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H1N1 subtype swine influenza virus and application thereof

A swine flu virus, H1N1 technology, applied in the direction of antiviral agents, viruses/phages, medical preparations containing active ingredients, etc., can solve the problem of increased mortality, complicated epidemic situation, concurrent or secondary infections of other viruses or bacteria in pig herds and other problems, to achieve the effect of good immunogenicity and high proliferative ability

Active Publication Date: 2012-07-18
兆丰华生物科技(南京)有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, swine flu is an immunosuppressive disease, which often leads to concomitant or secondary infection of other viruses or bacteria in pig herds, making the epidemic more complicated, increasing the mortality rate, and causing huge economic losses to the animal husbandry industry

Method used

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  • H1N1 subtype swine influenza virus and application thereof
  • H1N1 subtype swine influenza virus and application thereof
  • H1N1 subtype swine influenza virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Isolation and Identification of H1N1 Subtype Swine Influenza Virus A / Swine / Shanxi / D5 / 2011 (H1N1)

[0026] (1) Isolation of H1N1 subtype swine influenza virus A / Swine / Shanxi / D5 / 2011 (H1N1)

[0027] The lung disease materials of pigs suspected of having swine flu disease were collected from a pig farm in Shanxi and cut into pieces, then added sterilized saline containing penicillin (1000U / ml) and streptomycin (1000μg / ml) at a ratio of 1g:5ml, and repeated grinding with a tissue grinder Freeze and thaw 3 times, centrifuge at 4000r / min for 10min, take the supernatant, filter it through a disposable filter, inoculate 9-11 day-old SPF chicken embryos through the allantoic cavity, 0.2ml / embryo, incubate in a 35°C incubator, discard The chicken embryos that died within 24 hours were inoculated, and the allantoic fluid of the surviving chicken embryos was harvested 72 hours after inoculation.

[0028] (2) Detection of hemagglutination activity

[0029] According to t...

Embodiment 2

[0045] Example 2 H1N1 Subtype Swine Influenza Virus A / Swine / Shanxi / D5 / 2011 (H1N1 Whole Genome Sequence Determination and Evolution Village Mapping

[0046] (1) RT-PCR amplification of 8 gene fragments:

[0047] Referring to the published relevant information of H1N1 subtype swine influenza, design and synthesize the RT-PCR primers of 8 influenza virus genes HA, NA, NP, PB1, PB2, PA, M, NS, and the primer sequences are shown in Table 1. The RNA of the virus was extracted and reverse-transcribed into cDNA using random primers, and 8 gene fragments were amplified using the cDNA as a template. Among them, the long fragments PB2, PB1, and PA were amplified in segments to obtain the products at both ends of the amplified segments. The full length was then amplified by overlapping PCR. The remaining five fragments HA, NA, NP, M and NS were amplified to full length at one time.

[0048] Table 1 Primer sequences of 8 gene segments of swine influenza virus

[0049]

[0050]

[...

Embodiment 3

[0057] Example 3 Determination of biological characteristics of H1N1 subtype swine influenza virus A / Swine / Shanxi / D5 / 2011 (H1N1)

[0058] 1. Continuous passage and EID of H1N1 subtype swine influenza virus A / Swine / Shanxi / D5 / 2011 (H1N1) on chicken embryos 50 Determination of

[0059] (1) Continuous passage on chicken embryos

[0060] The F1 generation H1N1 subtype swine influenza virus A / Swine / Shanxi / D5 / 2011 (H1N1) was carried out with 1×PBS for 10 -3 Dilute, inoculate 9-11 day-old SPF chicken embryos, 0.2ml / embryo, incubate in an incubator at 35°C for 72 hours, collect chicken embryo allantoic fluid for hemagglutination detection. According to this method, the virus can be passed continuously for 12 generations on chicken embryos, and the hemagglutination value is stable, up to 2 9 , the allantoic fluid collected from each generation was stored at -80°C for later use.

[0061] (2)EID 50 Determination of

[0062] Will F 13 Generation H1N1 subtype swine influenza virus A / ...

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Abstract

The invention discloses an H1N1 subtype swine influenza virus A / Swine / Shanxi / D5 / 2011 (H1N1) with the preservation number of CGMCC No.5323 and also discloses a vaccine containing the virus. The H1N1 subtype swine influenza virus provided by the invention is not polluted by extraneous pathogeny, has higher multiplication capacity on both a chicken embryo and a cell, shows obvious clinical symptoms after infecting a BALB / c rat and a swine influenza negative piglet and has typical autopsy pathological changes and good immunogenicity, and an oil emulsion inactivated vaccine prepared by using the H1N1 subtype swine influenza virus has 100% of protective efficiency for the attack of a homologous virus after immunizing a pig and serves as a favorable candidate swine influenza vaccine strain.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a H1N1 subtype swine influenza virus and its application. Background technique [0002] Swine influenza (SI) is an acute and infectious porcine respiratory disease caused by type A influenza virus, which evolves through continuous antigenic drift or antigenic shift, resulting in numerous subtypes and difficult to eradicate. At present, there are 11 serotypes of swine influenza virus, including H1N1, H1N2, H3N2, H1N7, H2N3, H3N1, H3N3, H3N6, H4N6, H5N1, and H9N2. H1N2 and human-like H3N2 subtypes. At the same time, swine flu is an immunosuppressive disease, which often leads to concomitant or secondary infection of other viruses or bacteria in pig herds, making the epidemic more complicated, increasing the mortality rate, and causing huge economic losses to the animal husbandry. [0003] Pigs have both sialic acid α-2,3-galactose (SAα-2,3-Gal) and sialic acid α-2,6-galac...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K39/145A61P31/16C12R1/93
Inventor 杜金玲王丹娜孟珊珊吕超超王吉玮石磊赵亚荣
Owner 兆丰华生物科技(南京)有限公司
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