Influenza virus vaccine strain

Abstract

The invention discloses an influenza virus vaccine strain. The invention discloses a virus. The amino acid sequence of each of PB2 protein, PB1 protein, PA protein, NP protein, NA protein, M1 protein, M2 protein, NS1 protein, NS2 protein and HA protein are respectively shown as follows: SEQ ID No. 26, SEQ ID No. 34, SEQ ID No. 33, SEQ ID No. 21, SEQ ID No. 29, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 31, SEQ ID No. 32 and SEQ ID No. 16. The recombinant virus disclosed by the invention can be rapidly and stably proliferated in high yield. Compared with the method for preparing the vaccine by utilizing the chicken embryo, the recombinant virus has obvious advantage, and the inactivated vaccine of the recombinant virus can be used for protecting animals against the infection of influenza virus.

Description

technical field

[0001] The present invention relates to an influenza virus vaccine strain. Background technique

[0002] The H9N2 subtype avian influenza virus mainly infects chickens, ducks, turkeys, quails, wild ducks and other poultry, and can cause clinical symptoms such as low mortality and mild respiratory infections or decreased egg production rate. Although the H9N2 subtype avian influenza virus is a low-pathogenic avian influenza virus, it can cause a serious drop in the egg production of laying hens, and an increase in the combined respiratory disease and mortality rate of broiler chickens and young chickens, often causing serious economic losses. . It is worth noting that the H9N2 subtype avian influenza virus can not only infect birds, but also break through the interspecies barrier and infect mammals and humans without going through an intermediate host. Since the first case of human infection with H9N2 subtype avian influenza was discovered in the world in 19...

Examples

Embodiment 1

[0050] Example 1. Rescue and purification of recombinant virus using reverse genetic manipulation technology

[0051] 1. Extract the RNA of A / Chicken / Hebei / YT / 2010 and A / Puerto Rico / 8 / 34 viruses.

[0052] 2. Reverse transcription was carried out using the RNAs of the two viruses as templates respectively.

[0053] The reverse transcription system is as follows:

[0054]

[0055] Add the above-mentioned ingredients in turn to the reaction tube, mix well, and centrifuge briefly.

[0056] Wherein vRNA refers to the RNA of the virus obtained in step one.

[0057] Reverse transcription conditions: 42°C for 1h, 94°C for 5min.

[0058] The cDNA product after reverse transcription was used as a template for PCR reaction.

[0059] 3. PCR amplification

[0060] The PCR reaction system is as follows:

[0061]

[0062] Wherein the cDNA is the cDNA of the A / Chicken / Hebei / YT / 2010 or A / Puerto Rico / 8 / 34 virus obtained in step 2.

[0063] Add the above ingredients in sequence, cen...

Embodiment 2

[0174] Embodiment 2, passage, purification and identification of recombinant virus

[0175] One, the 18 strains of recombinant viruses that embodiment 1 obtains are carried out serial passage on MDCK cell, and specific method is as follows: the mixed solution that contains the cell of virus and supernatant is mixed with 10 respectively. -2 、10 -3 、10 -4 Infect MDCK cells at a dilution ratio (DMEM containing 1% double antibody), select the virus with the highest HA titer (that is, the highest dilution factor at which the virus can agglutinate red blood cells) at each dilution ratio, and then use the selected virus as Infect MDCK cells in the same way, and continuously select the virus with the highest HA titer (that is, the highest dilution factor at which the virus can agglutinate red blood cells) at each dilution. In this way, 18 strains of recombinant virus were continuously transmitted to the 10th generation.

[0176] 2. Plaque purification of passaged virus

[0177] (1...

Embodiment 3

[0191] Embodiment 3, passage poison TCID 50 / mL and determination of hemagglutination titer

[0192] 1. Preparation of MDCK monolayer cells

[0193] Transfer MDCK cells to a 96-well cell culture plate 24 hours before the experiment. When the cells grow to 70%-90%, remove the culture solution in the well with a 10mL pipette, wash the cells with PBS, discard the washing solution, and repeat the washing 3 times.

[0194] 2. Doubling dilution of virus solution

[0195] (1) Take out the purified virus solution of 18 strains obtained in Example 2 in a -80°C refrigerator 30 minutes before the experiment, put it in a 4°C refrigerator, and let it melt naturally.

[0196] (2) Each virus was sequentially diluted to 10 times with DMEM containing 1% double antibody. -8 , mark it.

[0197] (3) Store the doubly diluted virus solution on ice.

[0198] 3. Inoculate the virus

[0199] (1) Inoculate MDCK cells on a 96-well plate, inoculate 3 cell wells with each dilution of virus solution...

technical field

[0001] The present invention relates to an influenza virus vaccine strain. Background technique

[0002] The H9N2 subtype avian influenza virus mainly infects chickens, ducks, turkeys, quails, wild ducks and other poultry, and can cause clinical symptoms such as low mortality and mild respiratory infections or decreased egg production rate. Although the H9N2 subtype avian influenza virus is a low-pathogenic avian influenza virus, it can cause a serious drop in the egg production of laying hens, and an increase in the combined respiratory disease and mortality rate of broiler chickens and young chickens, often causing serious economic losses. . It is worth noting that the H9N2 subtype avian influenza virus can not only infect birds, but also break through the interspecies barrier and infect mammals and humans without going through an intermediate host. Since the first case of human infection with H9N2 subtype avian influenza was discovered in the world in 19...

Examples

Embodiment 1

[0050] Example 1. Rescue and purification of recombinant virus using reverse genetic manipulation technology

[0051] 1. Extract the RNA of A / Chicken / Hebei / YT / 2010 and A / Puerto Rico / 8 / 34 viruses.

[0052] 2. Reverse transcription was carried out using the RNAs of the two viruses as templates respectively.

[0053] The reverse transcription system is as follows:

[0054]

[0055] Add the above-mentioned ingredients in turn to the reaction tube, mix well, and centrifuge briefly.

[0056] Wherein vRNA refers to the RNA of the virus obtained in step one.

[0057] Reverse transcription conditions: 42°C for 1h, 94°C for 5min.

[0058] The cDNA product after reverse transcription was used as a template for PCR reaction.

[0059] 3. PCR amplification

[0060] The PCR reaction system is as follows:

[0061]

[0062] Wherein the cDNA is the cDNA of the A / Chicken / Hebei / YT / 2010 or A / Puerto Rico / 8 / 34 virus obtained in step 2.

[0063] Add the above ingredients in sequence, cen...

Embodiment 2

[0174] Embodiment 2, passage, purification and identification of recombinant virus

[0175] One, the 18 strains of recombinant viruses that embodiment 1 obtains are carried out serial passage on MDCK cell, and specific method is as follows: the mixed solution that contains the cell of virus and supernatant is mixed with 10 respectively. -2 、10 -3 、10 -4 Infect MDCK cells at a dilution ratio (DMEM containing 1% double antibody), select the virus with the highest HA titer (that is, the highest dilution factor at which the virus can agglutinate red blood cells) at each dilution ratio, and then use the selected virus as Infect MDCK cells in the same way, and continuously select the virus with the highest HA titer (that is, the highest dilution factor at which the virus can agglutinate red blood cells) at each dilution. In this way, 18 strains of recombinant virus were continuously transmitted to the 10th generation.

[0176] 2. Plaque purification of passaged virus

[0177] (1...

Embodiment 3

[0191] Embodiment 3, passage poison TCID 50 / mL and determination of hemagglutination titer

[0192] 1. Preparation of MDCK monolayer cells

[0193] Transfer MDCK cells to a 96-well cell culture plate 24 hours before the experiment. When the cells grow to 70%-90%, remove the culture solution in the well with a 10mL pipette, wash the cells with PBS, discard the washing solution, and repeat the washing 3 times.

[0194] 2. Doubling dilution of virus solution

[0195] (1) Take out the purified virus solution of 18 strains obtained in Example 2 in a -80°C refrigerator 30 minutes before the experiment, put it in a 4°C refrigerator, and let it melt naturally.

[0196] (2) Each virus was sequentially diluted to 10 times with DMEM containing 1% double antibody. -8 , mark it.

[0197] (3) Store the doubly diluted virus solution on ice.

[0198] 3. Inoculate the virus

[0199] (1) Inoculate MDCK cells on a 96-well plate, inoculate 3 cell wells with each dilution of virus solution...

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