Preparation method for triple inactivated vaccine of recombinant Newcastle disease, bird flu and infectious bronchitis

A technology for bronchitis and Newcastle disease, applied in the field of vaccines, can solve problems such as lack of vaccines

Inactive Publication Date: 2017-02-15
TIANJIN RINGPU BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The present invention aims at the technical defects of the prior art, and provides a preparation method of a recombinant Newcastle disease, avian influenza, and infectious bronchitis triple inactivated vaccine, so as to solve the technical problem of lacking o

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  • Preparation method for triple inactivated vaccine of recombinant Newcastle disease, bird flu and infectious bronchitis

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1 (construction of recombinant chicken Newcastle disease virus)

[0026] 1. Construction of auxiliary plasmid NP, P, L genes

[0027] The three genes NP, P, and L were amplified by RT-PCR, and then the three genes were cloned into the expression plasmid vector pTM1, and the constructed helper plasmid was identified by DNA sequence analysis, and the positive plasmid was named pTM-NP, pTM-P, pTM-L.

[0028] 2. Construction of recombinant Newcastle disease virus expressing S1 protein

[0029] Linearize the TOPO TA Cloning vector through a pair of specific primers pM-s and pM-r, use the IBV-M41 strain as a template, and amplify the S1 gene with a pair of specific primers S1-F and S1-R to obtain rLaSota / S1 recombinant subclone. Finally, the rLaSota / S1 subclone was linearized with the In-fusion PCR Cloning Kit and connected to the full-length LaSota cDNA plasmid, which was transformed into Stbl2 competent cells, cultured at 30°C for 24 hours, and then identified...

Embodiment 2

[0044] Embodiment 2 (propagation of recombinant Newcastle disease virus and preparation of triple inactivated vaccine)

[0045] 1. Virus reproduction

[0046] The chicken embryos are selected from the eggs produced by healthy chicken flocks with good feeding and management, and each batch of chicken embryos must be strictly screened. A total of 4 items of external inspection: white shell, size, damage and dirty embryos, a total of 9 items of internal inspection: removal of sand shells, partial air chambers, free air chambers, inverted embryos, sterile eggs, terminated embryos, weak embryos, polluted embryos, Crack embryo. Qualified chicken embryos can be used as seedling making materials. Dilute recombinant Newcastle disease virus rLaSota / S1-HA strain 1:8000 times, inoculate 10-day-old SPF chicken embryos in the allantoic cavity, 0.1 mL per embryo, set the relative humidity at 60% to 65%, and the temperature at 33 to 35°C Incubate in the lower position without turning the e...

Embodiment 3

[0063] Embodiment 3 (immune effect of recombinant Newcastle disease, bird flu, infectious bronchitis triple inactivated vaccine)

[0064] Sixty 21-day-old SPF chickens were randomly divided into two groups, of which 30 in group A were inoculated with recombinant chicken Newcastle disease, avian influenza and infectious bronchitis triple inactivated vaccine group, and 30 in group B were blank control group. 14 days after immunization, 1ml of Beijing strain (including 10 4 ELD50), infectious bronchitis M41 strain challenge dose is 10 4.0 EID 50 , Measuring AIV-HI antibody titer. The challenge group was observed for 10 days to observe its morbidity and death. Only the antibody titer was determined in the blood collection group. The specific results are shown in Table 3:

[0065] Table 3 Experimental results of the immune effectiveness of the dual vaccine

[0066]

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Abstract

The invention provides a preparation method for a triple inactivated vaccine of recombinant Newcastle disease, bird flu and infectious bronchitis. The technical scheme is realized by gene modification of a Newcastle disease virus (DNV) LaSota strain. A recombinant NDV low virulent strain (rLaSota strain) is taken as a vector, firstly an S1 gene of a chicken infectious bronchitis virus (IBV) is inserted between F-HN, a recombinant NDV rLaSota/S1 expressing an S1 protein is constructed by utilizing a reverse genetic technology, and a result shows that rLaSota can serve as a live vector for stably expressing the S1 protein; secondly a recombinant NDV rLaSota/S1-HA expressing an HA gene is constructed, namely, the HA gene is inserted between P-M of the recombinant NDV rLaSota/S1; and thirdly immunogenicity assessment is performed. A result shows that the triple inactivated vaccine of the recombinant Newcastle disease, the bird flu and the infectious bronchitis, prepared through a conventional inactivated vaccine process has relatively good protection effects for the Newcastle disease, the bird flu and the infectious bronchitis.

Description

technical field [0001] The invention relates to the technical field of vaccines, and at the same time relates to genetic engineering technology, in particular to a preparation method of a recombinant chicken Newcastle disease, bird flu and infectious bronchitis triple inactivated vaccine. Background technique [0002] Avian Influenza Virus (AIV) belongs to the genus Influenzavirus A in the Orthomyxoviridae family. Its genome consists of 8 single-stranded negative-strand RNA fragments. Fragments 1 to 8 are named PB2 and PB1 in descending order. , PA, HA, NP, NA, M, and NS genes are divided into subtypes according to their surface antigens hemagglutinin (HA) and neuraminidase (NA). So far, 16 HA and 9 NA subtypes have been found , the virus infects poultry from mild upper respiratory tract disease, reduced egg production, to acute systemic fatal disease, which also poses a threat to public health. [0003] IBV belongs to the genus Coronaviridae of the Coronaviridae family. Th...

Claims

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Application Information

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IPC IPC(8): A61K39/295A61K39/17A61K39/145A61K39/215A61P31/14A61P31/16C12N7/01
CPCA61K39/12A61K2039/5252A61K2039/552A61K2039/70C07K14/005C12N7/00C12N2760/16134C12N2760/18121C12N2760/18134C12N2770/20034A61K2300/00
Inventor 陈冰李亚杰付旭彬杨保收李守军
Owner TIANJIN RINGPU BIO TECH
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