 2, wherein the gene is introduced by homologous reciprocal recombination. Virus included. 4. The virus according to any one of claims 1 to 3, which is a bacteriophage. . 5. E. 5. The virus according to claim 4, which is E. coli phage T5. 6. Virus according to claim 5 selected from T5+ and T5sto. 7. A host cell infected with the lytic virus according to claim 1. 8. E. The host cell according to claim 7, which is E. coli. 9. The virus is E. It is a bacteriophage such as coli phage T5. The host cell according to claim 7 or 8. 10. Bacteriophage can exist autonomously in T5+ and derived from it The host according to claim 9, selected from stable deletion mutants, such as T5sto. Principal cell. 11. A method for producing a protein by expression in a host cell, the method comprising: a) introducing a heterogeneous gene into the chromosome of a lytic virus; b) the resulting infecting a host cell by introducing the recombinant virus into the cell; c) expressing a recombinant protein in the host cell, and d) A method consisting of the steps of recovering the protein thus produced. 12. A claim wherein the production gene is introduced in step a) by homologous reciprocal recombination. The method according to scope 11. 13. Claim 11 or 1, wherein the gene is introduced into a non-essential region of the viral chromosome. The method described in 2. 14. The production gene of step a) is transformed into E. coli. coli, T5+ or derived from Stable deletion mutant strains that can exist autonomously, such as T5sto, According to any one of claims 11 to 13, which is introduced into the chromosome of a teriophage. the method of. 15. In step b) E. 15. The method according to claim 14, wherein E. coli is infected. 16. The gene is introduced into a non-essential region of the phage chromosome and the phage promoter 16. The method according to claim 14 or 15, wherein the method is under the control of: 17. a) inserting the foreign gene into the chromosome of the lytic virus; b) in step a) Introducing the obtained recombinant virus into host cells to infect the cells, and c) recombinant genetics comprising the steps of analyzing the proteins expressed in said cells; Method of analyzing offspring products. 18. Claim 17, wherein the foreign gene is inserted downstream of a ribosome binding site. Method described.