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Construction method and application of recombinant chimeric Newcastle disease virus for expressing chicken infectious bursal disease virus variant VP2 gene

A technology for Newcastle disease virus and bursal disease, which can be used in applications, antiviral agents, genetic engineering, etc., and can solve problems such as the limitation of clinical application of recombinant live vector vaccines

Pending Publication Date: 2022-04-08
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the high level of anti-NDV LaSota strain maternal antibodies in chicken flocks in my country and the inability of vaccines based on LaSota strains to effectively control the infection of genotype VII NDV that is currently prevalent in my country, the clinical application of recombinant live vector vaccines based on LaSota strains restricted
Although my country already has a commercialized single inactivated vaccine for the prevention and control of genotype VII NDV (Yao Shunyu, Zhang Lilin. Research progress on Newcastle disease vaccine [J]. Biotechnology Progress, 2020,10(5):470-478.), However, due to the limitations of new veterinary drug certificates and safety issues, there are currently no multiple inactivated vaccines and attenuated live vaccines for the prevention and control of genotype VII NDV in China

Method used

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  • Construction method and application of recombinant chimeric Newcastle disease virus for expressing chicken infectious bursal disease virus variant VP2 gene
  • Construction method and application of recombinant chimeric Newcastle disease virus for expressing chicken infectious bursal disease virus variant VP2 gene
  • Construction method and application of recombinant chimeric Newcastle disease virus for expressing chicken infectious bursal disease virus variant VP2 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1 has the construction of the LaSota strain infectious clone of the F gene of gene VII type NDV epidemic strain HN gene and alkaline protease cleavage site amino acid mutation

[0053] 1.1 Construction of infectious clone plasmid of NDV LaSota vaccine strain

[0054] According to the sequence of the NDV LaSota strain published in GenBank (GenBank accession number AF077761), 7 cDNA fragments covering the entire genome were cloned into the transcription vector pOLTV5 by overlapping PCR to obtain the recombinant plasmid pLaSota. The recombinant plasmid pLaSota is as follows: figure 1 Shown; It should be noted that: the complete LaSota genome cDNA has T7 RNA polymerase promoter upstream, delta hepatitis virus (HDV) ribozyme sequence (ribozyme) and T7 transcription termination sequence downstream.

[0055] 1.2 Construction of recombinant vectors containing F and HN genes of type VII NDV epidemic strains

[0056] According to the genome sequence of the gene VII ty...

Embodiment 2

[0070] Embodiment 2 has the rescue of the recombinant LaSota strain of the F gene of gene VII type NDV epidemic strain HN gene and alkaline protease cleavage site amino acid mutation

[0071] 2.1 Construction of transcriptional helper plasmids expressing NDV LaSota vaccine strain nucleoprotein (NP), phosphoprotein (P) and large polymerase protein (L)

[0072] The cDNA sequence encoding the large polymerase protein (L) of the NDV LaSota vaccine strain was cloned into the downstream of the CMV promoter of the pCI-neo eukaryotic expression vector; The sequence was cloned into the downstream of the SV40 promoter of the pCI-neo eukaryotic expression vector after tandem, and the transcription plasmid pCI-NP-P-L, which simultaneously expresses NP, P and L proteins, was obtained. Specifically, the following schemes are adopted:

[0073] a. Preparation of NP-2A-P

[0074] Using the pLaSota plasmid as a template, using primers targeting NP:

[0075] NP-F: 5'-ATGTCTTCCGTATTTGATGAG-3';...

Embodiment 3

[0089] Example 3 Construction of recombinant chimeric Newcastle disease virus expressing chicken infectious bursal disease virus mutant strain VP2 gene

[0090] 3.1 Main reagents and materials

[0091] The plasmid pLaSota-7F-7HN of the chimeric NDV LaSota-7F-7HN full-length genome cDNA containing gene VII type NDV strain F and HN genes constructed by embodiment 1; the expression LaSota strain NP, P and L protein helper plasmid pCI-NP-P-L.

[0092] The pGEM-PM shuttle vector was constructed by the applicant. Specifically, the pGEM-PM shuttle vector was cloned into the P and M part genes of the NDV LaSota strain and the two Between the P and M sequences, additional NDV gene end (GE) and gene start (GS) sequences were introduced, and two Sap I restriction endonuclease sites were introduced downstream of GE and GS point to facilitate the insertion of foreign genes. The sequences cloned into the Apa I and Pml I restriction endonuclease sites in the pGEM commercial vector are sho...

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Abstract

The invention belongs to the field of animal genetic engineering vaccines, and particularly relates to a construction method and application of a recombinant chimeric Newcastle disease virus for expressing a VP2 gene of an infectious bursal disease virus variant strain, and the construction method specifically comprises the following steps: inserting a consensus sequence of the VP2 gene of the infectious bursal disease virus variant strain into a chimeric LaSota low virulent strain containing gene VII type F and HN genes; the recombinant chimeric Newcastle disease virus for expressing the chicken infectious bursal disease virus variant strain VP2 gene is obtained. The chimeric LaSota attenuated strain is obtained by respectively replacing HN and F genes of a LaSota vaccine strain with an HN gene of a gene VII type NDV and an F gene with mutated amino acid sequences near an alkaline protease cracking site. The strain can be used as a seed virus for multi-combined vaccine preparation, and a foundation is laid for research and development of cheap and efficient vaccines for effectively preventing and controlling IBDV variant strain and gene VII type NDV infection.

Description

technical field [0001] The invention belongs to the field of animal genetic engineering vaccines, and in particular relates to a construction method and application of a recombinant chimeric Newcastle disease virus expressing the VP2 gene of chicken infectious bursal disease virus mutant strain. Background technique [0002] Infectious bursal disease (IBD) is an acute and highly contagious disease caused by infectious bursal disease virus (IBDV). IBDV mainly infects chicks aged 3 to 6 weeks. After infection, the central immune organ of chicks, the bursa of Fabricius, is severely damaged, and the function of B lymphocytes is damaged, which leads to immunosuppression of the body, and can easily cause secondary infection of other pathogens and cause serious losses. . IBDV belongs to the family DiRNAviridae and the genus Avian BiRNAvirus, and its genome includes two segments, A and B. The A segment encodes four proteins VP2, VP3, VP4 and VP5. Among them, VP2 is the capsid pro...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N7/04C12N15/45C12N15/56C12N15/40C12N15/86A61K39/295A61K39/17A61K39/12A61P31/14
Inventor 赵军乔麒龙黄庆杨盼盼王白玉王增李永涛
Owner HENAN AGRICULTURAL UNIVERSITY
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