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Recombinant LaSota vaccine strain carrying gene VII type Newcastle disease virus F and HN genes as well as construction method and application of recombinant LaSota vaccine strain

A technology of Newcastle disease virus and its construction method, which is applied in the field of recombinant LaSota vaccine strain and its construction, can solve the problems of low efficiency, low efficiency of rescuing virus, and harm, and achieve the effect of high efficiency

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, in the process of rescuing the virus, three eukaryotic expression plasmids pCI-NP, pCI-P and pCI-L were co-transfected into BSR with a transcription vector (pA-NDV-LX / I4) containing the full-length NDV genome cDNA -T7 / 5 cells, that is, it needs to co-transfect cells with 4 plasmids, and the virus can only be rescued when 4 plasmids enter a cell at the same time, and the efficiency of rescuing the virus is low
Moreover, the traditional enzyme cutting and ligation method is used in the gene replacement process, which is not only inefficient, but also introduces additional non-viral sequences into the viral genome, resulting in unknown changes in the biological properties of the recombinant virus and potential hazards

Method used

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  • Recombinant LaSota vaccine strain carrying gene VII type Newcastle disease virus F and HN genes as well as construction method and application of recombinant LaSota vaccine strain
  • Recombinant LaSota vaccine strain carrying gene VII type Newcastle disease virus F and HN genes as well as construction method and application of recombinant LaSota vaccine strain
  • Recombinant LaSota vaccine strain carrying gene VII type Newcastle disease virus F and HN genes as well as construction method and application of recombinant LaSota vaccine strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] 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

[0047] 1.1 Construction of infectious clone plasmid of NDV LaSota vaccine strain

[0048] 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.

[0049] 1.2 Construction of recombinant vectors containing F and HN genes of type VII NDV epidemic strains

[0050] According to the genome sequence of the gene VII ty...

Embodiment 2

[0063] Example 2 Rescue of recombinant LaSota strain with gene VII type NDV epidemic strain HN gene and alkaline protease cleavage site amino acid mutation F gene

[0064] 2.1 Construction of transcriptional helper plasmids expressing NDV LaSota vaccine strain nucleoprotein (NP), phosphoprotein (P) and large polymerase protein (L)

[0065] 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:

[0066] a. Preparation of NP-2A-P

[0067] Using the pLaSota plasmid as a template, using primers targeting NP:

[0068] NP-F: 5'-ATGTCTTCCGTATTTGATGAG-3';

[0069] NP-R: 5'-...

Embodiment 3

[0082] The biological characteristic identification of embodiment 3 recombinant virus rLaSota-7F / HN

[0083] 3.1 RT-PCR identification of rLaSota-7F / HN

[0084] Total RNA was extracted from the allantoic fluid of LaSota strain infected chicken embryos and rLaSota-7F / HN infected chicken embryos with the virus genome RNA extraction kit. The identification primers 7F-F: 5′-GCTGCGTCTC TGAGATTGCG-3′ and 7F-R: 5′-GGCCTCTCTTACCGTTCTAC-3′ were designed for the gene sequences on both sides of the F gene; and the primers 7HN-F on both sides of the HN gene: 5′- GTAGAACGGTAAGAGAGGCC-3'; and 7HN-R: 5'-AGCACTGGCTGATTGTGGTC-3' were subjected to RT-PCR; the PCR products were sequenced and identified showing the correct insertion of the F and HN genes.

[0085] 3.2 Analysis of biological characteristics of rLaSota-7F / HN

[0086] According to the OIE standard, the mean lethal time (MDT) and half infection dose (EID) of chicken embryos in the 30th generation rLaSota-7F / -HN were determined. 50...

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Abstract

The invention belongs to the field of animal genetic engineering vaccines, and particularly relates to a recombinant LaSota vaccine strain carrying gene VII type Newcastle disease virus F and HN genes as well as a construction method and application of the recombinant LaSota vaccine strain. The recombinant LaSota vaccine strain is obtained by respectively replacing HN genes and F genes of the LaSota vaccine strain with HN genes of gene VII type NDV and F genes with mutated amino acid sequences near alkaline protease cracking sites. Specifically, the recombinant LaSota vaccine strain is prepared by using a homologous recombination technology and a reverse selection marking system in host bacteria. The recombinant LaSota vaccine strain provides a vaccine preparation strain and a suitable carrier for developing novel and efficient single-linked or multi-linked attenuated live vaccines and inactivated vaccines for preventing and controlling gene VII type NDV infection.

Description

technical field [0001] The invention belongs to the field of animal genetic engineering vaccines, in particular to a recombinant LaSota vaccine strain carrying gene VII type Newcastle disease virus F and HN genes, a construction method and application thereof. Background technique [0002] Newcastle disease (ND) is an acute, highly contagious and fatal infectious disease caused by Newcastle disease virus (NDV) infection that can infect various poultry, seriously endangering the health of poultry industry in my country and the world develop. The NDV genome encodes six structural proteins, namely nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase (HN) and large polymerase (L). Among them, F and HN are two functional envelope surface glycoproteins of NDV. F protein mediates the fusion of viral envelope and cell membrane, and the protease cleavage site in F protein is closely related to the virulence of NDV. HN a...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N7/04C07K14/125C12N9/24C12N15/85C12N15/65C12N15/64A61K39/17A61P31/14C12R1/93
Inventor 赵军杨盼盼王白玉乔麒龙黄庆
Owner HENAN AGRICULTURAL UNIVERSITY
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