CN1058751C - Full length CDNA clone of hepatitis virus G genome and its construction method - Google Patents
Full length CDNA clone of hepatitis virus G genome and its construction method Download PDFInfo
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Abstract
The present invention relates to clone of G hepatitis virus genome full-length cDNA and a construction method thereof, which belongs to the technical field of organism. Five smaller gene segments covering GBV-C/HGV genome full-length cDNA are used as starting materials, and the clone of the GBV-C/HGV genome full-length cDNA is completed through PCR overlapping extension, enzyme cutting, connection, conversion, etc. The success for the clone of the GBV-C/HGV genome full-length cDNA provides an important material for researching pathogenicity and pathogenic mechanisms of GBV-C/HGV, copying, transcription and translation mechanisms, state and state generation, specific gene and serology diagnosis, establishment of ribonucleic acid, genetically engineered vaccines and various animal models, etc.
Description
The present invention relates to biological technical field, particularly hepatitis G virus genome Full Length cDNA Cloning and construction process thereof.
Nineteen ninety-five, the U.S. has two research groups almost to report a kind of new Hepatitis virus simultaneously respectively, and called after GB virus C (GBV-C) and hepatitis G virus (HGV) abbreviate GBV-C/HGV as respectively.But the GBV-C/HGV menses are propagated, and analysing among patient, vein drug-addict and hepatitis B virus (HBV) and hepatitis C virus (HCV) the infected in commercial blood donor, hemodialysis all has higher infection rate, causes post-transfusion hepatitis and other acute and chronic hepatopathys.GBV-C/HGV belongs to flaviviridae, is the sub-thread positive chain RNA virus, and the about 9.4kb of genome total length only has an open reading frame (ORF) in the genome, and structure gene C, E1 and E2 are positioned at aminoterminal, coding structure albumen; Nonstructural gene NS2, NS3, NS4, NS5a and NS5b are positioned at carboxyl terminal, the coding Nonstructural Protein; Be respectively 5 ' non-coding region and 3 ' non-coding region (among Fig. 1 shown in the skeleton diagram) in the both sides of open reading frame.Though the GBV-C/HGV total order of report is shown more than 11 strains, reading of complete sequence is according to segmentation cloned genes fragments sequence, do not have the single clone of GBV-C/HGV genome full-length cDNA of allomeric function so far.Gene fragment clone can not reflect the real characteristic of virus on the whole, has influenced the further investigation to GBV-C/HGV.
The purpose of this invention is to provide GBV-C/HGV genome Full Length cDNA Cloning and construction process thereof.
Hepatitis G virus (GBV-C/HGV) the genome full length cDNA clone that the present invention makes up has comprised the GBV-C/HGV genome from 5 ' non-coding region, structure gene C, E1, E2, the full-length gene of nonstructural gene NS2, NS3, NS4, NS5a and NS5b and 3 ' non-coding region.
The structure of hepatitis G virus (GBV-C/HGV) genome full length cDNA clone adopts overlapping extension PCR spliced gene fragment to cut with enzyme, be connected the method that combines, and comprises the steps:
(1) primer of synthetic amplification and splicing GBV-C/HGV gene fragment;
(2) amplification and splicing GBV-C/HGV gene fragment;
(3) segmentation of GBV-C/HGV gene fragment clone;
(4) GBV-C/HGV genome Full Length cDNA Cloning.
The base mateiral that makes up GBV-C/HGV genome full length cDNA clone is that five recombinant plasmid I w5, Iwq2, Iwh6, Iw3 ' and Iw3 containing GBV-C/HGV genome different zones gene fragment (see document for details: Li Shao et al.Biochem.Biophys.Res.Commun.1996; 228:785-791).Five gene fragment from 3 ' to 5 ' end adjacent segment of the same name overlap each other, cover the whole genome (see figure 1), wherein two gene fragments of Iwq2 and Iwh6 are to increase from the patients serum with long PCR method, and Iw5, Iw3 ' and Iw3 gene fragment are then increased from same patients serum by 5 ' or 3 ' RACE method.
The present invention utilizes overlapping extension PCR (SOEing and PCR) method, and Iw5 and Iwq2 gene fragment are spliced into the Iw5-q2 gene fragment; After the splicing of Iw3 ' and Iw3 gene fragment obtained Iw3 '-3 gene fragment, obtain the Iwh6-3 gene fragment with the splicing of Iwh6 gene fragment again; Iwq2 and Iwh6 splicing are obtained the Iwq2-h6 gene fragment.During splicing Iwq2-h6 gene fragment, require Iwq2-h6 gene fragment 5 ' end and 3 ' end of Iw5-q2 gene fragment to overlap each other, and comprise common and unique restriction enzyme site; 3 ' end of Iwq2-h6 gene fragment overlaps each other with the 5 ' end of Iwh6-3, comprises common and unique restriction enzyme site equally.The restriction enzyme site that utilizes above-mentioned restriction enzyme site and in the downstream primer of the upstream primer of amplification Iw5 gene fragment and the Iw3 gene fragment that increases, add, carry out the segmentation clone of GBV-C/HGV gene fragment Iw5-q2, Iwq2-h6 and Iwh6-3 earlier, obtain recombinant plasmid pUC-5-q2, pUC-q2-h6 and pGEM-h6-3.After treating that segmentation is cloned successfully, more based on this, the method for utilizing digestion with restriction enzyme to be connected with ligase enzyme makes up GBV-C/HGV genome full length cDNA clone pHGVqz.
The present invention has following advantage and positively effect:
The present invention adopts overlapping extension PCR (SOEing and PCR) splicing, digestion with restriction enzyme to be connected the method that gene fragment combines with ligase enzyme, makes up GBV-C/HGV genome full length cDNA clone, and technical scheme is skillfully constructed.Both avoided initial by several minigene fragments, through digestion with restriction enzyme, the cumbersome procedure that connects by ligase enzyme again, again because adopt first segmentation clone to carry out full-length gene clone's approach again and reduced the experiment difficulty.The success of GBV-C/HGV genome Full Length cDNA Cloning provides important experiment material for studying this virus extensively and profoundly.Utilize GBV-C/HGV genome full length cDNA clone, can be from the cell levels to the molecular level, from the cell model to the animal model, multi-faceted from the part to integral body etc., launch pathogenic and mechanism of causing a disease with multi-angle to GBV-C/HGV, duplicate, transcribe and translating mechanism, extensive and deep researchs such as form and form generation.
Description of drawings:
Fig. 1 is the position of GBV-C/HGV genome structure among the present invention and cDNA gene fragment, and the skeleton diagram on top is represented the GBV-C/HGV genome structure and respectively distinguished the position of gene.Five gene fragments of digitized representation and the position in genome thereof of the thick line figure of bottom and thick line upper end.
Fig. 2 is the structure schema of recombinant plasmid pUC-5-q2.
Fig. 3 is the structure schema of recombinant plasmid pUC-q2-h6.
Fig. 4 is the structure schema of recombinant plasmid pGEM-h6-3.
Fig. 5 is the structure schema of recombinant plasmid pHGVqz.
Circle diagram in Fig. 2～5 is represented cyclic plasmid, and thick line is represented gene fragment, and Yx (x is 1-12) represents primer.This gene fragment position in the GBV-C/HGV genome of thick line digitized representation that mark at two ends.
Fig. 6 GBV-C/HGV genome full length cDNA sequence
The concrete construction process of GBV-C/HGV genome full length cDNA clone is seen following embodiment:
One, synthetic amplification and the segmental primer of spliced gene (seeing Table 1), primer design reaches synthetic size with reference to 5 gene fragments, will satisfy the general condition of design of primers simultaneously as far as possible.
The position polarity Y1 GCG AAT TCG CGG CCG CCC CCC CCC 1-26 of primer primer sequence (5 '-the 3 ') primer of table 1. amplification and splicing GBV-C/HGV gene fragment in genome+
CGG?CAC?TGG?GTG?CAA?GCY2 AGA?GAG?ACA?TTG?AAG?GGC?GAC 321-341 -Y3 GAC?AGG?GTT?GGT?AGG?TCG?TAA?ATC?C 109-133 +Y4 CCG?TCC?TTG?ATG?ATG?GAA?CTG?TC 4399-4421 -Y5 TAT?GGG?CAT?GGC?ATA?CCC?CT 4261-4280 +Y6 GAG?GGC?CAC?GAT?GAT?GTT?AG 8696-8715 -Y7 TTC?TGC?TCC?ACT?TGG?CTC?GCT?GAG?T 8416-8440 +Y8 CTG?GAT?GCC?ACA?ACA?GGC?CCC?T 8881-8902 -Y9 AGG?GGC?CTG?TTG?TGG?CAT?CCAG 8881-8902 +Y10 GCT?CTA?GAC?TCG?AGA?ATA?AAA?CCC
GGC?CTT?TGG?GCC?G 9353-9375 -Y11 TTG?TTC?GAT?CAT?ATG?GGG?TCC?TTC?TCG?C 2977-3004 +Y12 GAC?CGG?AGT?CCC?TTC?AAG?TAT?CTC?CTG?G 7737-7764 -
Two, be template with plasmid Iw5, Iwq2, Iwh6, Iw3 ' and Iw3, react 5 gene fragments of the same name that increase respectively through PCR, the PCR reaction conditions is adjusted according to the length of pre-amplification gene fragment and Tm (melting temperature(Tm)) value of the primer routinely.Pcr amplification and splicing reaction are all used Expand
TMLong Template PCR system.
1, the amplification of Iw5 gene fragment (Fig. 2)
(1) Iw5 plasmid (50ng/ul) 1ul
10mM?dNTP 1.75ul
Y1(30uM) 0.5ul
Y2(30uM) 0.5ul
Sterilization redistilled water 21.25ul
-----------------------------
(2) 10X damping fluid 1 5ul
Archaeal dna polymerase mixed solution (the 0.75ul of 3.5 units/ul)
Sterilization redistilled water 19.25ul mixes each composition in (1) (2) earlier respectively, and then (1) (2) are mixed, carry out the PCR reaction by following condition: 94 ℃ of pre-sex change in 2 minutes, 94 ℃ 30 seconds, 65 ℃ 30 seconds, 68 ℃ were carried out 35 circulations in 1 minute, extend before reaction finishes 68 ℃ 10 minutes.
2, the amplification of Iwq2 gene fragment (Fig. 2, Fig. 3)
(1) Iwq2 plasmid (50ng/ul) 1ul
10mM?dNTP 1.75ul
Y3(30uM) 0.5ul
Y4(30uM) 0.5ul
Sterilization redistilled water 21.25ul
-----------------------------
(2) 10X damping fluid 1 5ul
Archaeal dna polymerase mixed solution (the 0.75ul of 3.5 units/ul)
Sterilization redistilled water 19.25ul mixes each composition in (1) (2) earlier respectively, and then (1) (2) are mixed, carry out the PCR reaction by following condition: 94 ℃ of pre-sex change in 2 minutes, 94 ℃ 30 seconds, 65 ℃ 30 seconds, 68 ℃ were carried out 35 circulations in 6 minutes, extend before reaction finishes 68 ℃ 10 minutes.
3, the amplification of Iwh6 gene fragment (Fig. 3, Fig. 4)
(1) Iwh6 plasmid (50ng/ul) 1ul
10mM?dNTP 1.75ul
Y5(30uM) 0.5ul
Y6(30uM) 0.5ul
Sterilization redistilled water 21.25ul
-------------------------------
(2) 10X damping fluid 1 5ul
Archaeal dna polymerase mixed solution (the 0.75ul of 3.5 units/ul)
Sterilization redistilled water 19.25ul mixes each composition in (1) (2) earlier respectively, and then (1) (2) are mixed, carry out the PCR reaction by following condition: 94 ℃ of pre-sex change in 2 minutes, 94 ℃ 30 seconds, 60 ℃ 30 seconds, 68 ℃ were carried out 35 circulations in 6 minutes, extend before reaction finishes 68 ℃ 10 minutes.
4, the amplification of Iw3 ' gene fragment (Fig. 4)
(1) Iw3 ' plasmid (50ng/ul) 1ul
10mM?dNTP 1.75ul
Y7(30uM) 0.5ul
Y8(30uM) 0.5ul
Sterilization redistilled water 21.25ul
-------------------------------
(2) 10X damping fluid 1 5ul
Archaeal dna polymerase mixed solution (the 0.75ul of 3.5 units/ul)
Sterilization redistilled water 19.25ul mixes each composition in (1) (2) earlier respectively, and then (1) (2) are mixed, carry out the PCR reaction by following condition: 94 ℃ of pre-sex change in 2 minutes, 94 ℃ 30 seconds, 60 ℃ 30 seconds, 68 ℃ were carried out 35 circulations in 1 minute, extend before reaction finishes 68 ℃ 10 minutes.
5, the amplification of Iw3 gene fragment (Fig. 4)
(1) Iw3 plasmid (50ng/ul) 1ul
10mM?dNTP 1.75ul
Y9(30uM) 0.5ul
Y10(30uM) 0.5ul
Sterilization redistilled water 21.25ul
------------------------------------
(2) 10X damping fluid 1 5ul
Archaeal dna polymerase mixed solution (the 0.75ul of 3.5 units/ul)
Sterilization redistilled water 19.25ul mixes each composition in (1) (2) earlier respectively, and then (1) (2) are mixed, carry out the PCR reaction by following condition: 94 ℃ of pre-sex change in 2 minutes, 94 ℃ 30 seconds, 60 ℃ 30 seconds, 68 ℃ were carried out 35 circulations in 1 minute, extend before reaction finishes 68 ℃ 10 minutes.
Three, 5 of PCR gene fragments are behind the agarose gel electrophoresis purifying, respectively with the big fragment of dna polymerase i (Klenow) handle 37 ℃ 1 hour, remove " A " (adenosine) that gene fragment 3 ' end exists.
Example: the Klenow enzyme of Iw5 gene fragment is handled (Fig. 2)
Iw5 gene fragment (0.1ug/ul) 50ul
10X Klenow damping fluid 10ul
Klenow enzyme (the 1ul of 10 units/ul)
Sterilization redistilled water 39ul mixing rearmounted 37 ℃ 1 hour, phenol/chloroform extracting then, dehydrated alcohol precipitation.The Klenow enzyme of Iwq2, Iwh6, Iw3 ' and Iw3 gene fragment handle according to this example carry out (Fig. 2, Fig. 3, Fig. 4).
Four, overlapping extension PCR splicing Iw5-q2, Iwq2-h6, Iw3 '-3 and Iwh6-3 gene fragment, the PCR reaction conditions is adjusted according to the Tm value of pre-segmental length of spliced gene and the primer.
1, Iwq2 gene fragment (0.1ug/ul) 2ul of Iw5 gene fragment (0.1ug/ul) 2ul of the splicing of Iw5-q2 gene fragment (Fig. 2) (1) after the Klenow enzyme is handled after the Klenow enzyme is handled
10mM?dNTP 1.75ul
Y1(30uM) 0.5ul
Y4(30uM) 0.5ul
Sterilization redistilled water 18.25ul
------------------------------------
(2) 10X damping fluid 1 5ul
Polysaccharase mixed solution (the 0.75ul of 3.5 units/ul)
Sterilization redistilled water 19.25ul mixes each composition in (1) (2) earlier respectively, and then (1) (2) are mixed, carry out the PCR reaction by following condition: 94 ℃ of pre-sex change in 2 minutes, 94 ℃ 30 seconds, 60 ℃ 1 minute, 68 ℃ were carried out 35 circulations in 6 minutes, extend before reaction finishes 68 ℃ 10 minutes.
2, Iw3 gene fragment (0.1ug/ul) 2ul that handles through the Klenow enzyme of Iw3 ' gene fragment (0.1ug/ul) 2ul that handles through the Klenow enzyme of the splicing (Fig. 4) (1) of Iw3 '-3 gene fragment
10mM?dNTP 1.75ul
Y7(30uM) 0.5ul
Y10(30uM) 0.5ul
Sterilization redistilled water 18.25ul
-----------------------------------------(2) 10X damping fluid 1 5ul
Polysaccharase mixed solution (the 0.75ul of 3.5 units/ul)
Sterilization redistilled water 19.25ul mixes each composition in (1) (2) earlier respectively, and then (1) (2) are mixed, carry out the PCR reaction by following condition: 94 ℃ of pre-sex change in 2 minutes, 94 ℃ 30 seconds, 60 ℃ 30 seconds, 68 ℃ were carried out 35 circulations in 2 minutes, extend before reaction finishes 68 ℃ 10 minutes.
3, Iwh6 gene fragment (0.1ug/ul) 2ul that handles through the Klenow enzyme of Iw3 '-3 gene fragment (0.1ug/ul) 2ul that handles through the Klenow enzyme of the splicing of Iwh6-3 gene fragment (Fig. 4) (1)
10mM?dNTP 1.75ul
Y5(30uM) 0.5ul
Y10(30uM) 0.5ul
Sterilization redistilled water 18.25ul
----------------------------------------
(2) 10X damping fluid 1 5ul
Polysaccharase mixed solution (the 0.75ul of 3.5 units/ul)
Sterilization redistilled water 19.25ul mixes each composition in (1) (2) earlier respectively, and then (1) (2) are mixed, carry out the PCR reaction by following condition: 94 ℃ of pre-sex change in 2 minutes, 94 ℃ 30 seconds, 65 ℃ 30 seconds, 68 ℃ were carried out 35 circulations in 6 minutes, extend before reaction finishes 68 ℃ 10 minutes.
4, Iwh6 gene fragment (0.1ug/ul) 2ul that handles through the Klenow enzyme of Iwq2 gene fragment (0.1ug/ul) 2ul that handles through the Klenow enzyme of the splicing of Iwq2-h6 gene fragment (Fig. 3) (1)
10mM?dNTP 1.75ul
Y11(30uM) 0.5ul
Y12(30uM) 0.5ul
Sterilization redistilled water 18.25ul
----------------------------------
(2) 10X damping fluid 1 5ul
Polysaccharase mixed solution (the 0.75ul of 3.5 units/ul)
Sterilization redistilled water 19.25ul mixes each composition in (1) (2) earlier respectively, and then (1) (2) are mixed, carry out the PCR reaction by following condition: 94 ℃ of pre-sex change in 2 minutes, 94 ℃ 30 seconds, 65 ℃ 45 seconds, 68 ℃ were carried out 35 circulations in 6 minutes, extend before reaction finishes 68 ℃ 10 minutes.
Five, the Iw5-q2 gene fragment is cut through EcoR I+BmH I enzyme, reclaim the gene fragment of about 3301bp, be connected with the pUC18 carrier of cutting through EcoR I+BmH I enzyme, transformed into escherichia coli JM109, enzyme cut and identify that transformant obtains recombinant plasmid pUC-5-q2 (Fig. 2).The Iwq2-h6 gene fragment is cut through BamH I+SalI enzyme, reclaims the gene fragment of about 3298bp; Be connected with the pUC18 carrier of cutting through BamH I+Sal I enzyme, transformed into escherichia coli JM109, enzyme cut and identify that transformant obtains recombinant plasmid pUC-q2-h6 (Fig. 3).The Iwh6-3 gene fragment is cut through Sal I+XbaI enzyme, reclaims the gene fragment of about 2806bp, is connected with pGEM-3Zf (+) carrier of cutting through Sal I+XbaI enzyme, and transformed into escherichia coli JM109, enzyme cut and identify that transformant obtains recombinant plasmid pGEM-h6-3 (Fig. 4).
Six, recombinant plasmid pUC-5-q2 is cut with EcoR I+BamH I enzyme, reclaim the gene fragment of about 3301bp; PUC-q2-h6 cuts with the BamHI+SalI enzyme, reclaims the gene fragment of about 3298bp; PGEM-h6-3 cuts with Sal I+XbaI enzyme, reclaims the gene fragment of about 2806bp.Three gene fragments that reclaim are connected with pGEM-3Zf (+) carrier of cutting through EcoR I+XbaI enzyme, transformed into escherichia coli JM109, the picking transformant carries out enzyme and cuts evaluation, the preliminary recombinant plasmid pHGVqz (Fig. 5) that determines clone GBV-C/HGV genome full-length cDNA.
GBV-C/HGV full-length gene fragment is connected with carrier:
pGEM-3Zf(+)/EcoRI+XbaI (0.1ug/ul)
5-q2/EcoR?I+BamH?I(50ng/ul)
q2-h6/BamH?I+Sal?I(25ng/ul)
h6-3/Sal?I+Xba?I (60ng/ul)
10X ligase enzyme damping fluid 1.5ul
Ligase enzyme (5u/ul) 1ul
14 ℃ are spent the night behind the sterilization redistilled water 3.5ul mixing.Getting 1/3rd of connector next day transforms.
Seven, extract the pHGVqz plasmid and carry out nucleotide sequencing.The long 9373bp of GBV-C/HGV genome full length cDNA sequence (Fig. 6), with the GBV-C/HGV sequence height homology of report, but change has taken place in part Nucleotide, and the result shows that we have successfully made up GBV-C/HGV genome full length cDNA clone.
The success of GBV-C/HGV genome full length cDNA clone provides important experiment material for furtheing investigate this virus.With GBV-C/HGV genome full length cDNA clone is that many-sided research can be carried out in the basis, for example:
(1) serve as that genetic expression is carried out at protokaryon, eucaryon (containing insect) cell system in the basis with GBV-C/HGV genome full length cDNA clone.
(2) be that gene test, Detection of antigen and antibody test research are carried out in the basis with GBV-C/HGV genome full length cDNA clone.
(3) with GBV-C/HGV genome full length cDNA clone be the basis carry out relevant GBV-C/HGV the cell in vitro system duplicate, transcribe and translating mechanism, morphology of virus and form are studied.
(4) be that the basis is carried out pathogenic and immunity research with GBV-C/HGV genome full length cDNA clone.
(5) be that gene therapy, antisense nucleic acid treatment and biological (polypeptide) drug research are carried out in the basis with GBV-C/HGV genome full length cDNA clone.
(6) be that nucleic acid vaccine, recombination engineered vaccine and vaccine immunity research are carried out in the basis with GBV-C/HGV genome full length cDNA clone.
(7) be that various animal infection modals, transgenic animal model and gene knockout The Animal Model Study are carried out in the basis with GBV-C/HGV genome full length cDNA clone.
(8) be structure, the screening study that peptide storehouse and antibody library are carried out in the basis with GBV-C/HGV genome full length cDNA clone.
Claims (3)
1. hepatitis G virus (GBV-C/HGV) genome full length cDNA clone, it is characterized in that this clone has comprised the GBV-C/HGV genome from 5 ' non-coding region, structure gene C, E1, E2, the full-length gene of nonstructural gene NS2, NS3, NS4, NS5a and NS5b and 3 ' non-coding region 9373bp.
2. the construction process of a hepatitis G virus (GBV-C/HGV) genome full length cDNA clone, what it is characterized in that adopting is that overlapping extension PCR spliced gene fragment is cut with enzyme, is connected the method that combines, and comprises the steps:
(1) primer of synthetic amplification and splicing GBV-C/HGV gene fragment;
(2) amplification and splicing GBV-C/HGV gene fragment;
(3) segmentation of GBV-C/HGV gene fragment clone;
(4) GBV-C/HGV genome Full Length cDNA Cloning.
3. by the construction process of the described hepatitis G virus of claim 2 (GBV-C/HGV) genome full length cDNA clone, it is characterized in that utilizing overlapping extension PCR method, Iw5 and Iwq2 gene fragment are spliced into the Iw5-q2 gene fragment; After the splicing of Iw3 ' and Iw3 gene fragment obtained Iw3 '-3 gene fragment, obtain the Iwh6-3 gene fragment with the splicing of Iwh6 gene fragment again; Iwq2 and Iwh6 splicing are obtained the Iwq2-h6 gene fragment; During splicing Iwq2-h6 gene fragment, require Iwq2-h6 gene fragment 5 ' end and 3 ' end of Iw5-q2 gene fragment to overlap each other, and comprise common and unique restriction enzyme site; 3 ' end of Iwq2-h6 gene fragment overlaps each other with the 5 ' end of Iwh6-3, comprise common and unique restriction enzyme site equally, the restriction enzyme site that utilizes above-mentioned restriction enzyme site and in the downstream primer of the upstream primer of amplification Iw5 gene fragment and the Iw3 gene fragment that increases, add, carry out the segmentation clone of GBV-C/HGV gene fragment Iw5-q2, Iwq2-h6 and Iwh6-3 earlier, obtain recombinant plasmid pUC-5-q2, pUC-q2-h6 and pGEM-h6-3; After treating that segmentation is cloned successfully, more based on this, the method for utilizing digestion with restriction enzyme to be connected with ligase enzyme makes up GBV-C/HGV genome full length cDNA clone pHGVqz.
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| AU2001249928A1 (en) * | 2000-04-06 | 2001-10-23 | University Of Iowa Research Foundation | Full-length gb virus c (hepatitis g virus) rna transcripts are infectious in primary cd4 positive t cells and methods of treating hiv |
| KR100491995B1 (en) * | 2001-06-25 | 2005-05-30 | 가부시끼가이샤 도시바 | Polynucleotide probe and primer derived from hepatitis e virus collected from japanese, chip including the same, kit including the same, and method of detecting hepatitis e virus using the same |
| CN105085655B (en) * | 2015-09-18 | 2019-02-19 | 昆明理工大学 | With the human protein LT- α of HGV RNA E2 protein-interacting |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995032292A2 (en) * | 1994-05-20 | 1995-11-30 | Genelabs Technologies, Inc. | Detection of viral antigens coded by reverse-reading frames |
| WO1997019195A1 (en) * | 1995-11-21 | 1997-05-29 | Boehringer Mannheim Gmbh | Amplification of nucleic acids and detection of a new non-a, non-b, non-c, non-d, non-e hepatitis virus |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995032292A2 (en) * | 1994-05-20 | 1995-11-30 | Genelabs Technologies, Inc. | Detection of viral antigens coded by reverse-reading frames |
| WO1997019195A1 (en) * | 1995-11-21 | 1997-05-29 | Boehringer Mannheim Gmbh | Amplification of nucleic acids and detection of a new non-a, non-b, non-c, non-d, non-e hepatitis virus |
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