Porcine reproductive and respiratory syndrome virus attenuated strain, vaccine composition and preparation method and application thereof

A technology for respiratory syndrome and vaccine composition, which is applied in the field of constructing attenuated strains of porcine reproductive and respiratory syndrome virus, which can solve the problem of strong virulence, frequent inactivation vaccinations, inability to induce immunity of heterologous PRRS virus strains, etc. question

Active Publication Date: 2014-12-31
PU LIKE BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, the existing commercialized PRRS vaccines include inactivated vaccines and attenuated live vaccines, but inactivated vaccines are inoculated many times, the antibody production is slow, and the protective effect is unstable, which often leads to immune failure; it cannot induce strong immunity against heterologous PRRS virus strains. immunity
The attenuated live vaccine is made by weakening the strong virus strain through continuous passage, which requires a lot of manpower and material resources. Immune protection between strains of the

Method used

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  • Porcine reproductive and respiratory syndrome virus attenuated strain, vaccine composition and preparation method and application thereof
  • Porcine reproductive and respiratory syndrome virus attenuated strain, vaccine composition and preparation method and application thereof
  • Porcine reproductive and respiratory syndrome virus attenuated strain, vaccine composition and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1 Construction and biological characteristics of HP-PRRSV10FUJ-2 strain infectious molecular clone

[0075] 1.1 Construction of infectious molecular clone of HP-PRRSV10FUJ-2 strain

[0076] 1.1.1 Transformation of pBluescript II KS(+) vector

[0077] According to the restriction site analysis of the whole genome sequence of 10FuJ-2, the multiple cloning site region of pBluescript II KS(+) was modified to facilitate subsequent full-length cDNA splicing. The enzyme cutting sites in the transformed multiple cloning site region are in sequence: MluI-SfiI-XhoI-BamHI-BglII-NotI. The modified vector was identified by NotI digestion and then sent to Invitrogen for sequencing to verify the correctness of the modified multi-cloning site region. The transformed vector was named pBSMN.

[0078] 1.1.2 Primer design and synthesis

[0079] According to NCBI ( http: / / www.ncbi.nlm.nih.gov The complete genome sequence of highly pathogenic porcine reproductive and respirator...

Embodiment 2

[0099] Example 2 Construction and Identification of Porcine Reproductive and Respiratory Syndrome Virus Attenuated Strain (10FUJ-2-MLV)

[0100] 2.1 Construction of attenuated strain of porcine reproductive and respiratory syndrome virus (10FUJ-2-MLV)

[0101] 2.1.1 Primer design and synthesis

[0102] According to NCBI ( http: / / www.ncbi.nlm.nih.gov ) reported in The gene sequences of non-structural proteins Nsp9 and Nsp10 in the PRRS MLV strain (Accession No.: AF159149) (see sequence table SEQ ID No.3 and SEQ ID No.4 in turn) were synthesized including the gene sequences of Nsp9 and Nsp10. The 7591-9382bp sequence of the MLV genome was synthesized in TAKARA company (the 7601-7612bp position is the single enzyme cutting site BstXI shared by 10FuJ-2 and MLV, and the sequence between 7591-7600bp is replaced by the sequence corresponding to the 10FuJ-2 position; 9366 The -9372bp position is the single restriction site BamHI shared by 10FuJ-2 and MLV, the sequence between 937...

Embodiment 3

[0113] Example 3 Construction and Identification of Porcine Reproductive and Respiratory Syndrome Virus Attenuated Strain (10FUJ-2-CH-1R)

[0114] 3.1 Construction of attenuated strain of porcine reproductive and respiratory syndrome virus (10FUJ-2-CH-1R)

[0115] According to NCBI ( http: / / www.ncbi.nlm.nih.gov ) in the CH-1R strain (accession number: EU807840) reported in the gene sequence of the non-structural protein Nsp9, Nsp10 (see the sequence table SEQ ID No.5, SEQ ID No.6), construct the plasmid according to the method of Example 2.1 And the virus was rescued, and the rescued virus was named v10FuJ-CH-1R.

[0116] 3.2 Identification of rescued virus

[0117] 3.2.1 Gene identification

[0118] The virus supernatant RNA was extracted from the recombinant virus v10FuJ-CH-1R, identified by RT-PCR, and the fragment containing Nsp9 and Nsp10 was amplified, and sent to Invitrogen for sequencing. The results showed that the sequences of Nsp9 and Nsp10 in the recombinant vi...

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Abstract

The invention provides a porcine reproductive and respiratory syndrome virus attenuated strain. The porcine reproductive and respiratory syndrome virus is a novel recombinant chimeric virus obtained by respectively substituting a Nsp9 gene and a Nsp10 gene of a highly pathogenic porcine reproductive and respiratory syndrome virus strain with a Nsp9 gene and a Nsp10 gene of a classic porcine reproductive and respiratory syndrome virus attenuated strain. The porcine reproductive and respiratory syndrome virus attenuated strain can further contain a GP5 gene of the highly pathogenic porcine reproductive and respiratory syndrome virus attenuated strain. The invention also discloses a vaccine composition containing the attenuated strain and application thereof to preparation of drugs for preventing and / or treating porcine reproductive and respiratory syndrome. The attenuated strain overcomes the security problems of the artificial passage attenuated vaccine; the vaccine composition prepared from the attenuated strain not only can be used for the prevention of common porcine reproductive and respiratory syndrome but also can prevent highly pathogenic porcine reproductive and respiratory syndrome.

Description

technical field [0001] The invention belongs to the technical field of veterinary biological products, and in particular relates to constructing an attenuated strain of porcine reproductive and respiratory syndrome virus by reverse genetic technology and its application. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS), also known as "pig blue ear disease", is a disease of pigs caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV). Highly contagious disease, mainly characterized by reproductive disorders such as fever, anorexia, early abortion, stillbirth, mummified fetus, and weak piglets in sows, respiratory symptoms and high mortality in piglets (Yin Zhen, Liu Jinghua. Animal Virology. 2nd Edition . Beijing: Science Press. 1997). At the same time, PRRSV can cause immunosuppression in animals, and then secondary infections of various pathogens. [0003] The disease was ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01A61K39/12A61P31/14C12R1/93
Inventor 张许科孙进忠谭菲菲田克恭
Owner PU LIKE BIO ENG
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