Hog cholera vaccine infectious cDNA (Complementary Deoxyribonucleic Acid) as well as construction method and application thereof

A swine fever vaccine and swine fever technology, applied in recombinant DNA technology, applications, botanical equipment and methods, etc., can solve the problems of huge harm and high mortality of pigs, and achieve the effect of improving efficiency

Active Publication Date: 2015-05-27
WUHAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] On the other hand, CSFV infection causes immunosuppression in pigs, and pigs infected with CSFV are more prone to circovirus, porcine reproductive and respiratory syndrome virus, B Mixed infection with encephalitis virus, etc., leads to higher death rate of pigs and greater harm

Method used

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  • Hog cholera vaccine infectious cDNA (Complementary Deoxyribonucleic Acid) as well as construction method and application thereof
  • Hog cholera vaccine infectious cDNA (Complementary Deoxyribonucleic Acid) as well as construction method and application thereof
  • Hog cholera vaccine infectious cDNA (Complementary Deoxyribonucleic Acid) as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] [Example 1] A new infectious cDNA clone construction method for preparing swine fever vaccine seed virus

[0046] 1. Design and synthesis of primers

[0047] According to the full-length genome sequence of CSF vaccine C strain (Genbank No.AF091507), 6 pairs of specific PCR amplification primers were designed and synthesized for the in vitro synthesis of 6 cDNA fragments of the CSF vaccine C strain full-length genome. The primers were synthesized by Shanghai Shenggong Company. Genomic cDNA of strain C of swine fever vaccine was synthesized by in vitro reverse transcription using 9-nucleotide random primer pd(N)9, which is a product of Biocolor (UK).

[0048] The length of each cDNA fragment, the sequence and number of 6 pairs of specific primers from the 5' to the 3' end of the genome are as follows:

[0049] 1). Fragment 1 (CSf1: 1-2431nt) PCR amplification primer

[0050] Upstream primer P1F (SEQ ID NO.3):

[0051] 5′-GC GTC GAC TCTTAATACGACTCACTATAGTATACGAGGTTAG...

Embodiment 2

[0169] [Example 2] Construction of recombinant swine fever vaccine virus expressing exogenous protein luciferase

[0170] According to the full-length genome sequence of CSF vaccine C strain (Genbank No.AF091507), sea cucumber luciferase coding sequence (GenBank No.AF025846.2), foot-and-mouth disease virus (FMDV) 2A coding sequence (GenBank No.M95781.1), design Synthesize various RT-PCR or PCR amplification primers. The primers were synthesized by Shanghai Shenggong Company.

[0171] 1. Construction of the recombinant plasmid pASPI-CSf123 / RLuc fused to the 5' end of the luciferase gene CSF vaccine

[0172] A). Preparation of the restriction endonuclease PmlI site fragment near the 5' end of the swine fever vaccine genome (located at the 290-900 nt of the genome): design primers according to the genome sequence of the swine fever vaccine, and PCR amplify the restriction endonuclease near the 5' end of the genome Dicer PmlI site fragment:

[0173] Upstream primer CS-fPmF (SEQ...

Embodiment 3

[0220] [Example 3] Recombinant classical swine fever vaccine induces immune efficacy detection of pig body anti-CSFV

[0221] 1. Pig body immunization

[0222] Healthy 2-month-old piglets that were not inoculated with CSF, were negative for CSFV by immunofluorescence detection, and negative for CSFV serum antibodies by ELISA were selected and randomly divided into two groups for the vaccination experimental group and the non-vaccination control group.

[0223] Specifically, 6-week-old healthy piglets that had not been vaccinated against classical swine fever were taken, and blood was collected from the anterior vena cava, and divided into two parts. One part was used to separate serum for ELISA detection of CSFV antibodies; the other part was used for detection of CSFV virus. Specific detection method: Freeze-thaw lysed pig whole blood, centrifuge to separate the supernatant, perform 10-fold serial dilution with serum-free DMEM, inoculate PK15 cells, and perform indirect immun...

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Abstract

The invention belongs to the technical field of animal biological products, and in particular relates to construction of plasmid pASPITI-CS. The plasmid comprises hog cholera vaccine C strain infectious cDNA clone, fusion of a hog RNA (Ribonucleic Acid) polymerase I promoter sequence at the 5' tail end of the genome cDNA, and insertion of plasmid pASPITI-CS of a mouse RNA polymerase I termination sub-sequence at the 3' tail end. The plasmid is transfected to hog origin cells PK15, so that hog cholera vaccine C strain virus can be directly generated. Escherichia coli DH10B/pASPITI-CS transformed with the plasmid pASPITI-CS is preserved in the China centre for type culture collection; and the preservation number is CCTCC NO.M2015062. The hog cholera vaccine C strain infectious cDNA clone can be used for genetically modifying to prepare high-titre hog cholera vaccines or constructing bivalent vaccines based on a hog cholera vaccine vector.

Description

technical field [0001] The invention belongs to the technical field of veterinary biological products, and relates to the use of reverse genetic manipulation to obtain an infectious cDNA clone of a CSF attenuated vaccine C strain, as well as the use of the infectious cDNA clone in constructing high-potency recombinant CSF live vaccines and CSF vaccines application in the carrier. Background technique [0002] Swine fever is a highly contagious viral disease of pigs. It was called hog cholera (Hog Cholera, HC) in the early stage. It can infect pigs of all ages. It is prevalent throughout the year, with high morbidity and mortality. huge. The causative agent of swine fever is classical swine fever virus (CSFV). In the "International Animal Health Code" formulated by the International Office of Epizootics (OIE), swine fever is listed as one of the 16 legally notifiable infectious diseases in category A. . In recent years, swine fever has occurred frequently in our country, w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/40C12N15/85C12N15/66C12N7/00A61K39/187A61P31/14
Inventor 潘兹书吴锐李玲庞会宁赵昱
Owner WUHAN UNIV
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