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Recombinant herpesvirus of turkey (HVT) and preparation method thereof

A technology of turkey herpes virus and virus, applied in the field of recombinant turkey herpes virus and its preparation

Inactive Publication Date: 2018-03-20
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, the avian influenza vaccine with fowlpox virus as the carrier, however, due to the limitations of the current gene recombination technology, it is difficult for people to simultaneously insert two or more genes into the genome of an attenuated vaccine seed and obtain high-efficiency expression. As a result, there has been no report on the successful development of a triple recombinant poultry disease vaccine with HVT virus as a carrier

Method used

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  • Recombinant herpesvirus of turkey (HVT) and preparation method thereof
  • Recombinant herpesvirus of turkey (HVT) and preparation method thereof
  • Recombinant herpesvirus of turkey (HVT) and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] The construction of transfer vector pHVT-UL45-46 and pHVT-US1-10 of embodiment 1 recombinant HVT

[0080] Because the identified non-essential gene regions of HVT have only US10-2 region and UL45-46 region, and the turkey herpes vaccine virus (HVT) FC126 strain used in the present invention has inserted the framework gene of BAC in the US2 region of HVT, Therefore, only about 1.9kb US1-10 gene fragment can be used as the homology arm to construct the transfer vector pHVT-US1-10. The specific operation is as follows: First, use the genomic DNA of the turkey herpes vaccine virus (HVT) FC126 strain as a template to amplify the replication non-essential regions US1 and US10 of HVT respectively, and introduce a SalI restriction site downstream of US1 and upstream of US10 A HindIII restriction site is introduced. The specific primer sequences used are: the upstream and downstream primers of US1: 5'-aacgcaaaggcggccgctacatagaa-3', 5'-gaagcttcgtctcgtacaactgccatatcgagcattgtcgacc...

Embodiment 2I

[0081] Cloning and sequence optimization of embodiment 2 IBDV VP2 and NDV F gene

[0082] The viral total RNA of chicken infectious bursal virus (IBDV) L X strain and chicken Newcastle disease virus (NDV) PX02 / 3 strain in the allantoic fluid of chicken embryos were extracted by Trizol method, respectively, and the corresponding RNAs were obtained by QIAGEN reverse transcription kit. Then use specific primers (5'-ATGACA AACCT G CAAGATCAAACC-3', 5'-CCTTATGGCCCGGATTATGTCT-3') to amplify a 1.35kb IBDV VP2 gene fragment, and then use (5'-CTCGAGATGGGCCCCAAA TCT TCTACCAGGG -3', 5'-CTCGAGTCACGCTCTTGTAGTGGCTCTCATC-3') amplified the NDV F gene fragment with a length of 1.68kb, and analyzed the sequence and predicted the structure with the software DNAStar, indicating that the sequence contained a signal peptide and a transmembrane region, possibly To affect the expression of the protein, the codon was optimized and the kozak sequence was introduced at the 5' end of the gene, so as to be...

Embodiment 3

[0084] Example 3 The transfer vector construction of the recombinant HVT carrying the NDV F gene

[0085] The eukaryotic recombinant plasmid pCAGGS-F containing the NDV F gene and the transfer vector pHVT-US1-10 plasmid of the recombinant HVT were respectively digested with SalI and HindⅢ restriction endonucleases to obtain the chicken β-actin promoter pec-NDV F- The polyA expression cassette and the linearized pHVT-US1-US10 gene fragment with SalI and HindIII restriction endonuclease sites were ligated overnight at 16°C with T4 DNA ligase (NEB labs), and Transform TOP10 Escherichia coli competent cells to obtain the recombinant transfer vector pHVT-US-F. Wherein, the sequence of the expression cassette of pec-NDV F-polyA is shown in SEQ ID NO.2, wherein, the 1-1595bp part in SEQ ID NO.2 is the pec promoter sequence, and the 1966-3639bp part is the inserted NDV F Gene sequence, part 3646-4132bp is polyA gene sequence, gaattc and ctcgag are EcoRI and XhoI restriction site sequ...

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Abstract

The invention belongs to the technical field of biology and in particular discloses a recombinant herpesvirus of turkey (HVT). The recombinant HVT is capable of simultaneously expressing NDV and IBDVprotective antigen genes, and is a bacterial artificial chromosome (BAC) based on the HVT. The construction method comprises the following steps: inserting a VP2 gene expression cassette of an infectious bursal disease virus LX strain under regulation of a cytomegalovirus promoter CMV into a UL45-46 non-essential region of the HVT genome by utilizing a gene recombination technology; and insertingan F gene expression cassette of a newcastle diseases virus PX02 / 3 strain under regulation of a chicken beta-actin promoter pec into another non-essential region US1-10 of the HVT genome. The recombinant herpesvirus of turkey is a recombinant triplet poultry vaccine candidate strain capable of controlling Marek's disease of chickens and effectively resisting Newcastle disease and infectious bursaldisease.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant turkey herpes virus simultaneously expressing chicken infectious bursal virus VP2 gene and chicken Newcastle disease virus F gene and a preparation method thereof. Background technique [0002] At present, the annual production of poultry in China is about 12 billion, accounting for about 20% of the world's breeding volume, 8 billion chickens, 4 billion waterfowl, and an average of 1.408 billion commercial replacement layer chickens. The national poultry meat output is 18.67 million tons. Egg production is about 23.94 million tons. In fact, the number of chickens raised in my country is 9.6 billion every year, which means that at least 1.6 billion chickens are eliminated or die in the breeding process every year. Most of these direct and indirect mortality rates close to 20% are caused by infectious diseases. Therefore, the current situation of the prevent...

Claims

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Application Information

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IPC IPC(8): C12N15/869C12N7/01C12N15/66
CPCC07K14/005C12N7/00C12N15/66C12N15/86C12N2710/16021C12N2710/16043C12N2720/10022C12N2760/18122
Inventor 李永清许健江波黄秀芬
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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