CN1337463A - Breeding of hepatitis A virus Chinese strain and attenuated strain and its complementary DNA sequence - Google Patents
Breeding of hepatitis A virus Chinese strain and attenuated strain and its complementary DNA sequence Download PDFInfo
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Abstract
The present invention relates to hepatitis A virus Chinese strain and attenuated strain separation and cultivation and their complementary DNA sequence, in particular, it relates to separation and purification of hepatitis A virus Chinese strain, cultivation of series attenuated strain and its unique cDNA sequence. Said series cDNA series can provide basic material for further understanding genetic property and genetic stability of hepatitis A virus strain and expounding molecular mechanism of HAV attenuation, and can create condition for developing novel hepatitis A vaccine which can be quickly grown and possesses high titre and high antigenicity and can provide scientific basis for developing gene engineering polypeptide vaccine.
Description
Technical field
The present invention relates to hepatitis A virus (HAV), the complementary DNA (cDNA) of HAV Chinese epidemic strain Gkm and attenuated strain thereof (cDNA) sequence particularly, and the method for cultivation of attenuated strain, process and condition.
Background technology
Hepatitis A is the Pandemic infection disease, and it is to be on the hazard by about 4,000,000,000 people in the whole world that hepatitis A virus (HAV) causes.In China, among the grownup more than 35 years old, the hepatitis A rate of being contaminted reaches 90%, often causes seasonal popular.Because this sick high incidence and the long course of disease, bring to people ' s health and Economic development to have a strong impact on.According to China's reality, control hepatitis A popular effectively and essential measure be to develop vaccine, and the key of vaccine is good attenuated strain.
Once report obtained HAV F ' attenuated strain to people such as the Provost of U.S. MERCK company in 1986, people such as the Purcell of America NI H also obtain hepatitis A virus (HAV) HM175/7MK-5 attenuated strain in 1987 annual reports, and got its cDNA complete sequence clear, but people's test result proves, immune effect is very poor, so the work of further expansion people examination is not arranged so far.
Hepatitis A virus attenuated live vaccine (H2) is successfully developed through making great efforts for many years in the Zhejiang Academy of Medical Sciences, and inoculates proof through a large amount of crowds and can effectively prevent the infection of hepatitis A, and this disease is popular to provide effective measures in order to control.But the former strain virus of this strain and the sequence report of relevant strain are not arranged as yet so far.The present invention will fill up this blank, get complementary DNA (cDNA) (cDNA) sequence of HAV Chinese strain and attenuated strain thereof and vaccine strain clear, thereby further understand the hereditary property and the genetic stability of strain, illustrate the molecule mechanism of HAV attenuation, and for fast growth of development, high titre, the antigenic novel hepatitis A vaccine of height create conditions, for the development of gene engineering polypeptide vaccine provides scientific basis.
Summary of the invention
Summary of the invention
The present invention relates to the preparation of viruses of human hepatitis A's Chinese strain Gkm of isolating and basic purifying, the preparation of Gkm geneome RNA and the cDNA of Gkm.
The invention still further relates to cDNA nucleotide sequence and the varient thereof of viruses of human hepatitis A's Chinese strain Gkm.
The invention still further relates to preparation, the preparation of H2K5 geneome RNA and the cDNA of H2K5 of viruses of human hepatitis A's cell cultures adapted strain H2K5 of isolating and basic purifying.
The invention still further relates to cDNA nucleotide sequence and the varient thereof of viruses of human hepatitis A's cell cultures adapted strain H2K5, particularly the cDNA sequence in HAV genome 5 ' non-coding region (NCR), 2A district and 2C district.
The invention still further relates to preparation, the preparation of H2K20 geneome RNA and the cDNA of H2K20 of viruses of human hepatitis A's cell cultures adapted strain H2K20 of isolating and basic purifying.
The invention still further relates to cDNA nucleotide sequence and the varient thereof of viruses of human hepatitis A's cell cultures adapted strain H2K20.
The invention still further relates to the preparation of the viruses of human hepatitis A attenuated strain H2K25 of isolating and basic purifying, the preparation of H2K25 geneome RNA and the cDNA of H2K25.
The invention still further relates to cDNA nucleotide sequence and the varient thereof of viruses of human hepatitis A attenuated strain H2K25, particularly the cDNA sequence in HAV genome 5 ' non-coding region (NCR), 2A district and 2C district.
The invention still further relates to the preparation of the viruses of human hepatitis A attenuated strain H2M20K5 of isolating and basic purifying, the preparation of H2M20K5 geneome RNA and the cDNA of H2M20K5.
The invention still further relates to cDNA nucleotide sequence and the varient thereof of viruses of human hepatitis A attenuated strain H2M20K5, particularly the cDNA sequence in HAV genome 5 ' non-coding region (NCR), 2A district and 2C district.
The invention still further relates to based on above-mentioned all cDNA sequences, design primer and selective amplification hepatitis A gene fragment, thereby in genetic stability detection, toxicity detection and the detection of cell adapted property and the biology sample detection of hepatitis A virus, use.
The invention still further relates to based on above-mentioned all cDNA sequences any hepatitis A virus (HAV) attenuated strain that using gene engineering technique is made.
The invention still further relates to based on above-mentioned all cDNA sequences any hepatitis A virus (HAV) polypeptide vaccine that using gene engineering technique is made.
Detailed Description Of The Invention
The present invention relates to viruses of human hepatitis A (HAV) the Chinese epidemic strain Gkm of isolating and basic purifying.This virus strain is directly isolating from hepatitis A patient's ight soil in the suburbs, Hangzhou, and through the strain of F113 and PEG precipitation and differential centrifugation and ultracentrifugation purifying.
The invention still further relates to complementary DNA (cDNA) (cDNA) sequence of people HAV Gkm strain, it can instruct produce HAV protein or with the basic homologous protein of HAV protein.
The present invention relates to people HAV H2K5 strain through the cell cultures adaptation.This virus strain be HAV Gkm strain in human embryonic lung diploid fibroblast KMB17, cultivate the virus strain that obtains after 5 generations through 35 ℃.
The invention still further relates to the cDNA sequence of HAV H2K5, particularly the cDNA sequence in HAV genome 5 ' non-coding region (NCR), 2A district and 2C district.Below this group nucleotide sequence is shown in as SEQ NO.1, SEQ NO.2 and SEQ NO3 respectively.SEQ NO.1 (HAV H2K5 5 ' NCR district, 101bp-200bp) 5 ' TTCCCTTTCC CCTTCCCCTT CCTTGTTTTG ATTGTAAATA TAAATTCCTG CAGGTTCAGG GTTCTT GAA TTGGCATTAA ATTTGTTTCT CTATAAGAAC, 3 ' SEQ NO.2 (HAV H2K5 2A district, 3011bp-3110bp) 5 ' TATGTTATCC ACTGAATCAA TGATGAGCAG AATTGCGGCT GGAGACTTGG AGTCATCAGT GGATGATCCT AGATCAGAGG AGGATAAAAG ATTTGAGAGT, 3 ' SEQ NO.3 (HAV H2K5 2C district, 3986bp-4085bp) 5 ' CACAGAGTTT TTCCAACTGG TTAAGAGATA TCTGTTCAGG GATCACTATT TTTAAAAACT TCAAGGATGC AATTTATTGG CTCTATACAA AATTGAAGGA 3 '
The present invention relates to strain through the people HAV of low temperature cell cultures H2K20.This virus strain be HAV Gkm strain in human embryonic lung diploid fibroblast KMB17, cultivated for 15 generations through 35 ℃, cultivate the virus strain that obtains after 5 generations for 32 ℃.
The invention still further relates to HAV H2K20 and relate to the cDNA sequence, it can instruct produce HAV protein or with the basic homologous protein of HAV protein.
The present invention relates to strain through the people HAV of low temperature cell cultures attenuation H2K25.This attenuated strain be HAV Gkm strain in human embryonic lung diploid fibroblast KMB17, cultivated for 15 generations through 35 ℃, cultivated for 5 generations for 32 ℃, cultivate the virus strain that obtains after 5 generations for 35 ℃.
The invention still further relates to the cDNA sequence of HAV H2K25, particularly the cDNA sequence in HAV genome 5 ' NCR district, 2A district and 2C district.Below this group nucleotide sequence is shown in as SEQ NO.7, SEQ NO.8 and SEQNO.9 respectively.SEQ NO.7 (HAV H2K25 5 ' NCR district, 101bp-200bp) 5 ' TTCCCTTTCC CCTTCCCCTT CCTTGTTTTG ATTGTAAATA TAAATTCCTG CAGGTTCAGG GTTCTTTAAA TTGGCATTGA ATTTGTTTCT CTATAAGAAC, 3 ' SEQ NO.8 (HAV H2K25 2A district, 3011bp-3110bp) 5 ' TATGTTATCC ACTGAATCAA TGATGAGCAG AATTGCGGCT GGAGACTTGG AGTCATCAGT GGATGATCCT AGATCAGAGG AGGATAAAAG ATTTGAGAGT, 3 ' SEQ NO.9 (HAV H2K25 2C district, 3986bp-4085bp) 5 ' CACAGAGTTT TTCCAACTGG TTAAGAGATA TCTGTTCAGG GATCACTATT TTTAAAAACT TCAAGGATGC AATTTATTGG CTCTATACAA AATTGAAGGA
The present invention relates to strain through the people HAV of low temperature cell cultures attenuation H2M20K5.This attenuated strain is the HAVGkm strain in former generation monkey-kidney cells, cultivates for 15 generations through 35 ℃, cultivates for 5 generations for 32 ℃, cultivates the virus strain that obtains after 5 generations at the KMB17 cell in 32 ℃ again.
The invention still further relates to the cDNA sequence of HAV H2M20K5, particularly the cDNA sequence in HAV genome 5 ' NCR district, 2A district and 2C district.Below this group nucleotide sequence is shown in as SEQ NO.10, SEONO.11 and SEQ NO.12 respectively.SEQ NO.10 (HAV H2M20K5 5 ' NCR district, 101bp-200bp) 5 ' TTCCCTTTCC CCTTCCCCTT CCTTGTTTTG ATTGTAAATA TAAATTCCTG CAGGTTCAGG GTTCTTTAAA TTGGCATTGA ATTTGTTTCT CTATAAGAAC, 3 ' SEQ NO.11 (HAV H2M20K5 2A district, 3011bp-3110bp) 5 ' TATGTTATCC ACTGAATCAA TGATGAGCAG AATTGCGGCT GGAGACTTGG AGTCATCAGT GGATGATCCT AGATCAGAGG AGGATAAAAG ATTTGAGAGT, 3 ' SEQ NO.12 (HAV H2M20K5 2C district, 3986bp-4085bp) 5 ' CTCAGAGTTT TTCCAACTGG TTAAGAGATA TCTGTTCAGG GATCACTATT TTTAAAAACT TCAAGGATGC AATTTATTGG CTCTATACAA AATTGAAGGA 3 '
The invention still further relates to the varient of above-mentioned all cDNA nucleotide sequences because more than the dna sequence dna that provides only represented preferred example of the present invention.Because the degeneracy of genetic code, so can prepare many Nucleotide, they all are the dna sequence dnas that can instruct synthetic this virus protein or its analogue.Therefore, all be included within the present invention with the dna sequence dna of above-mentioned sequence equivalence on the function.
The invention still further relates to based on above-mentioned all cDNA sequences, design primer and selective amplification hepatitis A gene fragment, thereby in genetic stability detection, toxicity detection and the detection of cell adapted property and the biology sample detection of hepatitis A virus, use.
The invention still further relates to based on above-mentioned all cDNA sequences any hepatitis A virus (HAV) attenuated strain that using gene engineering technique is made.
The invention still further relates to based on above-mentioned all cDNA sequences any hepatitis A virus (HAV) polypeptide vaccine that using gene engineering technique is made.
The specific embodiment
Embodiment 1
The separation of hepatitis A virus Gkm strain and cDNA sequence thereof
Gather the suburbs, Hangzhou hepatitis A Patients with Acute stool sample, make 20% (V/V) with PBS (pH7.0) Suspension carries out extracting with F113～PEG precipitation and differential centrifugation, and extract is through Ultracentrifugation, Observe the hepatitis A virus particle that contains the 27nm size with the immuno-electron microscope detection, this is the HAV of preliminary purification The Gkm strain.
HAV Gkm strain virus extracts viral ribonucleic acid (RNA) through guanidinesalt/hot phenol method, closes with cDNA Become medicine box to cooperate high-fidelity polymerase chain reaction,PCR (PCR) technology, and molecule clone technology, HAV Gkm obtained The cDNA gene library of strain is used the dna sequence analysis medicine box at last, with the terminal cessation method of two deoxidations to The cDNA clone who has carries out sequence analysis, again by splicing, thus the cDNA of acquisition HAV Gkm strain Sequence. This sequence is shown in the literary composition with GkmNO.1.
Embodiment 2
Separation and the Partial cDNA Sequence thereof of hepatitis A virus H2K5 strain
HAV Gkm strain seed culture of viruses inoculation human embryonic lung diploid fibroblast KMB17 cell monolayer, 35 ℃ of absorption 2 are little The time, with Eagles and the lactoprotein maintenance medium of NBCS, put 35 ℃ of cultivations, change weekly maintenance medium one Inferior, to the 6th week, go former maintenance medium, with tryptose-EDTA liquid digestion, collecting cell, with freeze thawing, super Sonication extracting HAV carries out next round again and goes down to posterity take this HAV as seed culture of viruses. With said method at KMB17 Passed continuously for 5 generations in the cell, whenever substitute the propagation that direct immunofluorescence or ELISA monitor virus, thereby obtain The HAVH2K5 strain.
With reference to HAV Gkm strain cDNA sequence, synthetic HAV5 ' NCR district, 2A district and the 2C district of being positioned at 3 pairs of reverse-transcription polymerase chain reaction (RT-PCR) primer, the sequence of primer is as follows, from SEQ Primet NO.1 is until SEQ Primet NO.6. 1.SEQ Primer NO.1 (HAV5 ' NCR forward) 5 ' GGACTTGATACCTCACCG 3 ' 2.SEQ Primer NO.2 (HAV5 ' NCR reverse) 5 ' CTAATCATGGAGTTGACC 3 ' 3.SEQ Primer NO.3 (HAV 2A forward) 5 ' GTCACAGAGCAATCAGAGT 3 ' 4.SEQ Primer NO.4 (HAV 2A reverse) 5 ' AGCTTGTGAAAATAGTCC 3 ' 5.SEQ Primer NO.5 (HAV 2C forward) 5 ' CCAGAATGATGGAGCTG 3 ' 6.SEQ Primer NO.6 (HAV 2C reverse) 5 ' GGGTCAACTTGAGCCATT 3 '
HAV H2K5 strain virus extracts viral RNA through guanidinesalt/hot phenol method, utilizes above-mentioned 3 pairs of primers, uses The synthetic medicine box of cDNA cooperates the High fidelity PCR technology, obtains HAV H2K5 strain 5 ' NCR district, 2A district With 2C district genetic fragment, with the dna sequence analysis kit PCR fragment of purifying is carried out direct Sequencing, Obtain the Partial cDNA Sequence in HAV H2K5 strain 5 ' NCR district, 2A district and 2C district, sequence respectively Be shown in the literary composition, be SEQNO.1, SEQNO.2 and SEQNO.3.
Embodiment 3
Separation and the Partial cDNA Sequence thereof of hepatitis A virus H2K20 strain
HAV Gkm strain seed culture of viruses inoculation human embryonic lung diploid fibroblast KMB17 cell monolayer, 35 ℃ of absorption 2 are little The time, with Eagles and the lactoprotein maintenance medium of NBCS, put 35 ℃ of cultivations, change weekly maintenance medium one Inferior, to the 6th week, go former maintenance medium, with tryptose-EDTA liquid digestion, collecting cell, with freeze thawing, super Sonication extracting HAV carries out next round again and goes down to posterity take this HAV as seed culture of viruses. With said method at KMB17 Continuous passage in the cell, wherein 35 ℃ passed for 15 generations, and 32 ℃ passed for 5 generations, whenever substitute direct immunofluorescence or ELISA Monitor the propagation of virus, thereby obtain HAV H2K20 strain.
With reference to HAV Gkm strain cDNA sequence, synthetic HAV5 ' NCR district, 2A district and the 2C district of being positioned at 3 pairs of RT-PCR primers, the sequence of primer is seen appendix, from SEQ Primer NO.1 until SEQ Primer NO.6. HAV H2K5 strain virus extracts viral RNA through guanidinesalt/hot phenol method, utilizes above-mentioned 3 To primer, cooperate the High fidelity PCR technology with the synthetic medicine box of cDNA, obtain HAV H2K20 strain 5 ' NCR District, 2A district and 2C district genetic fragment are carried out the PCR fragment of purifying with the dna sequence analysis kit Direct Sequencing, the Partial cDNA Sequence in acquisition HAV H2K20 strain 5 ' NCR district, 2A district and 2C district, Sequence is shown in the literary composition, is SEQ NO.4, SEQ NO.5 and SEQ NO.6.
Embodiment 4
Separation and the Partial cDNA Sequence thereof of hepatitis A virus H2K25 attenuated strain
HAV Gkm strain seed culture of viruses inoculation human embryonic lung diploid fibroblast KMB17 cell monolayer, 35 ℃ of absorption 2 are little The time, with Eagles and the lactoprotein maintenance medium of NBCS, put 35 ℃ of cultivations, change weekly maintenance medium one Inferior, to the 6th week, go former maintenance medium, with tryptose-EDTA liquid digestion, collecting cell, with freeze thawing, super Sonication extracting HAV carries out next round again and goes down to posterity take this HAV as seed culture of viruses. With said method at KMB17 Continuous passage in the cell, wherein 35 ℃ passed for 15 generations, and 32 ℃ passed for 5 generations, and 35 ℃ passed for 5 generations again. Whenever substitute straight Connect the propagation that immunofluorescence or ELISA monitor virus, thereby obtain HAV H2K25 attenuated strain.
With reference to HAV Gkm strain cDNA sequence, synthetic HAV5 ' NCR district, 2A district and the 2C district of being positioned at 3 pairs of RT-PCR primers, the sequence of primer is seen appendix, from SEQ Primer NO.1 until SEQ Primer NO.6. HAV H2K5 strain virus extracts viral RNA through guanidinesalt/hot phenol method, utilizes above-mentioned 3 To primer, cooperate the High fidelity PCR technology with the synthetic medicine box of cDNA, obtain HAV H2K25 strain 5 ' NCR District, 2A district and 2C district genetic fragment are carried out the PCR fragment of purifying with the dna sequence analysis kit Direct Sequencing, the Partial cDNA Sequence in acquisition HAV H2K25 strain 5 ' NCR district, 2A district and 2C district, Sequence is shown in the literary composition, is SEQ NO.4, SEQ NO.5 and SEQ NO.6.
Embodiment 5
Separation and the Partial cDNA Sequence thereof of hepatitis A virus H2M20K5 attenuated strain
HAV Gkm strain seed culture of viruses inoculation cell monolayer, 35 ℃ adsorbed 2 hours, with the Eagles of NBCS And the lactoprotein maintenance medium, put 35 ℃ of cultivations, change weekly maintenance medium once, to the 6th week, go former maintenance medium, With tryptose-EDTA liquid digestion, collecting cell is with freeze thawing, ultrasonic processing extracting HAV, again with this HAV For carrying out next round, seed culture of viruses goes down to posterity. In former generation MK cells, passed continuously for 20 generations with said method, wherein 35 ℃ passed for 15 generations, 32 ℃ passed for 5 generations, and in the KMB17 cell, 32 ℃ passed for 5 generations more then. Whenever substitute directly Immunofluorescence or ELISA monitor the propagation of virus, thereby obtain HAV H2M20K5 attenuated strain.
With reference to HAV Gkm strain cDNA sequence, synthetic HAV5 ' NCR district, 2A district and the 2C district of being positioned at 3 pairs of RT-PCR primers, the sequence of primer is seen appendix, from SEQ Primer NO.1 until SEQ Primer NO.6. HAV H2M20K5 strain virus extracts viral RNA through guanidinesalt/hot phenol method, in the utilization State 3 pairs of primers, cooperate the High fidelity PCR technology with the synthetic medicine box of cDNA, obtain HAV H2K25 strain 5 ' NCR district, 2A district and 2C district genetic fragment are with the PCR sheet of dna sequence analysis kit to purifying The Duan Jinhang direct Sequencing, the part cDNA in acquisition HAV H2K25 strain 5 ' NCR district, 2A district and 2C district Sequence, sequence are shown in the literary composition, are SEQ NO.4, SEQ NO.5 and SEQ NO.6.
Compare with original velogen strain Gkm strain, the H2M20K5 attenuated strain is low virulent strain to people and monkey specie Reaction is the seed culture of viruses of attenuated live vaccine for hepatitis A (H2).
Claims (15)
1. the virus of an isolating and purifying is characterized in that it is hepatitis A virus (HAV) Chinese epidemic strain Gkm.
2. complementary DNA (cDNA) (cDNA) sequence of HAV Gkm strain is characterized in that this cDNA has the cDNA sequence as HAV GkmNO.1.
3. one kind through the HAV of passage virus strain H2K5, it is characterized in that its virus strain that to be HAV Gkm strain go down to posterity and obtain after five times through human embryonic lung diploid fibroblast KMB17 low temperature.
4. the cDNA sequence of HAV H2K5 strain is characterized in that, this cDNA has three sections cDNA sequences as SEQ NO.1, SEQ NO.2 and SEQ NO.3.
5. one kind through the HAV of passage virus strain H2K20, it is characterized in that its virus strain that to be HAV Gkm strain go down to posterity and obtain after 20 times through human embryonic lung diploid fibroblast KMB17 low temperature.
6. the cDNA sequence of HAV H2K20 strain is characterized in that, this cDNA has the cDNA sequence as HAV H2K20NO.1.
7. one kind through the HAV of passage virus strain H2K25, it is characterized in that its attenuated viral strains that to be HAV Gkm strain go down to posterity and obtain after 25 times through human embryonic lung diploid fibroblast KMB17 low temperature.
8. the cDNA sequence of HAV H2K25 strain is characterized in that, this cDNA has three sections cDNA sequences as SEQ NO.7, SEQ NO.8 and SEQ NO.9.
9. one kind through the HAV of passage virus strain H2M20K5, it is characterized in that, it is HAV Gkm strain in former generation monkey-kidney cells low temperature goes down to posterity 20 times, and the attenuated viral strains that human embryonic lung diploid fibroblast KMB17 low temperature goes down to posterity and obtains after 5 times is the seed strain of HAV attenuated live vaccine (H2).
10. the cDNA sequence of HAV H2M20K5 strain is characterized in that, this cDNA has three sections cDNA sequences as SEQNO.10, SEQ NO.11 and SEQ NO.12.
11. the cDNA sequence shown in claim 8 and 10 is characterized in that, this sequence is the necessary sequence of HAV attenuated strain.
12. the cDNA sequence shown in claim 2,4,6,8,10 is characterized in that, this series nucleotide sequence can be used for cultivation and the preparation of HAV attenuated strain.
13. a method of differentiating the hepatitis A virus (HAV) attenuated strain is characterized in that, attenuated strain can obtain cDNA sequence or its varient shown in claim 8 and 10 behind reverse transcription-polymerase chain reaction.
14. a method that detects hepatitis A virus (HAV) attenuated strain genetic stability is characterized in that, attenuated strain can obtain cDNA sequence or its varient shown in claim 8 and 10 behind reverse transcription-polymerase chain reaction.
15. a method of cultivating the hepatitis A virus (HAV) attenuated strain is characterized in that, HAV is through specific cells, under the specified temp, and continuous passage 20-25 generation, thus obtain attenuated strain.
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| CN 01140508 CN1206356C (en) | 2001-09-13 | 2001-09-13 | Breeding of hepatitis A virus Chinese strain and attenuated strain and its complementary DNA sequence |
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| CN 01140508 CN1206356C (en) | 2001-09-13 | 2001-09-13 | Breeding of hepatitis A virus Chinese strain and attenuated strain and its complementary DNA sequence |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102757941A (en) * | 2012-06-29 | 2012-10-31 | 北京健翔和牧生物科技有限公司 | Hepatitis A virus strain HAV-ZL2012, vaccine prepared by using same and application thereof |
| CN105349500A (en) * | 2015-11-26 | 2016-02-24 | 中国疾病预防控制中心病毒病预防控制所 | Novel hepatitis A virus strain and application thereof |
| CN109207638A (en) * | 2018-10-11 | 2019-01-15 | 天津国际生物医药联合研究院 | For detecting the real-time fluorescent quantitative RT-PCR method of Hepatitis A virus nucleic acid |
-
2001
- 2001-09-13 CN CN 01140508 patent/CN1206356C/en not_active Expired - Lifetime
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102757941A (en) * | 2012-06-29 | 2012-10-31 | 北京健翔和牧生物科技有限公司 | Hepatitis A virus strain HAV-ZL2012, vaccine prepared by using same and application thereof |
| CN102757941B (en) * | 2012-06-29 | 2014-01-29 | 北京健翔和牧生物科技有限公司 | Hepatitis A virus strain HAV-ZL2012, vaccine prepared by using same and application thereof |
| CN105349500A (en) * | 2015-11-26 | 2016-02-24 | 中国疾病预防控制中心病毒病预防控制所 | Novel hepatitis A virus strain and application thereof |
| CN109207638A (en) * | 2018-10-11 | 2019-01-15 | 天津国际生物医药联合研究院 | For detecting the real-time fluorescent quantitative RT-PCR method of Hepatitis A virus nucleic acid |
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| CN1206356C (en) | 2005-06-15 |
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