JPH074252B2 - DNA fragment used as a test agent for identifying human parainfluenza 4A virus - Google Patents

Description

TECHNICAL FIELD The present invention relates to a DNA fragment used for a test agent for identifying human parainfluenza 4A virus, more specifically, human parainfluenza 4A virus (hereinafter, PIV-4A). (Hereinafter abbreviated) hemagglutinin neuraminidase (hereinafter, abbreviated as HN) DNA fragment that is complementary to the gene RNA.

[Conventional technology and its problems]

Traditionally, viruses have been identified based on serological properties. The main methods include enzyme-linked immunosorbent assay method (hereinafter abbreviated as ELISA method), neutralization reaction method, complement fixation reaction method, hemagglutination inhibition reaction method, fluorescent antibody method, agar precipitation method, etc. However, in all of these methods, the determination is made by measuring the antibody against the virus in the sample, and in order to make a highly accurate determination, generally, the virus antibody titer increases one week after infection. Must be done at the beginning stage, and in some cases,
Furthermore, it is necessary to carry out remeasurement several weeks later for confirmation, and therefore there is a problem that pre-symptomatic and early diagnosis is difficult.

Usually, the ELISA method is used for the measurement of PIV-4A, this method is to measure the antibody against PIV-4A in the sample, for example, the plate on which PIV-4A is immobilized, the analyte After addition reaction and washing, anti-PIV-4A antibody is further added and reacted in the same manner, then enzyme-labeled antibody is added and reacted, and then color is developed.
This method also has a problem that it is difficult to perform pre-symptomatic and early diagnosis as in the above.

The present invention is intended to solve the above problems, and provides a DNA fragment useful as a test drug that enables early diagnosis of PIV-4A infection.

[Means for solving problems]

The present invention has the following [Expression], [formula]: GGGGAACACACTTCTCAGCCCTGATTGCTCAAGG CCCTTGCATGTGCAACCGAGACACCCCCCACAAGCACCGGAA TAAGACCTGACAACAAAGTAGCAGCCACCACGACCCAAAAAC AAAATTAAAAGGATCCGGTAACAGCCCATCAACCAGCAATCA TAGAATCCAACAATCCAGAGAGACGTCACATCAACTCATCCA CGAATCTTCGAAGGGAACATCCCAGACAAAATCACAGCCCAT TCCCTGATCACGGATAAACTGAGAAAGATCACAAGAATGCAA GACTCACATGGTAATACACAAATACTCAACCAGGCAAATTCA ATGGTGAAAAGAACATGGAGATTACTATTTCGAATTGCAACC TTAATATTACTTGTTTCAATATTTGTGTTATCGCTCATAATT GTATTACAGTCAACACCGGGGAATTTGCAAAACGATATCAAT ATAATTAGAAAGGAGCTCAATACAATTATGGAGAATTTTGAA ACTACATCTAAGTCACTGTTAAGTGTATCAAATCAAATCACT TACGATGTATCAGTACTTACTCCTATAAGACAAGAAGCTATT GAAACAAACATCATTTCAAAAATAAAAGATCATTGCAAAGAT AGAGTAATTAAAGAAGGAAGCACTTGCACATTGAATCGCAGC CCTTTGCATGATGTCTCTTTTTTAAATGGGTTCAATAAATTC TATTTCACATATAAAGATAATATGCAAATTAAGTTTAAATCA TTATTAGATTACCCCAATTTTATTCCAACTGCTACAACTCCC CACGGATGCATTCGAATTCCATCATTCTCCTTAGGTCAAACC CATTGGTGTTATACCCATAATATAAACCTACTAGGATGTGCA GACCCTGCATCTAGCAATCAATATGTATCACTAGGAACCTTA CAAGTCTTAAAAATGGGT GACCCTTATTTTAAAGTCGAGCAT AGTCATTATTTAAATGACGGGAGGAATCGAAAGAGTTGTTCA GTGGTTGCTGTCCCCGACGGATGCCTGCGGAATTGTGTGACC ATGACAAAAAATGAGACAGAGAATTTCAAAGACCTCAATTGG CAACACAATTACTTACATACATATCATATAATGGTACCATTA AAGACTCGTATAATAAATCCACCAGGATCATCCAGAGATTGG GTTCATATCGCACCAGGGGTAGGCTCGGGCCTTTTGTATGCC AAATTACTTATATTTCCTTTGTATGGGGGTCTCACGGAAAAA TCAGTGATACATAATAATCAATCAGGGAAATATTTTTTCCCT AATTCAACTAAATTGCAATGCCGTAACAGCACTATGGAAAAA ATAAAAGGAGCAAAAGATTCATACACAATAACTTACTTCTCA GGGAGACTTATACAGAGTGCATTTCTGGTTTGTGATCTAAGA CAATTTCTTTCTGAAGATTGTGAAATCTTAATTCCTAGTAAT GATTACATGATGGTCGGTGCAGAGGGTCGATTATATAACATT GAGAACAACATATTTTATTATCAGAGAGGATCCAGCTGGTGG CCTTATCCGAGCCTCTATAGAATCAGGTTAAACCTTAGTAAG AAATATCCTAGAATAACTGAAATTAAATTTACAAAAATTGAA ATCGCCCCAAGACCAGGCAACAAAGATTGTCCAGGAAATAAG GCTTGCCCAAAAGAATGTATAACGGGAGTCTACCAAGATATA TTGCCACTAAGTTATCCCAATACTGCATTTCCACACTTAAAA CAAGCGTATTATACAGGTTTTTATCTTAATAACTCGCTCGAG AGACGCAATCCAACATTTTATACTGCTGACAATCTAGATTAC CATCAACAGGAAAGATTAGGTAAATTCAATCTTACTGCTGGA TACTCTACTACAACTTGTTTTAAACAGAC CACTACTGCGAGG TTATACTGTCTCTACATAATTGAAGTGGGTGACTCAGTCATT GGGGACTTTCAGATCACCCTTTTTTTAGCAGCTTAATAGACC by AGACTGTTAATTAATCAACAAAGTTATTCTGTAATATAAACT GATCTTATAAGTGAAAAGATGCCTATCCAAGGAGGTTGATAG ACAAATAGTAAAAGTAGCAATTGTAACAAAACTCTAAGGAAA AAGTAATTCGAGAAATATTATAGACTGACTTCAGAGCAAACA CAACATCGATCCATAATAGTCAATATAATCAATAATACTCTA TGAGACCTTACCTATCAACAGCAAAAAACACAGTCCATCAAG CGGAACCCAACTCGCTCCATCCTTAATCATCCACTGAAAGAA AAAATATACGAAGGACCATCGGCCACCGGGTCCAAACAATCT AGCACAAAAATTCAAACAACCGCCAAACTCTGTTCGGCCTCA ACAAACAATCCGCCAAGCCATCTGTCATTCCTATACCAACAC ACAACCATCCCATTCCTCAAAAGCAATTCAATCCGCGACCCA AAGAAGACTCTCCACATATCCAGCTAATCCGTCGATCCGACA provides a DNA fragment represented by ShiATCATCGTATCTTTTAAGAAAAAAA, those which attained PIV-4A infection prior to the onset and early diagnosis of problems difficult Solving Is.

Since the DNA fragment provided by the present invention shows complementarity to the gene RNA of PIV-4AHN, when it is used as a test agent for identifying PIV-4A, PIV-4A can be directly identified. , Which enables the solution of problems by the conventional indirect identification method.

The method for producing the DNA fragment represented by the above-mentioned [formula] in the present invention is not particularly limited and can be according to a conventional method. For example, (a) a cDNA prepared from mRNA obtained from PIV-4A infected cells can be used. A method of screening by using nucleocapsid RNA as a probe by introducing into an appropriate vector such as Escherichia coli plasmid vector and cloning in E. coli, and using an oligonucleotide synthesized based on the nucleotide sequence represented by the above formula as a probe , A method of screening using an antibody against PIV-4A, a method of isolating a clone encoding the PIV-4A / HN gene, etc., and a method of separating the insert DNA from the plasmid of the isolated clone. , (B) based on the nucleotide sequence that is complementary to the gene RNA of PIV-4A.HN represented by the above [formula], Preparative method (Nature, 310, pp. 105 pp., 1984) according to a method of creating a synthetic nucleotide, can be prepared by methods, such as a combination of (c) above (a) and (b).

The DNA fragment of the present invention obtained by the above method can be used as follows.

For example, the DNA fragment obtained by the above method is further cleaved with an appropriate restriction enzyme (for example, BamHI, XbaI, etc.) to a dozen bases or more, if necessary, and then labeled with a radioactive isotope or the like to give a test agent. RN extracted from the test substance (pharyngeal fluid etc.)
After reaction with a nylon membrane or the like having A immobilized thereon, PIV-4A can be identified by a Northern hybridization method (hereinafter abbreviated as Northern method) determined by autoradiography or the like.

〔Example〕

Hereinafter, the present invention will be described in more detail based on examples.

(1) Preparation of RNA After growing primary monkey kidney cells in an essential eagle culture medium containing 5% fetal bovine serum, this essential culture medium containing actinomycin D (2 μg / ml) was newly prepared. Replaced with liquid. Then, PIV-4A (Toshiba strain) was inoculated and the cells were cultured in the same culture solution for 24 hours. After detaching the virus-infected cultured cells with a trypsin / EDTA mixture, centrifuge at 8000 rpm for 10 minutes to collect the cells, and collect the guanidine isothiocyanate solution (6 M guanidine isothiocyanate, 5 mM
Sodium citrate, 0.1M2-mercaptoethanol,
Chromosomal DNA was sheared by adding 0.5% sodium lauroyl sarcosinate), quickly dissolving in a homogenizer, and passing through a syringe equipped with a 21G needle 5 times 5 times. The obtained cell lysate was centrifuged again at 8000 rpm, 9 ml of the obtained supernatant was layered on another centrifuge tube containing 3 ml of a 5.7 M cesium chloride aqueous solution, and the Beckman rotor (Beckman SW4
Ultra-centrifugation was performed at 18 ° C for 18 hours at 37,000 rpm with a 0Ti rotor), and the RNA pellet was recovered after centrifugation.

From the RNA obtained as described above, an mRNA containing a poly A chain [hereinafter, abbreviated as poly (A +) RNA] was separated and purified using oligo dT cellulose Type7 [manufactured by Pharmacia].

(2) Preparation of cDNA library The cDNA library was synthesized according to the Okayama-Berg method.
That is, 5 μl of distilled water was added to 4 μg of the above poly (A +) RNA, heated at 65 ° C. for 10 minutes and then rapidly cooled, and then oligo dT was added to the KpnI site of the oligo dT primed vector [pUC118 vector (0.8 to 1.2). μg / μl)] 10 μl, synthetic reaction solution (500 mM Tris-hydrochloric acid buffer pH 8.3, 300 mM potassium chloride, 80 mM magnesium chloride, 3 mM dithiothreitol)
3μl and 20mMdATP, 20mMdCTP, 20mMdGTP, 20mMdT
Add 1 μl of TP each, and add 20units RNasin, 40uni
After adding ts reverse transcriptase, the total amount was adjusted to 30 μl with distilled water and reacted at 42 ° C. for 30 minutes to synthesize the first strand cDNA. After the first strand cDNA synthesis was completed, using terminal deoxynucleotidyl transferase in the presence of dGTP.
10-30 dG bases were added. Next, it was digested with a restriction enzyme HindIII, further annealed with a linker having a dC base chain, and then closed into a circular ring by E. coli ligase. Finally
The second strand was synthesized by DNA polymerase in the presence of RNaseH to prepare a complete plasmid DNA. Next, to 100-200 μl of the competent cells (DH1) obtained by applying calcium chloride treatment, 10 μl of 0.4 M magnesium chloride / 0.1 M calcium chloride mixed solution and 0.02 μg of the above DNA were added, and then at 40 ° C. for 40 minutes. Let stand for 90 seconds at 42 ° C, then culture medium (10 g bactotryptone, 5 yeast extract).
g, 1.5 ml of a solution prepared by dissolving 5 g of sodium chloride in 1000 ml of distilled water) was added, and the mixture was allowed to stand at 37 ° C. for 40 minutes to obtain a transformed cell.

As above, 0.02 was added to 200 μl of competent cells (DH1).
By introducing μg of plasmid DNA, 1500 independent clones were obtained in the presence of ampicillin.

(3) Preparation of nucleocapsid RNA probe 0.6% nonidet P-4 was added to PIV-4A-infected primary monkey kidney cells.
A solution containing 0, 10 mM vanadyl ribonucleotide complex (0.15 M sodium chloride, 0.05 M Tris-hydrochloric acid buffer solution) was added and solubilized by pipetting on ice for 1 hour.
It was centrifuged at 00 rpm for 10 minutes. Next, 1 ml, 2.5 ml and 1 ml of 40% aqueous solution, 30% aqueous solution and 25% aqueous solution of cesium chloride, respectively.
9 ml of the supernatant obtained by the above centrifugation was further layered on a centrifuge tube in which Beckman rotor SW40T
At i, equilibrium density gradient centrifugation was performed at 37,000 rpm for 18 hours at 16 ° C. After centrifugation, the nucleocapsid band of the virus generated in the 30% cesium chloride aqueous solution layer was collected. Then 0.1
% SDS and proteinase K (2.5mg / ml) were added, 56 ℃ 1
After proteolysis for 5 minutes, treat with phenol and phenol / chloroform mixed solution, and use nucleocapsid RN.
Got A. 50 mM Tris-hydrochloric acid buffer solution pH 9.7 was added to the obtained nucleocapsid RNA, heated at 95 ° C. for 10 minutes, and then gradually cooled to room temperature. Next, add 20 μl of this RNA solution to the buffer solution (2
50mM Tris-hydrochloric acid, 50mM magnesium chloride, 25mMDTT, 7.5m
M spermine, 500 mM potassium chloride) 10 μl, and ³² PATP
After adding 3 μl of (100 μCi / 600 pM) and 2 units of T ₄ -DNA kinase, the total amount was made up to 50 μl with distilled water and reacted at 37 ° C. for 1 hour to obtain a nucleocapsid RNA probe.

(4) Colony hybridization 100-200 μl of the transformed cells described above were treated with ampicillin (12
15 cm agar plate containing 10 μg / ml) (10 g bactotryptone, 5 g yeast extract, 5 g sodium chloride,
15 g of agar was added, and the whole was seeded with distilled water to a total volume of 1000 ml) and cultured at 37 ° C. for 12 hours. The colonies formed on the nitrocellulose membrane were copied and cultured at 37 ° C for 6 hours. The membrane is then treated with a 0.5N sodium hydroxide / 1.5M sodium chloride mixture for 10 minutes, further containing 1.5M sodium chloride.
After being placed in 0.5M Tris-hydrochloric acid buffer pH 8.0 for 10 minutes, it was rinsed in a 0.3M sodium chloride / 0.03M sodium citrate mixed solution for 5 minutes. After rinsing, the membrane was air-dried and baked at 80 ° C. for 1 hour while baking to immobilize the DNA on the membrane.

Denatured salmon RNA (1 mg) per 5 membranes thus obtained
/ ml) 1 ml, SSPE solution (3.6 M sodium chloride, 200 mM sodium primary phosphate, 20 mM EDTA) 7.5 ml, Denhard solution (2
% BSA, 2% Ficoll, 2% polyvinylpyrrolidone) 1.25m
l, prehybridization solution consisting of 1.25 ml of 10% SDS, reacted at 42 ° C. for 12 hours, and then the above prepared nucleocapsid RNA probe was added to the prehybridization solution to 5 million Ci / ml. In addition, 42 ℃
The reaction was carried out for 18 hours. After the reaction is complete, containing 0.1% SDS 20
Wash twice in double diluted SSPE solution for 5 minutes at 42 ℃, then 0.1% S
Washing was performed twice at 42 ° C. for 15 minutes in a 200-fold diluted SSPE solution containing DS. After washing, air-dry the membrane and use X-ray film (RX
(5, manufactured by Kodak Co., Ltd.) was used for autoradiography at −80 ° C. for 12 hours to obtain several positive clones.

(5) Northern method The Northern method was performed using the above positive clones. That is, derived from PIV-4A-infected cells obtained by the method of (1) above.
poly (A +) RNA, poly (A
+) RNA and rRNA markers were electrophoresed in 1.5% agarose gel, transferred to a nylon membrane (Hybond-N, Amersham), and air-dried and UV irradiated to covalently bind the RNA to the nylon membrane. Then, hybridization was performed using each of the above positive clones as a probe.

That is, the nylon membrane was reacted in the above prehybridization solution for 12 hours at 42 ° C., and then the probe labeled with ³² P prepared from each positive clone was added to the prehybridization solution at 1 million Ci / ml. In addition, the mixture was reacted at 42 ° C for 18 hours. After completion of the reaction, washing was carried out in the same manner as in the above (4) and autoradiography was carried out using an X-ray film, and as a result, one clone hybridizing at the position of 2600 base was obtained. This clone was named pM4H.

In addition, the probe of each positive clone
After digestion with III and EcoRI, insert DNA was separated by electrophoresis using low melting point agarose, and after extraction, it was obtained by random prime labeling with ³² PdCTP.

(6) Construction of restriction enzyme map of DNA showing complementarity to PIV-4A · HN gene and determination of nucleotide sequence The clone pM4H obtained in (5) above is cleaved with various restriction enzymes, and the restriction enzyme map is obtained. Was prepared and the results are shown in FIG. In the drawings, 1 and 3 are vector DNAs, 2 is an insert DNA, which is the DNA fragment of the present invention.

After cutting with each restriction enzyme according to FIG. 1, the obtained fragments were introduced into the multiple cloning sites of the plasmid pUC118, and sequenced using SEQUENASE ^™ kit (Toyobo Co., Ltd.). In addition, a part was prepared with a deletion kit for kilosequencing (manufactured by Takara Shuzo Co., Ltd.), which was sequenced to determine the nucleotide sequence.

The result was as shown in the above [formula].

(7) Application to test agents (identification of PIV-4A) The pM4H clone obtained in (4) above was cleaved with BamHI, and a part of the insert DNA was separated and extracted by electrophoresis using low melting point agarose. Then, a probe was prepared by random priming labeling using ³² P. Next, the PIV-4A-infected cell-derived RNA and the virus-non-infected cell-derived RNA obtained by the method (1) were plotted on a nylon membrane in an amount of 0.35 μg each, and then air-dried, UV-irradiated, and the subsequent treatment was performed as described in (5) )
Autoradiography was carried out in the same manner as in the Northern method described above. As a result, RNA derived from PIV-4A infected cells
PIV-4A viral RNA in the sample is plotted in
A positive signal generated by the same probe hybridized with was confirmed, and it was confirmed that PIV-4A was infected. In contrast, RNA derived from non-virus infected cells
No reaction was observed in the plotted area.

〔The invention's effect〕

The DNA represented by the above [formula] provided by the present invention
Since the fragment shows specific complementarity to the gene RNA of PIV-4A.HN, it can be very usefully used as a test agent for early diagnosis of PIV-4A infection.

[Brief description of drawings]

FIG. 1 is a restriction map of the cDNA region of DNA (pM4H) complementary to the PIV-4A.HN gene represented by the above [formula], 1 and 3 ... Vector DNA, 2 ... Insert DNA,

Claims (1)

[Claims] 1. A DNA fragment represented by the following [formula] and used as a test agent for identifying human parainfluenza 4A virus. [Expression]; GGGGAACACACTTCTCAGCCCTGATTGCTCAAGG CCCTTGCATGTGCAACCGAGACACCCCCCACAAGCACCGGAA TAAGACCTGACAACAAAGTAGCAGCCACCACGACCCAAAAAC AAAATTAAAAGGATCCGGTAACAGCCCATCAACCAGCAATCA TAGAATCCAACAATCCAGAGAGACGTCACATCAACTCATCCA CGAATCTTCGAAGGGAACATCCCAGACAAAATCACAGCCCAT TCCCTGATCACGGATAAACTGAGAAAGATCACAAGAATGCAA GACTCACATGGTAATACACAAATACTCAACCAGGCAAATTCA ATGGTGAAAAGAACATGGAGATTACTATTTCGAATTGCAACC TTAATATTACTTGTTTCAATATTTGTGTTATCGCTCATAATT GTATTACAGTCAACACCGGGGAATTTGCAAAACGATATCAAT ATAATTAGAAAGGAGCTCAATACAATTATGGAGAATTTTGAA ACTACATCTAAGTCACTGTTAAGTGTATCAAATCAAATCACT TACGATGTATCAGTACTTACTCCTATAAGACAAGAAGCTATT GAAACAAACATCATTTCAAAAATAAAAGATCATTGCAAAGAT AGAGTAATTAAAGAAGGAAGCACTTGCACATTGAATCGCAGC CCTTTGCATGATGTCTCTTTTTTAAATGGGTTCAATAAATTC TATTTCACATATAAAGATAATATGCAAATTAAGTTTAAATCA TTATTAGATTACCCCAATTTTATTCCAACTGCTACAACTCCC CACGGATGCATTCGAATTCCATCATTCTCCTTAGGTCAAACC CATTGGTGTTATACCCATAATATAAACCTACTAGGATGTGCA GACCCTGCATCTAGCAATCAATATGTATCACTAGGAACCTTA CAAGTCTTAAAAATGGGTGACCCTTATTTTAAAGTCGAGCAT AGTCATTAT TTAAATGACGGGAGGAATCGAAAGAGTTGTTCA GTGGTTGCTGTCCCCGACGGATGCCTGCGGAATTGTGTGACC ATGACAAAAAATGAGACAGAGAATTTCAAAGACCTCAATTGG CAACACAATTACTTACATACATATCATATAATGGTACCATTA AAGACTCGTATAATAAATCCACCAGGATCATCCAGAGATTGG GTTCATATCGCACCAGGGGTAGGCTCGGGCCTTTTGTATGCC AAATTACTTATATTTCCTTTGTATGGGGGTCTCACGGAAAAA TCAGTGATACATAATAATCAATCAGGGAAATATTTTTTCCCT AATTCAACTAAATTGCAATGCCGTAACAGCACTATGGAAAAA ATAAAAGGAGCAAAAGATTCATACACAATAACTTACTTCTCA GGGAGACTTATACAGAGTGCATTTCTGGTTTGTGATCTAAGA CAATTTCTTTCTGAAGATTGTGAAATCTTAATTCCTAGTAAT GATTACATGATGGTCGGTGCAGAGGGTCGATTATATAACATT GAGAACAACATATTTTATTATCAGAGAGGATCCAGCTGGTGG CCTTATCCGAGCCTCTATAGAATCAGGTTAAACCTTAGTAAG AAATATCCTAGAATAACTGAAATTAAATTTACAAAAATTGAA ATCGCCCCAAGACCAGGCAACAAAGATTGTCCAGGAAATAAG GCTTGCCCAAAAGAATGTATAACGGGAGTCTACCAAGATATA TTGCCACTAAGTTATCCCAATACTGCATTTCCACACTTAAAA CAAGCGTATTATACAGGTTTTTATCTTAATAACTCGCTCGAG AGACGCAATCCAACATTTTATACTGCTGACAATCTAGATTAC CATCAACAGGAAAGATTAGGTAAATTCAATCTTACTGCTGGA TACTCTACTACAACTTGTTTTAAACAGACCACTACTGCGAGG TTATACTGTCTCTACATAAT TGAAGTGGGTGACTCAGTCATT GGGGACTTTCAGATCACCCTTTTTTTAGCAGCTTAATAGACC AGACTGTTAATTAATCAACAAAGTTATTCTGTAATATAAACT GATCTTATAAGTGAAAAGATGCCTATCCAAGGAGGTTGATAG ACAAATAGTAAAAGTAGCAATTGTAACAAAACTCTAAGGAAA AAGTAATTCGAGAAATATTATAGACTGACTTCAGAGCAAACA CAACATCGATCCATAATAGTCAATATAATCAATAATACTCTA TGAGACCTTACCTATCAACAGCAAAAAACACAGTCCATCAAG CGGAACCCAACTCGCTCCATCCTTAATCATCCACTGAAAGAA AAAATATACGAAGGACCATCGGCCACCGGGTCCAAACAATCT AGCACAAAAATTCAAACAACCGCCAAACTCTGTTCGGCCTCA ACAAACAATCCGCCAAGCCATCTGTCATTCCTATACCAACAC ACAACCATCCCATTCCTCAAAAGCAATTCAATCCGCGACCCA AAGAAGACTCTCCACATATCCAGCTAATCCGTCGATCCGACA CATCATCGTATCTTTTAAGAAAAAAA