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High-yield recombinant influenza B virus strain and its application

An influenza B virus, influenza virus technology, applied in the direction of antiviral agents, viruses/phages, medical preparations containing active ingredients, etc., can solve the problems of low yield, increase the difficulty of transfection, and limit the use of cell lines, etc. Achieving the effect of high application value

Inactive Publication Date: 2009-04-29
中国疾病预防控制中心病毒病预防控制所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] But it is worth noting that both of the above two systems have disadvantages: that is, the yield is low, and most of them are defective viruses.
The disadvantage of this system is that the co-transfection of 12 plasmids increases the difficulty of transfection and limits the use of cell lines

Method used

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  • High-yield recombinant influenza B virus strain and its application
  • High-yield recombinant influenza B virus strain and its application
  • High-yield recombinant influenza B virus strain and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1 Obtaining of the entire genome of influenza B virus

[0076] Strains: B / Jingke / 96 / 01 is from the National Influenza Center, B / Sichuan / 379 / 99 and B / HongKong / 330 / 01 are from the US CDC.

[0077] RT-PCR: The screened strain B / Jingke / 96 / 01 was serially diluted, inoculated into MDCK cells, and the virus was harvested 72 hours later. Viral RNA was extracted using RNeasy KIT (Qiagen), dissolved in 30ul of water and diluted.

[0078] The RT-PCR reaction system was denatured at 94°C for 3 minutes and extended at 55°C for 1 hour. The reverse transcriptase was provided by Invitrogen, and then the PCR system was 94°C 45Sec 50°C 45Sec 72°C 420Sec, and the PFU enzyme was purchased from (Promega).

[0079] Amplify the entire genome of B / Jingke / 96 / 01, use BM-NS-1BM-NS-2 primers to amplify the HA and NA fragments of popular strains, and connect the PCR products to the PGEM-T vector (Promega Company) superior.

[0080] The result obtained through the method of the present inv...

Embodiment 2

[0086] Construction of embodiment 2 PIVVII plasmid

[0087] One: Construction of PIVV II plasmid:

[0088] see Figure 10 and Figure 11 Plasmid map and experimental rationale shown.

[0089] 1: Features of the PIVV II plasmid: The inventors constructed the PIVV II plasmid. The characteristic of the plasmid is that it has both polymerase I and polymerase II promoter and terminator sequences, and the direction of the promoter of polymerase I and polymerase II is opposite. The viral genome is inserted between the promoter of polymerase 1 and the BSMB1 site of the terminator sequence, and the genome of the virus is transcribed under the action of the polymerase 1 promoter, and the viral genome is transcribed under the action of the polymerase II promoter. mRNA, which translates the viral protein,

[0090] 2: Construction method of PIVV II plasmid: amplify the promoter and terminator sequence of PHH21 plasmid (gifted by Neumann G), and use primers:

[0091] Upstream: 5'ATCCC...

Embodiment 3

[0094] Example 3 The fragments of the viral genome are connected to the PIVV II vector respectively

[0095] The PIVV II plasmid vector obtained in Example 2 was digested with BSMB1 and linearized, and then the linearized PIVV II fragment was recovered with a Qiagen recovery kit. Similarly, the target fragment ligated to the PGEM-T vector was digested with BSMB1, and then the target fragment was recovered. Connect the fragments described in sequences 1-8 to the PIVV II vector respectively, the system is 3ul target fragment, 1ul vector, 1ulT 4 DNA ligase (Promega) 15ulddH 2 O, thereby obtaining the connection product PIVV II-BX.

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Abstract

The present invention relates to a kind of new influenza B virus complete gene sequence with mammalian cell high-yield characteristics and carrier containing said equence and strain. Said invention also relates to the influenza B vaccine prepared by using the described strain.

Description

technical field [0001] The present invention relates to a high-yielding recombinant influenza B virus strain and its use, as well as the nucleic acid and primers of the recombinant virus strain, especially the influenza B vaccine prepared through the strain. Background technique [0002] Influenza is a respiratory disease caused by influenza virus. According to the "Science" magazine, during the high flu season, due to the reduction of the labor force and the increase of medical expenses, the economic loss to the United States is more than 12 billion US dollars. In addition, more than 20,000 Americans die from the flu every year. [0003] The influenza storm that swept the world in 1918 caused a total of 20-40 million deaths, more than the total number of deaths in World War I. The last flu to sweep the world occurred in 1968 and claimed the lives of nearly 500,000 people around the world, most of them elderly, but many young people who were once healthy. Influenza outbre...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/44C12P19/34A61K39/145A61P31/16
Inventor 张智清齐立董婕徐红
Owner 中国疾病预防控制中心病毒病预防控制所
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