JPH0544269B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a method for isolating a recombinant vaccinia virus, and more particularly, to a method for isolating a recombinant vaccinia virus when an attenuated Lister temperature-sensitive mutant strain is used as a vector. (Prior art) In recent years, a method for constructing a recombinant vaccinia virus that incorporates foreign DNA into a vaccinia virus has been devised, and a recombinant vaccinia virus using, for example, infectious disease antigen-encoding DNA as the foreign DNA can be used as a live vaccine. Methods have been proposed to do this (for example, Japanese Patent Publication No. 129971/1982, Japanese Patent Publication No. 500518/1982). This method is performed, for example, according to the following steps. (See Figure 1). That is, first, a DNA region encoding thymidine kinase (TK) of vaccinia virus is cut out using a restriction enzyme, and this is inserted into a plasmid to prepare a first hybrid plasmid. Next, the TK DNA portion of this hybrid plasmid is
Incorporate the DNA and prepare a second hybrid plasmid. On the other hand, when animal cells infected with vaccinia virus are prepared in advance and a second hybrid plasmid is introduced into them, homologous recombination between the vaccinia virus and the second hybrid plasmid occurs within the infected animal cells. occurs, and a recombinant vaccinia virus incorporating foreign DNA is formed. Thereafter, TK-negative (TK ^- ) animal cells were inoculated with the formed culture supernatant and cultured for 3 days in a medium containing 25 μg/ml of 5-bromodeoxyuridine (BudR). The desired recombinant vaccinia virus is selected by plaque hybridization using part or all of the virus.
VoL2, No. 9, pp. 84-87, 1987, etc.). This method is extremely efficient as a method for preparing recombinant vaccinia virus. but,
Although this method can be applied to the WR strain, which has traditionally been used as a vector, the present inventors used the attenuated Lister temperature-sensitive mutant, which is known to be a much less virulent strain than the WR strain. When this method was applied to strains, it was found that selection of recombinants was impossible due to significant inhibition of virus proliferation. (Problems to be Solved by the Invention) Therefore, the present inventors conducted intensive studies to develop a method for selecting recombinant vaccinia viruses using the attenuated Lister temperature-sensitive mutant strain as a vector. The present inventors discovered that the selection of recombinants became possible by controlling the BudR concentration and culture time, and completed the present invention. (Means for Solving the Problems) Thus, according to the present invention, an attenuated Lister temperature-sensitive mutant vaccinia having a growth-inhibiting temperature of 40.5°C in rabbit kidney cells and a pox size of 1 mm or less on the chorioallantoic membrane of hatched chicken eggs. A mixture of virus (a) and recombinant vaccinia virus (b) in which foreign DNA has been integrated into the thymidine kinase-encoding DNA region of the virus is inoculated into thymidine kinase-negative animal cells, and 5-bromodeoxyuridine It is characterized by culturing in a medium with a concentration of 3 to 23 μg/ml for 5 days or more, and selecting the recombinant vaccinia virus (b) from the formed plaques by plaque hybridization using exogenous DNA. A method for isolating a recombinant vaccinia virus is provided. The vaccinia virus used to incorporate foreign DNA in the present invention is a weakly virulent temperature-sensitive mutant strain of the Lister strain virus, and its pock size on the chorioallantoic membrane of hatched chicken eggs is 1 mm or less compared to the Lister parent strain. The temperature at which rabbit kidney cells cannot proliferate is 40.5°C. Attenuated virulence here refers to the fact that the central nervous system pathogenicity in rabbits and monkeys is significantly weaker than that of the Lister parent strain, and the invasiveness of the central nervous system in mice by peripheral infection is virtually absent ( (See Reference Example 3), especially the amount of virus recovered from the 10% brain emulsion (log ₁₀ TCID50/dose) after 6 days of inoculating 5.8 (log ₁₀ TCID50/dose) to a 2.0-2.5 kg Japanese white rabbit. ml) is preferably 1 or less. Such attenuated temperature-sensitive mutant strains can be obtained by, for example, subculturing the Lister strain in rabbit kidney cells at 30°C, performing plaque purification, and then subculturing the Lister strain, which has extremely poor proliferation in Vere cells at 40°C. It can be obtained by selecting, forming a pock on the chorioallantoic membrane of a hatched chicken egg, and selecting relatively small clones of the pock. A specific example of the attenuated temperature-sensitive mutant strain used in the present invention is produced according to the following method.
Examples include LB strain (CNTM-I-423). Generation of LB strain The original Lister strain was passaged for 36 generations in rabbit kidney cells at 30°C, and plaque purification was performed three times to isolate 50 clones. Among the 50 clones, we selected a temperature-sensitive mutant strain that showed the worst growth in Bellow cells established from green monkey kidney cells at 40°C. When this temperature-sensitive mutant strain was compared with the original strain, its growth in rabbit skin was better than the original strain, but its growth in the central nervous system of rabbits was poor, and its pathogenicity in the central nervous system of monkeys was DI.
It was confirmed that the strain was significantly weaker than the original strain, and was found to be significantly weaker than the original strain, so we conducted an inoculation test on a small number of cases.
As a result, the fever rate was 14% and the systemic reaction was mild, but crust formation tended to be delayed, so we attempted to isolate a clone with poor skin proliferation. The size of the pock on the chorioallantoic membrane of hatched chicken eggs was used as a marker for clonal separation. That is, after passage of the above-mentioned temperature-sensitive mutant strain for 6 generations in rabbit kidney cells, plaque purification was performed twice, and relatively small (2 to 3 mm) homogeneous clones with a pock on the chorioallantoic membrane of a hatched chicken egg were isolated.
Then, after 3 passages in rabbit kidney cells at 30°C,
An extremely small clone (less than 1 mm) was isolated on the chorioallantoic membrane of a hatched chicken egg and named the LB strain. Table 1 shows a comparison of the properties of the Lister strain and the LB strain.

[Table] Conventional methods can be used to incorporate foreign DNA into these temperature-sensitive mutant strains of vaccinia virus, and this can be achieved by using the DNA region encoding thymidine kinase (TK) of vaccinia virus. That is, a first recombinant vector containing part or all of the TK DNA region is constructed, and a second recombinant vector is further constructed in which foreign DNA is inserted into the TK DNA region integrated into the first recombinant vector. However, if the second recombinant vector is introduced into animal cells that have been previously infected with the above-mentioned temperature-sensitive vaccinia mutant strain using a conventional method such as the calcium phosphate method, homologous recombination can occur within the infected animal cells via the DNA region. Recombination occurs, and a recombinant vaccinia virus containing foreign DNA is obtained (for example, Japanese Patent Publication No. 500518/1983). The vector used to construct the first recombinant vector in the present invention is not particularly limited as long as it can insert the TK DNA region, and specific examples thereof include pBR322, pBR322,
pBR325, pBR327, pBR328, pUC7, pUC8,
Plasmids such as pUC9, lambda phage,
Examples include phages such as M13 phage and cosmids such as pHC79 (Gene, 11 , 291, 1980). Construction of the first recombinant vector may be carried out according to a conventional method, for example, using a vaccinia virus.
After completely decomposing the DNA with an appropriate restriction enzyme,
Separate and purify the DNA fragment corresponding to the TK DNA region, and cut the same sticky end as the restriction enzyme-cleaved end into the DNA.
It may be enzymatically linked to a vector that has been cut with a restriction enzyme that can be generated. Successfully obtaining the desired vector can be determined by transforming (or transducing) cells such as E. coli with a DNA mixture containing the cut vector and the recombinant vector enzymatically linked, and the resulting transformation. This can be confirmed by a method such as selecting a strain carrying a vector into which the desired TK region DNA has been inserted from among the strains (or transduced strains). A second recombinant vector is constructed from the thus obtained first recombinant vector and foreign DNA. The foreign DNA used is not particularly limited, but it is preferably DNA that encodes an antigenic protein of an infectious disease pathogen (e.g., virus, bacteria, parasite, protozoa, etc.) for which the development of a live vaccine is expected. Specific examples include herpes virus, vesicular stomatitis virus, hepatitis B virus, hepatitis A virus, non-A non-B hepatitis virus, foot-and-mouth disease virus, poliovirus, rabies virus, Japanese encephalitis virus, Vibrio cholerae, Salmonella enterica, and tetanus. Examples include DNA regions encoding antigen proteins of bacteria, malaria parasites, schistosomiasis, and the like. The construction method of the second recombinant vector may also be carried out according to a conventional method. For example, the vector is cut with a restriction enzyme having a cutting site in the vaccinia virus TK DNA region inserted into the first recombinant vector. Exogenous with a sticky end that is the same as the restriction enzyme cut end
The DNA fragments can be linked together enzymatically. The desired second recombinant vector can be obtained in the same manner as the first recombinant vector. A recombinant vaccinia virus is then formed by introducing the second recombinant vector into animal cells that have been previously infected with the vaccinia virus. The animal cells used here need only be those in which vaccinia virus can propagate, and specific examples include TK-143 (derived from human osteosarcoma), FL (derived from human amniotic membrane), Hela (derived from human cervical cancer). ), K.B.
(derived from human nasopharyngeal carcinoma), CJ-1 (derived from monkey kidney),
BSC-1 (derived from monkey kidney), RK13 (derived from rabbit kidney),
Examples include L929 (derived from mouse binding tissue). The recombinant vector may be introduced into animal cells by any conventional method, such as the calcium phosphate method, liposome method, microinjection method, and the like. In the present invention, special measures are required to selectively separate the thus obtained recombinant vaccinia virus from a virus mixture containing non-recombinant vaccinia viruses. That is, a virus mixture containing a non-recombinant vaccinia virus obtained by introducing a second recombinant vector into animal cells previously infected with vaccinia virus is used as a TK.
Negative (TK ^- ) animal cells were inoculated and cultured in a plaque-forming medium containing BudR at a concentration of 3 to 23 μg/ml, preferably 5 to 20 μg/ml for 5 days or more, preferably 2 to 4 days. Thereafter, an agar medium of the same composition is layered and cultured for another 2 to 4 days. At the later stage of culture, plaques can be detected by adding a dye such as 0.01% neutral scarlet to stain according to a conventional method. At this time, if the concentration of BudR used is too high, the recombinant vaccinia virus will become unable to grow, and if the concentration of BudR is too low,
When using this method, not only recombinant vaccinia virus but also non-recombinant vaccinia virus plaques will appear, making it extremely difficult to selectively isolate recombinant vaccinia virus. Furthermore, unless the culture time is prolonged for 5 days or more, it will be extremely difficult to detect plaque formation even when staining with a dye. However, culture conditions other than these are not particularly limited and may be carried out according to conventional methods. The thus obtained recombinant vaccinia virus
In order to prove that the plaque is a recombinant vaccinia virus plaque containing foreign DNA, perform plaque hybridization using a foreign DNA fragment labeled with ³² P as a plaque according to a conventional method. Just do it. (Effects of the Invention) Thus, according to the present invention, a recombinant vaccinia virus using an attenuated Lister temperature-sensitive mutant strain as a vector can be efficiently obtained by a simple operation. The obtained recombinant vaccinia virus was judged to be equivalent to the parent strain or less neurovirulent than the parent strain based on the results of a rabbit central nervous system pathogenicity test, suggesting that the recombinant virus is highly safe. . (Example) The present invention will be described in more detail below by giving Examples and Reference Examples. Reference example 1 (1) Preparation of the first recombinant vector (plasmid pCZ02) containing the vaccinia virus TK gene (see Figure 2) After digesting 5 μg of pBR328 with EcoRI, extracted with phenol chloroform (1:1). The cleaved pBR328 was recovered by ethanol precipitation, and then treated with S1 enzyme to make both ends blunt. This DNA fragment is ligated,
Plasmid without EcoRI cleavage site (pCZ01)
I got it. pCZ01 is selected from competent E. coli
The transformation was carried out by transforming the C600 strain with the ligated DNA and selecting the transformed strain sensitive to chloramphenicol. Next, 15 μg of plasmid pCZ0 was treated with Hind, extracted with phenol/chloroform, precipitated with ethanol, and then the DNA was recovered. The 5′-terminal phosphate was removed by alkaline phos- tase treatment,
After extracting the DNA again with phenol/chloroform,
Recovered by ethanol precipitation. 0.5μg cleaved
pCZ01DNA was ligated to 0.1 μg of purified vaccinia virus (WR strain) Hind fragment containing the vaccinia virus thymidine kinase (TK) gene,
The resulting hybrid plasmid was named pCZ02. The presence of the vaccinia virus TK gene in pCZ02 was confirmed by the following procedure. That is, a competent Escherichia coli HB101 strain was transformed with the ligated DNA, ampicillin-resistant and tetracycline-sensitive bacteria were selected, and then the method of Birnboim and Doly [also Kraytske Assisted Research, 7 ] , 1513-, (1979)], digested with Hind, and then examined by electrophoresis to see if a fragment with the same length as the original vaccinia virus HindJ fragment was present. did. (2) Preparation of the second recombinant vector (plasmid pCZ3) containing the HBsAg gene Plasmid pBRHBadr72 containing the gene encoding hepatitis B surface antigen (HBsAg) (Nuclide Research, VOL11.No.13, 4601~
4610, 1983) was digested with BamHI and XhoI, and a DNA fragment of approximately 1.27 kb was separated by low melting point electrophoresis (40 volts, 16 hours). Next, after confirming DNA fragments with ethidium bromide staining, the gel was cut, phenol extracted, and about 1.27 kb of DNA containing the HBsAg gene was extracted with ethanol precipitation.
Fragments were recovered. The recovered DNA fragment was 10mM
Tris-HCl (PH8.0), buffer containing 1mM DNA
It was dissolved in 10 μg, treated with DNA polymerase to make it blunt-ended, extracted with phenol and chloroform, and recovered by ethanol precipitation. On the other hand, 1 μg of EcoRI linker was phosphorylated at the 5′ end with 10 units of kinase in kination buffer (37°C, 30 min), and this was followed by HBsA
DNA fragments were added and ligated (12°C, 16°C).
time). The reactants were treated with phenol and chloroform, and the DNA was recovered by ethanol precipitation. After further digestion with 20 units of EcoRI, phenol,
It was treated with chloroform and precipitated with ethanol. Meanwhile, add 2 units of plasmid pCZ02 in buffer solution.
Linearization was performed by treatment with EcoRI/μg DNA at 37°C for 2 hours. Linearized plasmid
50mM Tris-HCl (PH9.0), 1mM _MgCl2 , 0.1mM
The 5' end of the plasmid was dephosphorylated with 0.1 units of bovine intestinal alkaline phostase in a buffer containing ZnCl ₂ and 1 mM spamicin by standing at 37° C. for 30 minutes. DNA was recovered by extraction with equal volumes of phenol and chloroform and ethanol precipitation.
0.1μg of this linear plasmid was added to 66mM Tris−
HCl (PH7.5), 66mM _MgCl2 , 10mM DTT,
After treatment in 0.5mM ATP at 12℃ for 15 hours,
0.2μg of 1.27kb EcoRI fragment containing HBsAg gene
combined with. This plasmid was used to transform E. coli strain HB101, and the transformed E. coli was cultured in LB medium containing 1.5% agar and 50 μg/ml ampicillin at 37° C. for 15 hours. Transformed E. coli grown on agar was cultured in LB liquid medium supplemented with 50 μg/ml ampicillin at 37°C for 15 hours.
Purified by method. 10% of each DNA sample was digested with 5 units of EcoRI in buffer for 1 hour at 37°C, and plasmid DNA was analyzed by agarose electrophoresis.
A 1.27kb EcoRI fragment was screened. Since the 1.27 kb EcoRI hepatitis B virus DNA fragment can be inserted in the forward or reverse orientation within each plasmid, the orientation of the inserted gene was screened. screening is a plasmid
Plasmid derived from pCZ02 in buffer
The DNA fragments were digested with Xhol for 1 hour, and the generated DNA fragments were analyzed by agarose electrophoresis and their lengths were compared. The plasmid in which the HBsAg gene was inserted in the forward direction relative to the vaccinia virus TK gene promoter was named pCZ03, and the plasmid in which it was inserted in the opposite direction was named pCZ04. (3) Creation of recombinant vaccinia virus RK-13 cells cultured in a 6 cm Petri dish were incubated with an attenuated variola virus strain (attenuated variola strain LB, the temperature incapable of growth in rabbit kidney cells at 40.5°C, and incubated chicken egg chorioallantoic membrane). Pocket size on top is approx. 0.9mm, CNCM-I-
423, the acquisition method from the parent stock is as described above) 0.1 PFU
45 minutes after inoculation with plasmid pCZ03 in which the HBsAg gene was inserted into the vaccinia virus TK gene.
Dissolve 12 μg in 2.2 ml of sterile water and
DNA by the method of Nucleic Acids, Enzymes 27 , 340, 1985)
-Create calcium phosphate coprecipitate and its 0.5
ml was dropped onto infected RK-13 cells. 37℃ for 30 minutes
The mixture was left standing in a 5% CO ₂ incubator, and 4.5 ml of Eagle MEM containing 10% fetal bovine serum was added. Three hours later, the culture medium was replaced, and after culturing for 48 hours, the cultured cells were frozen and thawed three times and subjected to ultrasonication (1 minute). Example 1 100 μg of the virus solution obtained in Reference Example 1 was inoculated into TK ^- 143 cells cultured in a 6 cm Petri dish, and after 45 minutes, 1% agar, 12% fetal bovine serum, and a predetermined amount of BudR were inoculated.
The cells were layered with 4 ml of Eagle MEM medium supplemented with 100 ml of Eagle MEM medium, and cultured for 3 days at 37° C. in the presence of 5% CO ₂ . Some Petri dishes were stained with 0.01% neutral scarlet.
Another Petri dish was overlaid with 4 ml of agar medium of the same composition, and after culturing for an additional 3 days, it was stained with 0.01% neutral scarlet. For comparison, a recombinant vaccinia virus using the original Lister strain instead of the LB strain was prepared according to Reference Example 1, and experiments were conducted in the same manner using the resulting virus solution. The results are shown in Table 2. From this result, it can be understood that isolation of recombinants is only possible by controlling the BudR concentration and the number of days of culture.

[Table] Example 2 The virus solution obtained in Reference Example 1 was mixed with BudR15μg/ml.
After culturing for 6 days as in Example 1 under the following conditions, the agar medium was gently removed and the cells were stored at 4°C. A sterile nylon membrane was pressed onto the cell surface remaining at the bottom of the Petri dish to transfer the virus. ,
10 min in 0.5M NaOH, 5 min in 1M Tris-HCl buffer
After repeating the treatment for 3 times, 1.5M NaCl0.5M
It was treated with Tris-HCl buffer for 5 minutes. 2x SSC (1
x SSC, 0.15M, NaCl, 0.015MC ₃ H ₄ (OH)
(COONa) ₃ ) and baked at 80°C for 2 hours. 4x SET (0.6M NaCl, 0.08Tris−HCl,
4mM EDTA, PH7.8) - 10x Denhardt - 0.1%
It was treated with SDS at 68°C for 2 hours. 4x SET, 10x
Denhardt-0.1% SDS-50μg/ml denatured salmon sperm
Hybridization was carried out at 68°C for 14 hours with DNA labeled with ^32P by DNA translation. After washing, a nylon membrane was overlaid with X-ray film and autoradiography was performed to detect the presence of spots. After confirming this, an X-ray film was placed on agar that had been stored at 4°C, and a plaque that matched the spot was identified as a recombinant and isolated using a sterile Pasteur pipette. Reference Example 2 Expression of recombinant in cells RK-13 cells cultured in a 25 cm ² culture bottle
0.1 PFU of the recombinant or parent strain was inoculated, and after virus adsorption for 1 hour, Eagle MEM medium containing 10% fetal bovine serum was added and cultured at 37°C for 14 days. As a control, uninfected RK13 cells were placed. After culturing, freeze-thaw three times, sonicate, centrifuge (3000 rpm, 10 minutes) to remove cell debris, and measure the amount of HBsAg in the supernatant using enzyme-linked immunosorbent assay (EIA, Abbott).
Lab.). The beads coated with anti-HBsAg antibody were allowed to stand for 15 hours at room temperature with 0.2 ml of centrifuged supernatant, and 0.2 ml of peroxidase-labeled anti-HBsAg goat antiserum was added and allowed to react at 40°C for 1 hour. 5ml
After washing the beads three times with distilled water, transfer them to an enzyme reaction test tube, add 0.3 ml of hydrogen peroxide solution containing oxyltophenylenediamine dihydrochloride, and react for 30 minutes.
The enzymatic reaction was stopped by adding 1 ml of 1N sulfuric acid. The absorbance at a wavelength of 492 nm was measured, and the amount of HBsAg antigen was calculated with reference to HBsAg positive and negative controls that were performed at the same time. The results are shown in Table 3.

[Table] Reference example 3 iv Marker confirmation test In order to confirm the difference between the in vivo marker of the parent strain and the in vivo properties of the recombinant with the HBsAg gene inserted, a rabbit central nervous system pathogenicity test was conducted. did. Inject the parent strain and recombinant strain into healthy Japanese white rabbits weighing 2.0 to 2.5 kg with 10 ^6.8 , 10 ^5.8 TCID ₅₀ virus solution 0.25
After 6 days of clinical observation, the surviving rabbits were sacrificed, the virus was recovered from the brain, and histopathological examination was performed. The results are shown in Table 4.

【table】 [Brief explanation of drawings]

FIG. 1 is a drawing showing an outline of the operating procedure of the present invention, and FIG. 2 is a drawing showing the operating procedure in the first embodiment.

Claims (1)

[Claims] 1 The temperature at which rabbit kidney cells cannot proliferate is 40.5℃
A weakly virulent Lister temperature-sensitive mutant vaccinia virus (a) with a pox size of 1 mm or less on the chorioallantoic membrane of chicken eggs incubated in chicks and a recombinant vaccinia virus in which foreign DNA has been integrated into the DNA region encoding thymidine kinase of the virus. The mixture in (b) was inoculated into thymidine kinase-negative animal cells and cultured in a medium with a 5-bromodeoxyuridine concentration of 3 to 23 μg/ml for 5 days or more.
1. A method for isolating a recombinant vaccinia virus, which comprises selecting the recombinant vaccinia virus (b) by plaque hybridization using DNA.