JPH0279980A - Human papilloma virus 52b gene - Google Patents

Abstract

PURPOSE:To obtain human papilloma virus 52b gene having a specific restriction enzyme cleavage map among genes of human papilloma virus and useful in diagnosis of cervical carcinoma. CONSTITUTION:A cyclic gene being a gene of new type human papilloma virus separated in 52nd position from cervical carcinoma and containing one XhaI cleaving site, two BamHI cleaving sites, three Bg1II cleaving sites, two EcoRI cleaving sites, one EcoRV cleaving site, two KpnI cleaving sites, six PstI cleaving sites, two PvuII cleaving sites, one SalI cleaving sites and one SmaI cleaving site and having about 8kbp length. The aimed gene has preferably restriction enzyme cleavage map shown in the right figure.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to human papillomavirus type 52b (hereinafter referred to as HPV).
52b).

This gene is expected to be particularly useful for the diagnosis of cervical cancer in clinical obstetrics and gynecology.

[Conventional technology]

Papillomaviruses have been isolated from humans and various other animals as a virus that causes benign tumors on the skin and mucous membranes. Human papillomavirus isolated from humans (
HP V) is one of the few viruses, along with EB virus, hepatitis B virus, and adult T-cell leukemia virus, that are thought to be closely related to human cancer.

Typing of human papillomaviruses that host humans is based on the degree of complementarity of the viral DNA (high stringency).
A separate type of hybridization with a hybridization efficiency of 50% or less under stringent conditions (1, Coggin et al. Cancer Res,
39+545 (1979)), and currently more than 50 types are known.

Human cysts have been clinically classified based on their site of occurrence, morphology, Mi tissue image, etc. On the other hand, HPV typing has been carried out, and the relationship between each type and clinical image has been clarified. IIPV 1 is isolated from plantar subsidence.
, and HPV4, and from P. vulgaris) IPV 2
, and HPV 1 were isolated. epidermodysplasia verruc
i-formis; EV), various types of HP V
) More than 20 types of HP in addition to HPV5 have been obtained.
There are reports of V.

Genital warts (condyloma acumi)
natum) contains 1lPV6, and 1(
PVII, and HPV16, which was cloned from cervical cancer.

+1PV1B, HPV31, HPV33. HP
There are V35 etc. Epidemiological studies have shown that HPV DNA is found at a high frequency in cervical cancer, and in Europe and America,
PV 16 is 60-90% [mouth.

J, MaCance et al. Br, J, 0bstet, Gy
naecol, 92+ 1101 (1986);
M. Durst et al. Proc. Natl. Acad. Sc.
i, IJSA80, 3812 (1983)), HPV
It has also been reported that 18% is 15-36% (Boshart et al. EMBOJ, Yu, 1151 (1984)). Also HPV31. 1 (PV33, and 1lPV35
When adding this, the HPV positivity rate for cervical cancer is 90%.
It is said that it can be used as a stand. The entire base sequence of some of the HPV DNA has been determined (IIPVI,
HPV5.

11PV6, IPVB, HPVII, HP
V16, HPV18, and 11PV33).

[Problem to be solved by the invention]

In recent years, reports have been made suggesting that human papillomavirus (HPV) is closely related to cervical cancer.

Currently, there are over 50 types of HPV, but
Among them, 1 (PV16, 1B.

31, 33, and 35 are commonly found in cervical cancer tissue. According to reports from Europe and America, these types of HPV make up the majority, but the results are not necessarily the same in Japan.

HPV31, 33, and 35 have hardly been investigated, but HPV16 and 18 have only been reported in about 20-30%. By the way, there are reports that these two types account for almost 70% of cases in West Germany. It was predicted that a new type of HPV might exist in Japan. In clinical obstetrics and gynecology, known types, especially types 16 and 18, are used as diagnostic materials, and other types of HP are used in the diagnosis.
V may be overlooked.

Therefore, the present invention provides the gene of a new type of human papillomavirus that is closely related to cervical cancer, thereby contributing to the diagnosis of cervical cancer with a high probability.

[Means to solve the problem]

The present inventors have developed a new type of HP from cervical cancer tissue.
An attempt was made to isolate V, and the isolated gene was combined with the known H
As a result of comparison with PV, the Reference Center forllum was identified as the 52nd HPV isolated.
an Pathogenic Papilomavi
A new HPV named HPV52b was obtained from H. rus.

Therefore, the present invention provides the HPV52b gene and its parts,
The present invention also provides vectors containing these.

[Specific explanation]

In the present invention, cancer tissues were obtained from four patients with cervical cancer, and viral DNA was extracted from them. These DNAs were digested with restriction enzymes EcoRI or 1IindlI.
After digestion with I, high stringet
nt) conditions (Tm -10°C) with human papillomavirus HPV16 DNA as a probe. As a result, DNA from one specimen hybridized with the probe, and a band appeared at the same position as HPV16 DNA.

The results suggested that the DNA from this specimen was identical to that of liver V16. Furthermore, the restriction enzyme-digested DNA sample was subjected to low stringency conditions (Tm
When hybridized with the probe at -37°C), in addition to the positive DNA, two other DNA samples hybridized with the probe, and these showed bands at different positions from the former. Therefore, the latter two types of DNA
The sample was suggested to contain human papillomavirus DNA, which is different from IPV16 DNA.

Next, when these two types of DNA were digested with various restriction enzymes and their digestion patterns were compared, it was suggested that they contained the same viral DNA. Therefore,
One of these DNA samples was digested with Xba I and the Xba I fragment was digested with Charomid 9-36.
Vector DNA (1,5aito and G,R,5
tark, Proc, Natl, Acad, Sci,
USA 83.8664 (1986)) and HPV under low stringency conditions (Tm -37°C).
After selection by hybridization with the DNA of plasmid plJc19, a 7.9 Kb insert was excised from the positive clone with Xba I, and the Xba of plasmid plJc19 was
I cleavage site.

Next, this Xba I DNA fragment was cut with various restriction enzymes to create a restriction map, and the results shown in Figure 4 were obtained.This result was compared with the known restriction map of human papillomavirus DNA. It was confirmed that this DNA was a novel human papillomavirus.

Therefore, the human papillomavirus gene of the present invention is
In one embodiment, one Xba I cleavage site, two BamHI cleavage sites, three BglI cleavage sites,
2 EcoRI cleavage sites, 1 EcoRV cleavage site, 2 Kpn I cleavage sites, 6 PstI cleavage sites, 2 PvU cleavage sites, 1 5all cleavage site,
and one 5and I cleavage site, approximately 8 Kbp
It is a circular gene with a length of .

However, human papillomaviruses generally exist in a state where they are integrated into the host animal's chromosomes as a linear gene produced by cutting a circular virus gene at one location, and the present invention is directed to It also includes linear genes. Furthermore, in order to use for diagnosis, it is possible to use a linear gene in which a circular gene is cut at any one position, and also gene fragments produced by cutting a circular gene in multiple positions. can be used. Therefore, the present invention also encompasses linear genes and gene portions produced by cutting the circular gene at one or more arbitrary positions. The cleavage site is one of the various restriction enzyme cleavage sites listed above.
One location or a combination of multiple locations can be used.

Furthermore, the human papillomavirus genes of the present invention are not limited to those defined by the above characteristics. In other words, taking into account that viruses tend to mutate naturally,
It is distinguished from other viruses based on the degree of complementarity of its DNA. That is, a virus with a hybridization efficiency of 50% or less under high hybridization conditions is defined as a different type of virus. Therefore, DNAs that hybridize under high stringency conditions with the genes characterized as described above are included within the scope of the genes of the present invention. The present invention therefore covers, in addition to the previously defined viral genes, naturally mutated viral genes which hybridize under high stringency conditions with the previously defined genes, as well as artificially mutated or chemically synthesized viral genes. Also within the scope of the present invention are DNA fragments that hybridize with the previously defined genes under high stringency conditions.

Next, the method for cloning the human papillomavirus gene of the present invention will be specifically explained with reference to Examples.

1. Four patients with viral DNA spread and cervical cancer (T, H, M,
.

Cancer tissues were collected from patients (F, H, and S, N), and DNA was extracted from the cancer tissues. A little finger-sized section was cut from the cancer tissue, and while it was soaked in PBS (phosphate buffer), it was cut into 0.5 m pieces with a razor blade. PBS was added for washing and centrifugation was performed twice for 10 minutes at 11000 rpm.

5-10ml 10mM Tris-HC per g
l (pH 7.8), 150mM NaCl, 2
mM EDTA, 0.5% SDS.

The mixture was shaken overnight at 37°C with 400 n/ml pronase (Boehringer Mannheim). Phenol extraction 1
After 3 times of phenol/chloroform extraction and 2 times of chloroform extraction, TE buffer (10mM Tr
is-HCl (pt17.5)/1mM ED
TA) for 24 hours. 100 by the above operation
DNA of tzg-2■ was obtained.

Each of these DNAs (T, H, , M, ,
F, H, and S, N,) 100 for 10Ig
The restriction enzyme EcoRI or old ndl [l] was added to the mixture and the mixture was digested overnight in a total volume of 400 pl. Another 1■/-
RNaseA (manufactured by Boehringer Mannheim) 8
I and 50 units of the above-mentioned restriction enzyme were added, and after several hours, ethanol precipitation was performed.

This precipitate was dissolved in 14m TE, 4111 running buffer was added, and electrophoresis was performed on an agarose gel.

Agarose gel is Seachem Agarose (manufactured by Takara Shuzo Co., Ltd.)
was used at 0.6%. After electrophoresis, the gel is
．． After slow shaking for 10-12 min in 25 M 11cI solution (1), 0.2 M NaOH/0.6
Rinse 1-2 times with M NaCl solution (2). The gel was then slowly shaken in the solution (2) for 30 minutes, and 0.2M Tris-HCI (pH 7.4
)/0.6M NaCl buffer (3) for 1-2 rinses. Finally, the gel was immersed in the buffer solution (3) twice for 30 minutes each while changing the buffer solution.

Next, the DNA was immobilized on the filter by subjecting it to a nitrocellulose filter overnight and heating at 80° C. for 2 hours.

Next, the DNA was transformed into human papillomavirus 11PV16 DNA labeled by two-fold translation.
(probe) and hybridized with (Rightb
y et al., J. Mo1. Biol, 113.237 (1
977)]. Hybridization was performed under high and low stringent conditions. As a control, DNA extracted from lymphocytes of human adult T-cell leukemia (ATL) patients was also treated in the same manner.

Hybridization under high stringency conditions (Tm -10°C) was carried out at 42°C in 50% formamide, 5x Denhardt's solution, 10% in 5x SSC buffer.
Dextran sulfate, 50mM Tris II
cI (pH 7,4), 0.1% SO5, and 100
The reaction was carried out in a solution of g/- calf thymus DNA or salmon sperm DNA. Thereafter, it was washed with 0.2x SSC buffer and 0.1% SOS at 68°C. The washed filter was exposed to an XvA film (X-Omat AR, Kodak) at -80° C. and subjected to autoradiography along with an intensifying screen.

The results are shown in Figure 1. In this figure, lane 2.4゜6.8
and 10 were digested with the restriction enzyme EcoR [,
and lanes 3.5, 7.9 and 11 are restriction enzyme old nd
It was digested with ul. Lane 1 shows the results for HPV16 DNA used as a probe.

The position of the fragment (molecular weight standard) generated by digesting the λ phage DNA with the restriction enzyme old ndllll is shown on the left. As a result, it was revealed that (7) DNA samples from patients S and N contained DNA of IPV16.

Next, the above filter was 0.01xSSC, 10%S
Washed with O3 at 95°C for 30 minutes to remove radioactive DNA. Next, to perform hybridization under low stringency conditions (Tm 37°C) using the same probe as above, the filter was heated at 41°C for 1. OM NaCl, 28% formamide, 50I
IM TES [N-)lis(hydroxymethyl)-
Methyl-2-aminoethanesulfonic acid, 10x Denhardt's solution, 0.5mM EDTA, 20mM sodium phosphate (pH 7,4), 100g/ml tR
Incubated in a solution of NA and 100 n/- salmon sperm DNA. Thereafter, the plate was washed with 1.1×SSC buffer 0, 1 mM EDTA, 10 mM sodium phosphate, and 0.1% SDS at 52°C. Next, autoradiography was performed in the same manner as above. The results are shown in FIG. As a result, D from the above S, N,
In addition to NA, DNA samples from M, and T, H, also hybridized with IPV16 DNA. From this, it was deduced that the DNA samples from MJ, T, and H (7) contained viral genes that were not identical to HPV16 DNA, but had some degree of homology thereto.

Next, 10 pieces of DNA from each of the positive samples M, T, and H were digested with various restriction enzymes, separated by electrophoresis, and using the DNA of III''V16 as a probe. Stringet (Tm -37℃)
Southern blotting was performed. The results are shown in FIG. In this figure, lanes 2 and 10 are labeled with the restriction enzyme BamHI.
lane 3 by EcoRT; lanes 4 and 1
1 by old ndI[I; lane 5 by PstI;
Lane 6 with Sac I; lanes 7 and 12 with Xb
a with I; lane 8 with EcoRI + BamHI; and lane 9 with EcoRI + HindI[I
The results of digestion are shown below. DNA from M, to, and T,) 1. DNA from BamHI, HindI
Since they showed the same pattern when digested with [[ and Xbal, it was presumed that these DNAs contained the same viral DNA.

2. Loning of viral DNA 60x DNA from the positive sample M was digested with restriction enzyme Xba 1 and then electrophoresed on a 0.6% agarose gel. The part of the agarose gel corresponding to the position of the 7.9 Kb linear DNA was cut out and
DNA was extracted using an e clean kit (manufactured by Bio 101). The extracted DNA was treated with restriction enzyme Xba
Charomid 9-36 DNA digested with I
(Sait.

et Proc, Natl, Acad, Sci, USA 8
3.8664 (1986)) for 8 hours. The ligated DNA was packaged into lambda phage using an in vitro packaging kit (manufactured by Amajam) and introduced into E. coli DHI strain. Infected E. coli were grown on nitrocellulose membranes on agar medium containing 50 n/ml ampicillin. 4×10
h colonies were obtained. The method of Hanahan et al. (G
Colony hybridization was performed according to the method of EnelO, 63 (1980)), and 23 colonies were obtained that hybridized with liver V16 DNA under low stringency conditions. All six colonies picked showed the same results in analysis using several restriction enzymes. 7. from one of those clones. lb
The inserted fragment was cut out using the restriction enzyme Xbal and pH
It was recloned into the Xba I recognition site of c19.

Furthermore, Escherichia coli E containing a plasmid containing this inserted fragment
scherichia coli JIB 10Hp19
) IPV52b') was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, Microbiological Research Institute No. 1o) g1 (FER1'I P-/
It has been deposited as υg7>.

Xbal 7.9 Kb cloned into pUc19 of 3
The fragment was digested with restriction enzymes BamHI, BglIT, E
coRI, EcoRV, KpnI.

Pst I, Pvu II, Sal I and Sm
A restriction enzyme map was created by digesting with aI alone or in combination of two types, and combining the obtained fragment lengths. Note that the restriction enzyme old ndll [, Sac I,
The recognition sites for Sph I and Xho I enzymes were not present in the Xba 1 fragment of 7.9b. The reaction conditions for each restriction enzyme are shown below. The reaction was carried out by adding 5 units of restriction enzyme to the reaction solution for ln DNA and incubating at 37°C for 2 hours.

BamHI 10+wM Tris-HCI (pH 8
,0), 7mM MgClg, 100mM NaCl,2
mM 2-Mercaptoethanol Bgl II
, 10mM Tris-11cI (pt17.5),
7mM MgC1z, 100mM NaC1, 7mM
2-Mercaptoethanol EcoRI 100
nM Tris-HCI (pH 7,5), 7mM
MgCh.

50mM NaCl, 7mM 2-mercaptoethanol EcoRV 10mM Tris-HCI
(pH 7,5), 7mM MgCIz, 150mN
aC1, 7mM 2-mercaptoethanol Hind
llr 10++M Tris-FICI (pH 7,
5), 7mM Mgc+, 6o mMaCI Kpn I 6mM Tris-HC
I (pH 7,5), 6mM MgCl,.

5mM 2-mercaptoethanol Pst I 20mM Tris-HCI
(pf+7.5), 10mM MgCIz, 100a
+M NaCI Pvu II 10mM Tris-HCI (
pH7.5), 7mM MgC1z, 60mN
aC! , 7mM 2-mercaptoethanol Sac
I 10mM Tris-HCI (pH 8,0
), 7mM MgC1z, 7mM 2-mercaptoethanol Sat I 10mM ↑ris-)Ic
I (pH 7,5),? ff1M MgCh+ 17
5mM NaC1,711IM 2-mercaptoethanol, 0.2n+M EDTASma I
10mM Tris-HCI (pH 8,0), 7m
M MgC1z, 20mM MCl, 7mM2
-Merkabutethanol Sph I 10mM
Tris-HCI (p) 18.0), 7mM MgCl
z, 150mM NaCl, 7mM 2-mercaptoethanol Xba I 10o+M Tris-H
CI (pH 7,5), 7n+M MgC1z, 100m
MNaCl, 7mM 2-mercabutoetha/-ICX
ho I 10mM Tris-HCI (pH 7
,5), 7mM MgC1z, 100mM NaC1,7
The results obtained in mM 2-mercaptoethanol were
As shown in the figure. Distances between restriction enzyme recognition sites are shown in kilobases. The HPV restriction enzyme map of the present invention is a known HPV restriction enzyme map.
P V (HPV6゜+1PV11.HPV16,1I
PV18. HPV31. and HPV35) (Boshart et al. EMBOJ,
3.

1151 (1984); Lorincz et al. J, Vi
rol, 58.255 (1986); Beauden
on et al. Nature 321.246 (1986)s
Lorincz et al.
of Cervical Cancer+ Bunbu
ryReport 2L 225+ New York
: Co1d Spring Harbor Labora
tory (1986); de Villiers et al. J, Virol.

Parallel, 932 (1981); Gissman et al. J, V
irol, 44.393 (1982); and Du
rst et al. J, gen, Virol, 66, 1515 (
(1985)), the novel human papillomavirus thus obtained was named HPV52b.

Contains the HPV52b gene, cloned into the Xba I cleavage site of the HPV52b vector pUc19. , 9Kb Xb
After digesting the a1 fragment with each of the above restriction enzymes, ptl
c118, the dideoxy method developed by Sanger et al. (Proc, Natl, Acad, S
ci, USA? C5463 (1977)).

The results are shown in Figures 5-1 to 5-5. Figures 5-1 and 5-2 show 2. in the restriction map shown in Figure 4. I
Figures 5-3 to 5-5 show the nucleotide sequence of the XbaI-BamHI fragment of Kbp, and Figures 5-3 to 5-5 show the restriction map shown in Figure 4,
The base sequence of 3.2 bp from the Kpn+ I site on the left to the Pvo It site on the right is shown.

〔Effect of the invention〕

The present invention makes it possible to reliably identify HPV (HPV52b), which has not been able to be identified in cervical cancer until now. This is due to the previous 11P
This will provide valuable material for a diagnosis that will go beyond diagnosis based solely on the presence or absence of V DNA to distinguishing between types of HPV that can lead to cervical cancer.

[Brief explanation of the drawing]

Figure 1 shows D extracted from cancer tissue of a cervical cancer patient.
FIG. 5 is a 5outhern plot diagram when a DNA lilr fragment obtained by digesting NA with EcoRI or old ndI was detected under high stringet conditions using human papillomavirus 1IPV16 DNA as a probe. Figure 2 shows the first sample obtained under low stringet conditions.
FIG. FIG. 3 shows that the 5outherFn protein shown in FIG.
D derived from M, which is positive in n protecting
NA and T,)1. It is a Southern plot diagram when the derived DNA was digested with various restriction enzymes. Figure 4 shows human papillomavirus HPV52 of the present invention.
It is a restriction map of the gene DNA of b. Figures 5-1 to 5-5 show partial base sequences of the gene DNA of the human papillomavirus HPV52b of the present invention.

Claims (1)

[Claims] 1. Human papillomavirus gene, including one Xha I cleavage site, two BamH I cleavage sites, three BglII cleavage sites, and two EcoR I cleavage sites, 1.
EcoRV cleavage sites, 2 Kpn I cleavage sites,
6 Pst I cleavage sites, 2 Pvu II cleavage sites,
A circular gene containing one Sal I cleavage site and one Sma I cleavage site and having a length of approximately 8 Kbp, and a gene fragment produced by being cleaved at one or more positions. 2. The gene and its fragment according to claim 1, wherein the cleavage sites of the various restriction enzymes are represented by the restriction enzyme map shown in FIG. 3. The gene and its fragment according to claim 1 or 2, wherein the base sequence portion is as shown in FIG. 4. DNA that can hybridize with the gene according to any one of claims 1 to 3 under high stringet conditions. 5. A vector containing the gene according to any one of claims 1 to 3. 6. A vector containing the DNA fragment according to claim 4.