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Recombinant newcastle disease vaccine candidate rAI4-penton to express avian adenovirus penton protein and construction method thereof

An avian adenovirus and a construction method technology, which are applied in the field of expression of avian adenovirus penton protein recombinant Newcastle disease vaccine candidate strain rAI4-penton and construction

Inactive Publication Date: 2018-11-02
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The protective antigens of avian adenovirus include: hexon, penton and fibril protein. At present, there is no newcastle disease virus as a carrier to express foreign genes of avian adenovirus to construct a double Precedents for Valuable Vaccines

Method used

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  • Recombinant newcastle disease vaccine candidate rAI4-penton to express avian adenovirus penton protein and construction method thereof
  • Recombinant newcastle disease vaccine candidate rAI4-penton to express avian adenovirus penton protein and construction method thereof
  • Recombinant newcastle disease vaccine candidate rAI4-penton to express avian adenovirus penton protein and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Using pNDV / rAI4 as a carrier

[0043] Step 1: Construction of recombinant Newcastle disease vaccine candidate rAI4-penton full-length expression clone expressing avian adenovirus penton protein

[0044] The full-length expression vector pNDV / rAI4 of NDV AI4 strain (Hu Z, Hu S, Meng C, et al. Generation of a Genotype VII Newcastle Disease Virus Vaccine Candidate with High Yield in Embryonated Chicken Eggs. Avian Diseases 2011,55(3):1759- 1769) was constructed and preserved by the Key Open Laboratory of Livestock and Poultry Infectious Diseases of the Ministry of Agriculture of Yangzhou University. Blunt vector: purchased from Transgen; Dephospharylation (BAP) Kit: purchased from Bao Biological Engineering (Dalian) Co., Ltd.; AMV reverse transcriptase, Highfidelity DNA polymerase, T4 DNA ligase, Agarose Gel DNA Extraction Kit were purchased from Roche; transfection Reagents SuperFect and plasmid extraction kit (QIAprep Spin MiniPrep Kit) are products of QIAGEN...

Embodiment 2

[0102] Example 2: Using pNDV / rAI4-T4FHN as a carrier

[0103] The deposit number of the plasmid pNDV / rAI4-T4FHN is CGMCC No: 12987, which has been disclosed by Chinese patent application 201710596136.7.

[0104] Step 1: Construction of recombinant Newcastle disease vaccine candidate rAI4-2FHN-hexon full-length expression clone expressing avian adenovirus hexon protein

[0105] 1. Construction of intermediate plasmid pAI4-2FHN-blunt (AgeⅠ-BstZ17Ⅰ)

[0106] Design primers with reference to pNDV / rAI4-T4FHN as follows:

[0107]

[0108] The bold part of the primer sequence is the restriction site sequence, and the italic part is the overlapping sequence, and the primers were synthesized by Jinsirui Company.

[0109] Use pNDV / rAI4-T4FHN as template for PCR reaction:

[0110]

[0111] PCR reaction program: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 4 min, and 15 cycles; denaturation at 94°C for 30 s, ...

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Abstract

The invention relates to recombinant newcastle disease vaccine candidate rAI4-penton to express avian adenovirus penton protein and a construction method thereof. newcastle viral strain rAI4-penton iscollected under CGMCC No: 15492. The construction method includes: using a reconstructed reverse genetics operating platform of a newcastle attenuated strain to penton gene sequence of avian adenovirus into genome full-length transcription vector pNDV / rAI4 of the strain AI4 so as to obtain recombinant newcastle viral genome full-length cDNA clone pNDV / rAI4-penton, and performing transfecting to obtain recombinant viral rAI4-penton that successfully express penton protein. The recombinant virus rAI4-penton has high breeding titer on chicken embryos, can stabilize the penton protein even aftercontinuous passage, is suitable for large-scale production of vaccines, and is suitable for making vaccines.

Description

technical field [0001] The invention relates to the application of reverse genetic technology to rescue a recombinant virus strain rAI4-penton expressing poultry adenovirus penton protein with Newcastle disease virus as a carrier, and is used for vaccine development. Background technique [0002] Reverse genetics technology is the process of reverse-transcribing the RNA virus genome into cDNA, and then the cDNA interacts with various auxiliary proteins to assemble infectious RNA [Neumann G, Kawaoka Y. A decade after the generation of a negative-sense RNA virus from cloned cDNA - what have we learned? [J]. TheJournal of general virology, 2002, 83 (Pt 11): 2635-62.], this process is also called "virus rescue". Since the RNA virus finally "rescued" by reverse genetics technology is derived from cDNA clones, various in vitro artificial manipulations can be performed on the genome of RNA viruses at the DNA level, such as gene mutation, gene knockout (deletion), gene The transfo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/861
CPCC12N7/00C12N15/86C12N2710/10222C12N2710/10243
Inventor 刘秀梵程昊胡顺林刘晓文王晓泉
Owner YANGZHOU UNIV
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