JPH0231679A - Recombinant vector - Google Patents

Abstract

PURPOSE:To obtain an antigenic polypeptide by forming a recombinant vector with a fragment containing the DNA coding p-21 of adult T cell leukemia virus coat protein gene origin with a specified site of this DNA nicked. CONSTITUTION:Lymphocytes are separated from the peripheral blood of an adult T cell leukemia patient, and from the DNA extracted from said lymphocyte a fragment (HTLV-15'-3' fragment) containing the DNA coding p21 and nicked at a site within 17 base pairs upstream from the 5' end of this DNA is taken out. This fragment is incorporated into the cloning site of a silkworm manifestation vector (pBF vector) to form a recombinant vector. Thence, using this vector, the HTLV-15'-3' fragment is put to recombination with part of the polyhedral protein structure gene of BmNPV, thus producing the objective recombinant virus.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention is directed to adult T cell leukemia (Adult T cell leukemia).
Adult T-cell leukemia virus (Iluman Tc) is the cause of leukemia; hereinafter abbreviated as ATL).
pHleukemiavirusS-1H or less +1TLV
The present invention relates to a novel recombinant vector for producing a polypeptide having antigenicity against an antibody (abbreviated as -1).

[Prior Art] It is known that the etiology of ATL is infection with IITLV-1, and 5aHTLV-1 has antigenicity against ITLV-1 antibodies, which are useful as vaccines, diagnostic agents, etc. It has been desired to develop a method for producing polypeptides.

Conventionally, in order to produce polypeptides having antigenicity against HTLV-1, HTLV-1 coat protein (env
Attempts have been made to grow Escherichia coli that has been recombined with the gene for elope protein (hereinafter abbreviated as env protein) and to recover the polypeptide produced.

However, the polypeptide obtained by the above method had low antigenicity to HTLV-I antibodies. This is presumed to be due to the fact that polypeptides produced in bacteria such as E. coli are not modified by glycosylation reactions or the like.

On the other hand, there is a method for producing a polypeptide in which a recombinant virus in which the structural gene of a protein, which is a useful substance, is recombined with the polyhedral protein structural gene portion of the DNA of the silkworm nuclear polyhedrosis virus is grown in silkworm denule cells or in the living body of the silkworm. , Japanese Patent Publication No. 6
2-208276 and JP-A-61-9288.

[Problems to be Solved by the Invention] However, these publications do not contain any specific description regarding the application of the above method to the production of the polypeptide that is the antigen protein of HTL Sea 1. do not have. In order to produce a polypeptide that is an antigenic protein of HTLV-I by such a method, it is necessary to recombine the polyhedral protein structural gene of the silkworm nuclear polyhedrosis virus DNA with which part of the HTLV4 structural gene, and also to make the polyhedron protein structural gene of the silkworm nuclear polyhedrosis virus DNA. Much further research was required to determine whether the peptide could be produced and, if a polypeptide was obtained, whether the polypeptide would be antigenic to HTL Sea 1 antibodies.

The present inventors have conducted extensive research in order to develop an effective method for producing a polypeptide that has high antigenicity against ITLV-1 antibodies. As a result, Bombyx Nuclear Polyhedrosis Virus (Bombyx Nuclear Polyhedrosis Virus)
We have discovered that this objective can be achieved by propagating a recombinant virus in which the gene of drosis Virus (hereinafter abbreviated as BmNPV) has been recombined with a specific fragment of DNA derived from the enν protein gene of HTLV-1. . The object of the present invention is to provide a recombinant vector that can be used to achieve this objective.

[Means for Solving the Problems] The present invention provides that the silkworm expression system Pekku is
A fragment of DNA derived from the v protein gene that contains the DNA encoding p21 and is cleaved within 17 base pairs upstream from the 5' end of the DNA (hereinafter referred to as HTLVr
This is a recombinant vector that has been modified with a 5'-3' fragment. As a silkworm expression vector, p
Examples include BF vectors.

The method for producing the above recombinant vector of the present invention is not particularly limited. As a typical production method, lymphocytes are separated from the peripheral blood of ATL patients, the 1ITLV45'-3' fragment is excised from the DNA extracted from the lymphocytes, and this is inserted into the cloning site of a silkworm expression system vector (pBF hector). Examples include a method for producing a Mi recombinant vector. Next, using this recombinant vector, a recombinant virus is produced by recombining the HTLV-15'-3' fragment with a part of the Ba+NPV polyhedral protein structural gene.

The above method will be explained in detail below.

1-I RmNPV In the present invention, BsNPV that is recombined with the ITLV-15'-3' fragment is widely known to sericulture workers, and is the representative strain isolated by Furusawa et al. There is a T3 strain, and the virus I) NA of this strain is
US ATCC (A@erican Type Cu1
ture Co11ection) to ATCCN [L4
It has been deposited as No. 0188 and can be easily obtained manually. It can also be isolated from silkworms infected with BaNPV by known methods.

The BdPV DNA described above can be represented by the restriction enzyme map shown in FIG.

Of this BaNPV DNA (7), the part that is recombined with the IITLVl 5'-3' fragment, which is relevant to the present invention, is the polyhedral protein structure of the polyhedral protein gene shown in FIG. 1, excluding a portion of the promoter. It's part of the gene.

1-2 11TI, V-15'-3'
'. Method 11TLV-1 is a retrovirus that has RNA as a gene, and this gene RNA is transmitted in infected cells.
As for the base sequence centered on the env gene of DNA synthesized from A, 5eiki et al.
Natl.

8cad, Sci, USA 80” (1983) No. 36
The one published on page 21 is known.

In the present invention, the method for obtaining the IT, LV-15'-3' fragment is not particularly limited. Typical methods include AT
L DNA located in lymphocytes in the patient's peripheral blood)
Examples include methods for obtaining the ITLV-15''-3' fragment.

To illustrate a typical method, first, lymphocytes are separated from the peripheral blood of an ATL patient, DNA is extracted from the lymphocytes,
Next, the DNA was cut with the restriction enzyme EcoRI to obtain a DNA containing about 20 kilograms of salt fragments.
phage containing the HTLV4 provirus (after ligation to the restriction enzyme cleavage site) of the 4A vector was isolated by screening, and the provirus was subtracted from the provirus using an E. coli vector group represented by the pUC vector. This is a method for obtaining an HTLV-15''-3' fragment by cloning and cleavage with restriction enzymes.

The HTLV-15'-3' fragment
Among DNA derived from protein genes, DNA encoding p21, a protein located in and inside the membrane
(See Figure 9) and is cleaved within 17 base pairs upstream from the 5' end of the DNA.

As shown in Figure 9, the DNA sequence encoding p21 is the IITL sequence published by 5eiki et al.
V-1env protein gene sequence 6116th to 664th
The third sequence corresponds to this, and the 17 base pairs upstream from the 5' end correspond to the 6099th sequence.

According to the findings of the present inventors, the above ITLV-15'-
Even if the 3' fragment contains DNA encoding p21, if it contains more than 17 base pairs upstream from the 5' end of the 4g DNA, it can be isolated by the method described below. Even if an attempt is made to produce a polypeptide by propagating a recombinant BsNPV virus obtained using a fragment, the polypeptide may not be obtained, or even if it is obtained, it may not be antigenic to HTLV-1 antibodies. However, the purpose of the present invention cannot be achieved.

If a suitable site is indicated, the DNA encoding the p21
GAT from a position 17 base pairs upstream from the 5' end of
The Baa+HI restriction enzyme cleavage site is sequenced as C.

In the present invention, the HTLV45'-3' fragment is p
After being recombined into the BmNPV vector, the virus is recombined with a part of the polyhedral protein structural gene of BmNPV using the knee recombination vector to produce a recombinant virus. The pBF vector used to obtain the recombinant virus is characterized by the restriction enzyme map shown in FIG. That is, the pBF vector is B
This is a vector containing the promoter region of the polyhedral protein gene of mNPV DNA, the vicinity before and after the polyhedral protein structural gene, and the gene of pUC hector, which is an E. coli vector.

The above pap vectors include Xbal, EcoRI,
There is a cloning site for the 5tul restriction enzyme cleavage site.

There are various types of such pBF vectors depending on the extent to which they contain the polyhedral protein structural gene starting from the base sequence ATG upstream of the cloning site, and pBF4 to pBF133 as shown in FIG. 3 exist.

In the production of a recombinant vector, the above-mentioned vector information, inserted I! TLV-r 5'-3' fragment and pB
Any vector may be used as long as it shares a reading frame with the upstream portion of the F hector. However, in order to create a recombinant virus that can efficiently express the 5'-3' fragment in established cultured silkworm cells and silkworm larvae, it is necessary to use BiNPV D starting from the base sequence ATG.
pBF hector containing many gene parts encoding part of the polyhedral protein structural gene derived from NA, such as ρ
BF124. pRF129. pB such as pBF133
Preferably, the F vector is used.

The IITLV-15'-3' fragment may be inserted into the pBF vector using the Eco[if, Xbal, and 5tul restriction enzyme cleavage sites present in the cloning site of the pBF hector. To insert the 11TLV-15'-3' fragment into the pBF vector cloning site, use DNA synthesis or the E. coli vector system pUc.
Either EcoRI, Xbal or 5tuI restriction enzyme cleavage sites are connected to both ends of the fragment by known means such as insertion into Hector, cleavage, etc., or EcoRI is added to the 5' end of the fragment.
, a 5jul restriction enzyme cleavage site is connected to the 3' end of the fragment, or Xbal is added to the 5' end of the fragment, and Stu! is added to the 3' end of the fragment. Any operation that connects restriction enzyme cleavage sites is generally performed. Among the above methods, the method of connecting EcoRI OI2 or Xbal restriction enzyme cleavage sites to both ends of the HTLV-15'-3' fragment is preferred.

When EcoRI restriction enzyme cleavage sites are connected to both ends of the 5'-3' fragment, the fragment converts the pBF vector into an Eco
Connect with the one cut with RI restriction enzyme. Also, +1TL
Similarly, when Xbal restriction enzyme cleavage sites are connected to both ends of the V-15'-3' fragment, the fragment is connected to the pBF vector cut with XbaT. In this case, when making the connection, the pBF hector that has been previously cut with a restriction enzyme is treated with alkaline phosphatase to allow self-ligation of the pBF hector.
on) is preferably avoided.

In addition, EcoRI was added to the 5' end of the HTL sea 15'-3' fragment.
, if a 5jul restriction enzyme cleavage site is connected to the 3' end, the fragment
1 (TLV-15"-3')
When connecting the Xbal restriction enzyme cleavage site at the 5' end and the 5jul restriction enzyme cleavage site at the 3' end, the fragment
Just connect it to the one you disconnected with bal and Stu[.

Such a connection method can be performed by a known method using ligase. For example, pBF cut with a restriction enzyme
5'-3' to be inserted relative to the amount of DNA in Hector.
In this method, the amount of DNA of the fragments is adjusted to 3 to 8 times, and the fragments are ligated using, for example, T4 DNA ligase.

pBF into which the above HTL Sea + 5'-3' fragment was inserted
Isolation and confirmation of Hector can be performed by the following method.

That is, for separation, the above-mentioned connecting reaction solution was used for strain JM109 (No. 9052, manufactured by Zenshuzo ■) and strain MV11B4 (No. 9052, manufactured by Zenshuzo ■).
In addition to E. coli competent cells, such as those typified by E. coli (manufactured by J.D. Co., Ltd.), E. coli was transformed using a known method, and since pBF hector contains an ampicillin resistance gene, the transformed solution was transformed into LB agar containing ampicillin. This can be done by inoculating a medium and culturing at a suitable temperature above room temperature, for example 37°C, for 12 to 20 hours to obtain a single colony that appears as a transformed strain.

In addition, pBF into which the HTLV-15”-3” fragment was inserted
Hector can be confirmed using the boiling method or alkaline vector recombinant vector present in the transformed strain.
Mini-prevention axis ini preparation using alkali lysis method
) L, a recombinant vector suspension was obtained, and the recombinant vector suspension thus prepared was transformed into IITLV-15′.
-Cut with any restriction enzyme that can confirm that the -3' fragment has been inserted, such as EcoR1+ This can be done by checking whether the In addition, 5
The '-3' fragment with EcoRl or Xbal restriction enzyme cleavage sites at both ends is the EcoRl of the pBF vector.
, in the case of a recombinant vector inserted into the Xbal restriction enzyme cleavage site, the upstream part of the pBF vector and the inserted 5'
-3' fragments are cleaved with restriction enzymes such as Bamtll, Xhol, etc. that can confirm whether they are bound in the correct direction, and the cleaved products are subjected to agarose gel electrophoresis in the same manner, and after staining with ethidium bromide, the expected positions are determined. It is a good idea to check at the same time whether or not there is a hand.

It is preferable to increase the amount of the recombinant vector isolated as a transformed strain by the above method by propagating the transformed strain before use. For example, a transformed strain possessing a knee recombinant vector is transformed into an LB containing ampicillin.
Inoculate into a liquid medium and keep at a suitable temperature above room temperature, e.g.
The culture was cultured with shaking for 12 to 20 hours at 40°C, and the culture was cultured using the alkali lysis method to obtain medium-prevalence (o+edium lysis).
preparation) This can be done by obtaining a recombinant vector suspension.

It is preferable to confirm whether the obtained recombinant vector is the desired recombinant vector again using a method similar to the method described above.

The obtained recombinant vector suspension was treated with RNase to obtain Mi for recombinant virus.
It is preferable to use the vector as a suspension.

1-3- In the present invention, the recombinant BgeNPV in which a part of the polyhedral protein structural gene has been recombined with the HTLV-T 5'-3' fragment is a combination of BmNPV DNA and the recombinant vector. were simultaneously transfected (co-transfected) into silkworm established cells using the calcium precipitation method, and the recombinant vector and Ba + NPV D N
It can be obtained by replacing alleles between A.

Specifically, the above-mentioned co-transfection is carried out using 0.
BmNPV DNA and recombinant vector [)NA were mixed at a molar ratio of 1:100 in the presence of 25M calcium chloride and carrier DNA, and then, into the mixture,
HEPES buffer containing 0.28M sodium chloride (
A preliminary step of adding a mixture of pH 7.1) and phosphate buffer, mixing, and then adding the mixture into Ba+ cultured cells;
It is preferable to carry out the method according to Furusawa et al. (Japanese Patent Publication No. 61-9297).

After co-transfection, the reaction solution containing the recombinant virus is cultured at a temperature around room temperature, e.g. 27°C, for 5 to 6 days. After culture, the medium is collected, and after centrifugation, the supernatant is used for cloning of the recombinant virus. used for. Cloning of the recombinant virus from the supernatant of the reaction mixture obtained by co-transfection was performed using the plaque assay method (J, 5eri
This may be accomplished by isolating a recombinant virus using the limiting dilution method (C Sci, Jpn, 53547 (1984)) or the limiting dilution method. Either method may be used, but the method is easier to operate, requires fewer separations (
It is better to use the limiting illusion method because it saves a lot of time.

Cloning of a recombinant virus using the above-mentioned limiting dilution method involves diluting the virus solution obtained by co-transfection, and combining the virus dilution solution with lXlOs ~ 1 x 10h silkworm cell number/@l Infection is achieved by mixing 1:1 of silkworm culture medium, preferably TC1O medium (see Table 2) with a silkworm established cell suspension whose concentration is adjusted, and this mixture is injected into wells in microtiter trays. and at a temperature near room temperature, e.g.
After 2 to 7 days of culture, the wells in the microtiter tray are examined under a microscope, and the presence or absence of the recombinant virus is determined based on the shape and morphology of the silkworm cells observed in the wells. The morphology of silkworm cells found through microscopy includes:
As shown in Figure 6, three types can be confirmed.

Collecting and centrifuging the medium in the well containing only the silkworm cells shown in FIG. 6 that are infected with the virus and in which no polyhedral protein is detected;
A recombinant virus solution is obtained by collecting the supernatant. When wild strain BmNPV and recombinant virus coexist in a well, it is preferable to collect the medium in the well and repeatedly perform limiting dilution to separate the recombinant virus.

(2) Method for producing polypeptide 11-1 Silkworm In the present invention, any established cultured silkworm cell to be infected with the recombinant virus may be used as long as it is capable of proliferating Ba+NPV.

The established cultured silkworm cells capable of propagating BmNPV include Vo
lkman, L.E., and Golds@i
th+ P, A, (1982): ^ppl.

Environ, former crobio1. ,44.227-
BIN shown in 233, (^TCC 隘CRL-8
910) and Bm-N2., which was cloned from B1.N by the aforementioned et al. Cell lines such as Be-N4 are known. In terms of good propagation of kNPV and ease of handling, B.
It is appropriate to use established cultured m/N4 silkworm cells. In addition, it is appropriate to use established cultured silkworm cells used for infection that have been cultured under known culture conditions, for example, in TC-10 medium containing lθ% calf fat serum at 27°C for 4 days. It is.

H-2 virus No. 7 injected into silkworms
In the present invention, the polypeptide of interest is expressed by infecting established cultured silkworm cells or silkworm larvae with the recombinant virus and allowing them to multiply. As a method for infecting established cultured cells of silkworms with the knee recombinant virus, known methods can be used without particular limitations. For example, after putting the prepared culture solution of silkworm cultured cells into a container and allowing the cells to settle on the bottom of the container, remove the old culture solution so that the cultured silkworm cells attached to the bottom of the container do not come off. It is common to extract the virus, add fetal bovine serum as a stabilizer to a silkworm culture medium, and drop the recombinant virus into the culture. The recombinant virus is propagated at a temperature around room temperature, for example, 27°C, after infection with the recombinant virus.
This can be done by culturing in C for several days.

After culturing, the infected silkworm established cell culture was centrifuged, and the precipitated cells were used for confirming the expression of HTLV-1env protein and purifying tlTLV-1env protein, which will be described later. It may also be used as a recombinant virus solution for infection.

Furthermore, the method of infecting silkworm larvae with the recombinant virus is not particularly limited. Generally, the silkworm larvae that infect
It is preferable to use fifth instar silkworm larvae. For infection of silkworm larvae, a recombinant virus solution with increased virus titer obtained as a supernatant by infecting established silkworm cells or a recombinant virus solution without the above procedure is administered percutaneously at 10 to 1 ml.
This can be done by injecting about 00 μl. After infecting the recombinant virus, the infected silkworms are bred to multiply the recombinant virus, and the polyhedral protein and HTLV
A fusion protein of -1env protein is accumulated in the fat body of silkworm larvae.

The method of rearing silkworms is not particularly limited, but either mulberry leaves or mulberry leaves are homogenated, sterilized and freeze-dried, and a paste-like sample (manufactured by Kyodo Sample Co., Ltd., etc.) is soaked in distilled water. A general method of culturing at a temperature around room temperature, for example, 27°C may be adopted.

The breeding period is preferably continued until just before the silkworm larvae die. Although the breeding period varies somewhat depending on the virus titer of the recombinant virus solution to be infected, the breeding period can be set to 3 to 5 days after infection.

The fat body was removed from the silkworm larva, and the fat body was used to confirm the expression of HTLV-1enν protein and to confirm the expression of IITLV-
Used for purification of 1enν protein.

The method for obtaining the fat body described above is to dissect the silkworm larva infected with the recombinant virus by carefully cutting the epidermis without cutting the midcolumn, remove the midcolumn and other organs, and then collect the fat body in the lower abdomen with a spatula. The recommended method is to scrape off the remaining fat pads.

In the present invention, the method for separating polypeptides from the recombinant virus-infected silkworm established cells obtained by the above method and the fat body of the recombinant virus-infected silkworm larvae is not particularly limited; A preferred method is to suspend the silkworm established cells or fat bodies in a solution, disperse by sonication, add an aqueous urea solution, sonicate again, and then centrifuge to collect the precipitate.

The polypeptide obtained by the above method is preferably dissolved in an aqueous SDS solution and purified by conventional methods such as purification using an ion exchange resin and purification by gel filtration.

In addition, the above polypeptide is a part of the polyhedral protein encoded by the polyhedral gene portion of pBF133 etc.
tlTLV-1 encoded by the LV-15'-3' fragment
It is a fusion protein with the ρ21-containing part of the env protein, which is a protein located in and inside the membrane, and it can be further purified by removing the polyhedral protein as it is or by combining chemical or enzymatic methods depending on the purpose. It may be used after purification.

Specifically, the method for removing polyhedral proteins is to use a proteolytic enzyme that recognizes the amino acid sequence of the peptide part that connects polyhedral proteins and p21, cut that part, and then use gel filtration to detect the difference in molecular weight. It is best to purify using the purification means used.

〔Effect of the invention〕

The recombinant vector of the present invention is useful for preparing a recombinant virus that efficiently produces a polypeptide that exhibits good antigenicity against antibodies of H. It is something.

Furthermore, polypeptides obtained by infecting established cultured cells of silkworms with recombinant viruses are useful for creating vaccines against ATL and as antigens for ATL diagnostic agents. For example, as an example of application to an ATL diagnostic agent, an antigen is attached to latex particles, the particles are reacted with a test serum in a microtiter plate, and HTLV-1 antibody positivity and negativity are determined by agglutination and non-aggregation of the particles. Examples include the manner in which the determination is made.

[Example] Example 1 (Obtaining DNA derived from IITLV-I env protein gene) Lymphocytes were separated from ATL injured peripheral blood 10- according to a known method, and the lymphocytes were treated with proteinase K.
(PO390 manufactured by Sigma), then phenol.
Chloroform extraction and ethanol precipitation were carried out to obtain 1.5 ml of lymphocyte DNA from an ATL patient.

0 μg of the DNA was added to E in a solution having the composition shown in Table 4.
Cut with coRr restriction enzyme (N111040 manufactured by Zenshuzo ■), precipitate with ethanol, and add 200μ of TE buffer! dissolved in.

Then, the solution was subjected to sucrose density gradient centrifugation (sucrose 10-4
0%-t/vol, 26,000 rpm, 18 hours), and a DNA fragment corresponding to 20 kilobase pairs was obtained as confirmed by agarose gel electrophoresis. Next, this DNA
1.0 μg of the fragment was transferred to Sharon 4A vector (vector DN
A, Yoshiyuki Mikami, Kodansha 1986) was connected to the IEcoRI restriction enzyme cleavage site, and the DNA fragment was inserted into the EcoRI cleavage site present in the Sharon 4A vector. For this connection, use T4 DNA ligase (Zen Shuzo No.
, 2011), and the connection reaction was carried out at 15°C for 112 hours in a solution having the composition shown in Table 5. Next, the obtained DNA was subjected to in vitro pancasing, and the treated solution was centrifuged (7000 rpm,
2 hours), and the supernatant was used as a recombinant phage solution for plaque hybridization. After infecting indicator bacteria LE392 (manufactured by Zenshuzo ■) with the knee recombinant phage solution and forming plaques, HTLV-1pol was infected with 32P.
Plaque hybridization was performed using the DNA-containing fragment as a probe, and +1T1. A phage containing DNA from the V-1 gene was isolated. The obtained recombinant phage was incubated with Hin in a solution having the composition shown in Table 4, No.
dlII (manufactured by Zenshuzo ■), EcoRI (manufactured by Zenshuzo ■)
m1040) was cleaved with a restriction enzyme to obtain 400 μg of a DNA fragment of approximately 3.9 kilobase pairs, which corresponds to the env-px-LTR region of the DNA derived from the +1TLV-1 gene and contains DNA encoding ρ21. And vector pUc19 for Escherichia coli (Full 3219, manufactured by Zenshuzo ■)
was cleaved with old nd [[I + EcoR1 restriction enzyme under similar conditions, and the above DNA fragment was ligated to the cleavage site. The obtained connecting reaction solution was prepared by H
Ba5ll l-Ba containing the TL sea 15'-3' fragment
Similarly to the ligation reaction solution of m+ old fragment DNA and pUc19, Escherichia coli JM109 (manufactured by Zenshuzo ■, No. 9052) was used.
) was used for a series of operations, including transformation, mini-prevention, and medium-preparation. As a result of the above operations, HTLV-1env-px-LT
A transgenic vector in which a DNA fragment derived from the R region gene is inserted into plJc19 in the correct direction P) IT 3
．． 400 μg of 9Kb/pUC19 was obtained.

To the above recombination') TarI'llT 3.9 to +3/p
Uc19200 u g was added to Pstr (manufactured by Takara Shuzo Co., Ltd., No. 107, in a solution having the composition shown in Table 4, 3).
3), Hind [I (manufactured by Zenshuzo ■, No. 1060)
to obtain the Hindllr-Pstr fragment (approximately 1700 base pairs).

On the other hand, the Escherichia coli vector puc19 was incubated at 5ails (Nci manufactured by Zenshuzo ■) in a solution having the composition shown in Table 4 No. 1.
1080), treated with mung bean nuclease (N[12420, manufactured by Zenshuzo ■), and ligated to obtain pUc19 without A c c I +5all, ) I inc II restriction enzyme cleavage site. Next, the plJc
19 in a solution with the composition shown in Table 4 Nα2.
+ PstI to cut the old ndl of the vector.
l, the above old nd III-Pstl at the Pstr cleavage site
Connected the pieces.

Next, the ligation reaction solution was used for a series of operations including transformation of E. coli JM109 and miniprevention.
d 400 μg of recombinant pUc19 hector in which the I[I-Pstl fragment was inserted into the pUc19 vector in the correct orientation was obtained. Furthermore, the recombinant pUc19 vector was added to Acal (Zen Shuzo ■) in a solution having the composition shown in Table 4 Nα2.
Manufactured NO.

fool), Xbal (manufactured by Zenshuzo ■, No. 1093
) and cut Accl corresponding to the latter half of the DNA derived from the IITL sea 1 env protein gene.
-Xbal fragment (approximately 1060 base pairs) was obtained.

On the other hand, m call vector PIT 3.9 and b/pUc19
．． 200 μg was added to Nc in a solution having the composition shown in Table 4, Floor 5.
The Ncol-Ncol fragment (approximately 610 base pairs) was obtained by cleaving using the oI restriction enzyme (Takara Shuzo A-Kasei No. 1160). On the other hand, Escherichia coli vector pUc18 (No. 3218, manufactured by Zenshuzo ■) was added to linell restriction enzyme (Na, manufactured by Zenshuzo ■) in a solution with the composition shown in Table 4, No. 3.
1059), and the above Ncol-Ncol fragment was ligated to the Hinall restriction enzyme site of the vector by fill-in ligation. The ligation reaction mixture was then subjected to a series of operations including transformation of E. coli JM 109, mini-prevention, and medium pre-preparation to ensure that the Ncol[-Ncoll fragment was inserted into the above pHc18 vector in the correct orientation. 400 μg of recombinant pUc18 vector was obtained. The vector was mixed with Xba[, Acc
Xba I-^ which corresponds to the first half of the DNA derived from the 11TLV-1env protein gene by cutting with the r restriction enzyme.
A ccl fragment (approximately 500 base pairs) was obtained.

Next, pUcl 19 (manufactured by Zenshuzo ■, No. 3319), which is an E. coli vector, was added to Xbal restriction enzyme (Na10, manufactured by Zenshuzo ■) in a solution having the composition shown in Table 4, N0.6.
93) and the obtained vector and the above AccL
Connections were made with the Xbal fragment and the Xbal-Accl fragment. Then, the ligation reaction solution was used for a series of operations including transformation of E. coli JM109, mini-prevalence, and medium-prevalence.
-1env protein gene-derived DNA is pUcl 19
Dark blue vector e inserted into the vector in the correct direction
200 μg of nv/pUc119 was obtained. The above steps are shown in FIG.

(Production of recombinant vector) Recombinant vector env/pUc1 obtained by the above method
19.200 μg was dissolved in a solution with the composition shown in Table 4 Na6, and then BamHI restriction enzyme (Zen Shuzo ■ NO,
1010) was added intermittently for 9 hours to carry out a cleavage reaction. After cleavage, phenol extraction and ethanol precipitation were performed sequentially, and the precipitated DNA was added to TE buffer (pH 8).
, 0), and the DNA solution was subjected to agarose gel electrophoresis. Then, an agar piece of the band corresponding to the Bawl I-Bao+old fragment containing the HTI, V-15'-3' fragment was cut out, and the fragment was extracted by electrophoretic elution. Next, the extract is further extracted with phenol,
A BamH-BamHI fragment containing the IITLV-15''-3'' fragment was obtained by ethanol precipitation.

On the other hand, E. coli vector pUc1910μg @Ba+
It was cut with 5lll restriction enzyme under the same cutting conditions as above. The resulting reaction solution was then mixed with 1 u of alkaline phosphatase suspension (No. 2120, manufactured by Zenshuzo ■).
After stopping the reaction with alkaline phosphatase, the reaction solution was extracted with phenol and precipitated with ethanol.
I got a 9.

The Bae+HI-cleaved p thus obtained
Hc190.2 ug and the HTLV-I 5'-3'
0.25 μg of the BamH-BaIIHI fragment containing the fragment was mixed, and a ligation reaction was carried out at 16° C. for 3 hours or more using T4 DNA ligase in a solution having the composition shown in Table 5.

Then, 25 μl of the connection reaction solution obtained by this operation was added to 200 μl of a competent cell suspension of E. coli JM109, and the mixture was left on ice for 30 minutes. Thereafter, heat shock was performed at 42°C for 2 minutes, and after the mixture was returned to room temperature, 800 μl of LB liquid medium was added and cultured with gentle shaking at 37°C for 1 hour.

100μ2 of the liquid medium was mixed with ampicillin lOOμg/−
After inoculating 15 ml/plate of LB agar medium containing
Cultured at °C for 12 hours. Take out 20 single colonies that appeared after culturing, and treat each with 30μ of ampicillin.
The cells were inoculated into LB liquid medium 15- containing g/@l and cultured at 37°C for 8 hours. One sample was collected from each liquid medium, and the plasmid present in E. coli in each collection medium was extracted by the mini-preview method. Each of the obtained plasmids was subjected to a cleavage reaction using KpnT restriction enzyme (k1068, manufactured by Zenshuzo ■) in a solution having the composition shown in Table 4, No. 4. After the reaction, each reaction solution was subjected to agarose gel electrophoresis, and B containing the HTLV-15'-3' fragment was
A plasmid in which the am1l I-Ban old fragment was inserted into pUcl9 in the correct direction was confirmed.

0.2111/ was collected from the liquid medium containing E. coli possessing this confirmed plasmid, inoculated into LB liquid medium 50@1 containing 100 μg/d of ampicillin, and cultured at 37° C. for 12 hours.

The plasmid present in E. coli in the obtained liquid medium was extracted by the medium precipitation method, and the recombinant Hector Bam old Env(600)/pUc19400
μg was obtained.

Knee recombinant vector Bam1lXEnv(600)/pL
200 μg of lc19 was dissolved in a solution having the composition shown in Table 4, Na6, and then Xbar restriction enzyme was added intermittently for 9 hours to perform a cleavage reaction. By subjecting the obtained cut product to agarose gel electrophoresis, 11 liters of V-15'
-3” fragment (approximately 600 b
p) Obtained 0.15 ug.

Further, 133.10 μg of the silkworm expression vector ρBP was cut with Xba I restriction enzyme under the same cutting conditions as above, and then treated with alkaline phosphatase.

Xbal-X containing the above +1TL sea 15'-3'' fragment
1.25 μg of the balDNA fragment and 0.2 ug of p8F1333 cut with Xbalte were mixed, and a ligation reaction was performed using T4 DNA ligase in the same manner as described above.

Then, E. coli JM109 was transformed using this ligation reaction solution and the transformed bacteria was isolated by the same method as described above. A series of mini-prevention operations were then performed for each isolated culture, and plasmids were extracted from each liquid medium.

Next, each plasmid was subjected to a cleavage reaction with BaaHI, and agarose gel electrophoresis revealed that HTLV
-15'-3' fragment containing Xbar-Xbal D N
A plasmid in which A was inserted into pBF133 in the correct direction was confirmed.

0.2 m of the liquid medium containing E. coli possessing this confirmed plasmid was collected and treated with 30 ampicillin.
After inoculating 50 m/B liquid medium containing μg/wI, 37
It was incubated at °C for 12 hours.

BS? The plasmid present in E. coli in the f1 body culture medium was extracted by the medium precipitation method, and the recombinant vector Ba5iFlr Env(600)/pBF was extracted.
13320 Qug was obtained. This recombinant vector BamH
T Env(600)/pBF133 was introduced into E.coli, and the resulting microorganism was transformed into [i.coli HTLVl
-Envl was deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology. The deposit number is FRE deposit number 10072 (FRE
MP 10072). The above steps are shown in FIG.

Reference Example 1 Production of Recombinant Virus Composition solution 12 in Table 1 in which the viral DNA of BsNPV T 3 strain and the recombinant vector BamHI Env (600)/pBF133 were mixed at a molar ratio of 1:100.
45μ2 was mixed with 255μ2 of composition liquid (1) shown in Table 1.

(Margins below this page) Table 1 0.5-ml of the resulting suspension was mixed with 8 mNd solution (4 x 105 Biece) of cultured silkworm cells in TC-10 (Table 2) medium.
Ba1HrEnv(600)/pBF133 was added to 5ml/d) and cultured at 27"C for 20 hours.
and B+aNPV DNA was introduced into established silkworm cells. The obtained culture was further cultured at 27°C for 6 days after exchanging the TC10 medium. This culture was then centrifuged (1500 rpm, 10 minutes), and the resulting supernatant was used as a reaction solution for cloning the recombinant virus.

The cloning reaction solution was diluted with lo-61 in TC-10 medium.
The mixture was diluted to 0-' and 10-' to obtain 10-' diluted reaction solutions. For the diluted reaction solution, 8 mNd solution of established cultured silkworm cells (105 Bamcells/l1
if) Mix 10@l, dispense the mixed solution into 96-sized microtiter trays in 200 μi portions, and
Cultured at °C for 4 days. After culturing for 4 days, the microtiter tray was examined under a microscope, and wells were found that were established cultured silkworm cells in which the cell surface was rough and deformed, indicating a virus-infected morphology, and in which no polyhedral protein was detected within the cells;
Cultures were collected from there. The resulting culture was centrifuged (1500 rpm, 10 minutes), and 150 μffi of the supernatant was
was used as a reaction solution for expressing a recombinant viral polypeptide.

The polypeptide expression reaction solution was tested by plaque assay with IX
It was a recombinant virus solution showing a titer of IO' PFU/a1. Using this polypeptide expression reaction solution, silkworm established cells BmN4 were infected with the recombinant virus and cultured to propagate the knee recombinant virus. This recombinant virus propagation procedure involves centrifuging the culture! (1500rp
The procedure was repeated until the supernatant 401d obtained by the test (10 minutes) had a titer of 3 XIO'' PFtl/d in the plaque assay.
% (-8) sucrose aqueous solution (5 tel) and ultracentrifuged it (2500 Orpm, 15 °C, 2
time), and the recombinant virus was precipitated as virus particles. After washing the virus particles with distilled water, TE
The virus particles were dissolved in 200 μl of buffer (pH 7.5) to obtain a suspension of virus particles. The suspension was further treated with protease in the presence of SOS, treated with phenol, treated with phenol/chloroform, and treated with chloroform/isoamyl alcohol (50:1) to obtain 50 gg of virus I) NA.

The medium for the above TC-10 is the medium 900 relative in Table 2 at pH 6.
Adjust to ℃30~6.35, sterilize by filtration, add fetal bovine serum 1
00m! It is prepared by adding.

(Materials below the true text) KO1EshikoL -Arginme L-Aspatic acid L-Asparagine -H20L -Alan
ine β-8 anine L-Glutamic acid L-Glutamine Glycine L -Hlstidine L 4soleucine L -Leucine L-Lysine -HCI L -Methiouine L-Proline L -Phenyla anine DL-5erune L -Threonme 5.79g 3.5g 3 .98g 2, 25g 2.0g 6, Oc 3, Og 6.5g 25.0g 0.5g 0.75g 6.25g 0.5g 3.5g 1.5g 11.0g 1.75g Make a total of 11000Ml with H2O 2nd Cap-"L" L-Cl aCI Cl CaC1z ・ 2HzO MgC12 ・ 6H20 MgC1z ・ 7H20 Tryptose Dextrose (glucose) L-glutamine soln A" 5oln B'"1 so1nC* *11 NaH2PO4・28zO (0,891g/100Id ) NaHCO.

(0,35g/100d) 0.5g 2.87g 1.32g 2.28g 2,78g 2.0g 1.1g 0.3g 100 relative00rnl d 00d Gate widening work with a total amount of 900- at 100HzO J shoulder 1 night L-Cystine L-Tryptophane L -Tyros 1ne HzO to make total amount 1000d 0, 25g 1.0g 0.5g ↓Upper coin top rainbow m Th1aa+ine -HCI Riboflavine D-Ca pantoth enatePrydoxi
ne -HCI Para-aminobenzoic acid Fo
lic acid Nicotinic acid Iso-Iuositol 1otin 2.0mg 2, O, mg 2.0■ 2.0■ 2.0■ 2.0■ 2.0■ 2.0mg 1.0■ Total amount 10001d with H2O! Reference Example 2 Production of polypeptide 100 μl of the reaction solution for polypeptide expression obtained in Reference Example 1
ffi was added to silkworm cultured cell BaN4 solution (10J3a+ce
lls/m) for 30 days and cultured at 27°C for 5 days. After culturing for 5 days, the culture was collected and centrifuged at M (15
00 rpm, 15 minutes).

Wash the precipitate (virus mature cells) with PBS buffer 5.
Axis M Tris-HCI (pH7,4) 400ul
After suspension and sonication, centrifugation (8000 rpm)
+, 20 minutes). The polypeptide obtained as a precipitate was suspended in 200 uffi of Laemmli buffer, boiled and centrifuged, and the supernatant was used as a sample for SDS gel electrophoresis.

As a result of SDS gel electrophoresis, this sample was pBF133
A part of the polyhedron protein (approximately 5 Kd) encoded by the polyhedron protein gene portion possessed by
A hand was detected at a position of approximately 25 Kd, which corresponds to the total molecular weight of d) (as shown in FIG. 4). In addition, FIG. 4 shows B+i
Intracellular tlTVL-1enν protein expression was confirmed by SOS gel electrophoresis. 1an in Figure 4
e 1 is a size marker, 1ane 2 is a protein of uninfected silkworm cells, 1ane 3 is F3araHI Env (0
, 6)/pBF133, proteins from silkworm cells infected with the recombinant virus were electrophoresed.

Anti-polyhedra protein antibody and IITLV-1p as primary antibodies
Western blot experiments using a monoclonal antibody against 21 showed that the 25Kd protein was isolated from the polyhedral protein and HT.
It was confirmed that it was a fusion protein containing LV4 p21. At the same time, as a primary antibody, normal human emotion or ATL
Perform Western blot experiments using patient serum;
It was confirmed that the 25Kd protein had good antigenicity to the IITLV-1 antibody (as shown in FIG. 5). In addition, FIG. 5 shows HTLV-1en in B-cells.
v protein expression was confirmed by Western flot method. 1ane 1 is a size marker, 1ane 2~
1ane 5 is a flow old Env (600) as an antigen
/pBF 133 is an electrophoresed protein of silkworm cells infected with a recombinant virus.

Western plotting was carried out using the primary and secondary antibodies shown in Table 3 and a color reaction with peroxidase using avidin and biotin as substrates.

Reference example 3 () shows the dilution rate 100 μff of the polypeptide expression reaction solution obtained in reference example 1
i to silkworm established culture cell solution (10'B+++cells
/@1) and cultured at 27°C for 5 days. After culturing for 5 days, the culture was collected and centrifuged (1500r
pm, 15 minutes). The supernatant was injected subcutaneously into 100 silkworms of 5 instars and 1 day old at a dose of 0.1-0.
After being reared for 5 days with paste pieces of the complete collection, the animals were dissected.
Fat bodies were collected. The fat body was suspended in 10 wkl of PBS buffer, sonicated, and then centrifuged (8000 rpm).
pm, 20 minutes), and a precipitate 5- was obtained. 200 μ2 of the precipitate was sonicated again, and the protein amount was measured by Bio-RAD protein assay. The result was 60
It was μg.

After measuring the amount of fluorescence, 50 μ2 of the suspension was suspended in 50 μ2 of Laemmli buffer, boiled, and centrifuged. The supernatant was used as a sample for SDS gel electrophoresis.

As a result of SDS electrophoresis, a band was detected at the molecular position of 25Kd.
In 5, the protein d is a polyhedral protein, flTl, and V-r p21.
It was confirmed that the fusion protein contains the following and has good antigenicity against IITLV-I antibodies.

(Margins below this page) No. table table 1mM ＾TP

[Brief explanation of the drawing]

Figure 1 is a restriction enzyme map of the DNA of silkworm nuclear polyhedrosis virus, Figure 2 is a restriction enzyme map of pBF vector, Figure 3 is the type of pBF vector, and Figure 4 is the restriction enzyme map of the DNA of Bombyx morivirus.
Figure 5 shows the expression of TLV protein confirmed by SOS gel electrophoresis, Figure 5 shows the expression of HTLV-1 env protein in B1 cells confirmed by Western blotting, and Figure 6 shows the results of silkworm nuclear polyhedrosis virus and bacterial infection. Silkworm cells infected with the recombinant virus, Figure 7 shows the method for preparing DNA derived from the ITLV-1env protein gene, Figure 8 shows the method for preparing the recombinant vector, and Figure 9 shows the method for preparing DNA derived from the HTLV-1env gene sequence. The gene and amino acid sequence diagram of the p21 protein portion of . 11dl[I...former ndln, N・”Nco I
, ^c-Acc I, Ec・”EcoRI, 5p
-5ph I, 5a=-5alI, old 11...
H4ncB ”・BamHT % X・!・xbal Drawing engraving Figure 4

Claims (2)

[Claims] (1) The silkworm expression system vector contains a p21-encoding DNA derived from the adult T-cell leukemia virus coat protein gene, and is a fragment cleaved within 17 base pairs upstream from the 5' end of the DNA. Recombinant vector recombined with. (2) The recombinant vector according to claim 1, wherein the silkworm expression system vector is a pBF vector.