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Novel bacterial artificial chromosome of turkey herpes virus, construction method and application

A turkey herpes virus and artificial chromosome technology, applied in the field of veterinary medicine, can solve the problems of loss of proliferation ability, weakened virus proliferation ability, inconsistent virus growth characteristics, etc., and achieve the effects of high stability, simple method and efficient construction

Pending Publication Date: 2017-01-25
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reported construction of HVT BAC is to insert the mini-F sequence into the US2 region of the HVT genome. Although infectious clones have been obtained, the mini-F recombinant virus is very unstable, and some recombinant viruses lose their ability to proliferate after passage. Moreover, the growth characteristics of viruses rescued from infectious clones were inconsistent, and some viruses had greatly weakened proliferation ability. These results indicated that the insertion of mini-F into the US2 site may interfere with the normal replication of HVT.

Method used

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  • Novel bacterial artificial chromosome of turkey herpes virus, construction method and application
  • Novel bacterial artificial chromosome of turkey herpes virus, construction method and application
  • Novel bacterial artificial chromosome of turkey herpes virus, construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Construction and Identification of Transfer Vector pUC19(HVT)-H1-H2-miniF

[0033] 1. Experimental materials

[0034] SPF chicken embryos were purchased from Beijing Meria Verton Experimental Animal Technology Co., Ltd. HVT FC-126 virus strain purchased

[0035]From the Central Prison Institute (CVCC AV19). The pUC19 plasmid was purchased from Takara Company. The mini-F plasmid was donated by Professor Nikolaus Osterriender, Institute of Virology, Free University of Berlin (disclosed in: Tischer B, Smith G, Osterrieder N. En passant mutagenesis: a two step markerless red recombination system. Methods Mol Biol. 2010; 634:421-430. ). Enzymes EcoR I, Hind III, Sma I, Sca I, BamHI and Pac I and Ex Taq DNA polymerase, as well as ligase and PCR reagents were purchased from Takara; DMEM medium, trypsin and newborn calf serum were purchased from Sigma company; other reagents were purchased from Nanjing Sangon Biotechnology Co., Ltd. Plasmid extraction reagent: s...

Embodiment 2

[0054] Construction of the bacterial artificial chromosome of embodiment 2HVT Fc-126 strain

[0055] 1. Experimental materials

[0056] SPF chicken embryos were purchased from Beijing Meria Verton Experimental Animal Technology Co., Ltd. HVT Fc-126 strain

[0057] From the Central Prison Institute (CVCC AV19). Plasmid Mini Kit and Midi Kit were purchased from QIAGEN. DMEM medium, trypsin and newborn bovine serum were purchased from Sigma; other reagents were purchased from Nanjing Sangon Biotechnology Co., Ltd. DH10B competent cells were purchased from Invitrogen.

[0058] 2. The method and identification of the bacterial artificial chromosome of strain HVT Fc-126

[0059] 2.1 Obtaining of Mini-F recombinant HVT

[0060] Take 10~20uL (about 1~3ug) of DNA of HVT FC-126 strain and 3~5uL (about 3~5ug) of transfer vector pUC19(HVT)-H1-H2-mini-F, mix them together, and use the calcium phosphate precipitation method to co- Primary chicken embryo fibroblasts (Chickenembryofibr...

Embodiment 3

[0065] Embodiment 3 virus rescue

[0066] 1. Materials

[0067] The HVT FC-126 strain comes from the Central Prison Institute (CVCC AV19) to prepare SPF chicken embryos from chicken embryo fibroblasts

[0068] Bought from Beijing Meria Weitong Experimental Animal Technology Co., Ltd. The recombinant bacteria BAC-HVT (C1), BAC-HVT (C2) and BAC-HVT (C3) carrying the bacterial artificial chromosome of turkey herpes virus were prepared in Example 2. Plasmid MiniKit and Midi Kit were purchased from QIAGEN. DMEM medium, trypsin and newborn calf serum were purchased from Sigma. Other reagents were purchased from Nanjing Sangon Biotechnology Co., Ltd.

[0069] 2. Experimental methods and results

[0070] The plasmid DNA of BAC-HVT (C1) was extracted using Qiagen plasma Midi kit according to the instructions, and about 500ng of DNA was taken to transfect freshly prepared monolayer CEF by calcium phosphate precipitation method. The next day, the culture medium was replaced with a ...

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Abstract

The invention provides a novel bacterial artificial chromosome of a turkey herpes virus, a construction method and application and relates to the field of animal medicine. The bacterial artificial chromosome is obtained by knocking out gC gene in HVT genome and replacing with a mini-F plasmid sequence. The construction method includes: amplifying upstream and downstream homologous arms of the gC gene in the HVT genome, and inserting into a pUC19 vector to obtain a recombinant vector A; inserting a mini-F plasmid segment into the recombinant vector A to obtain a recombinant transfer vector B, performing cotransfection (CEF) with DNA of HTV, and screening to obtain mini-F recombinant HVT; extracting DNA of the mini-F recombinant HVT to convert DH10B cells, adopting chloromycetin for screening to obtain positive recombinant bacteria, culturing, and then extracting plasmid DNA. The bacterial artificial chromosome has high stability, and the construction method is simple, efficiency and applicable to construction of recombinant vector live vaccines.

Description

technical field [0001] The invention relates to the field of veterinary medicine, in particular to a novel bacterial artificial chromosome of turkey herpes virus, a construction method and application thereof. Background technique [0002] Herpesvirus of turkey (HVT) is an alphaherpesvirus belonging to chicken Marek's disease virus serotype III. Early studies have found that HVT is genetically and serologically related to Marek's disease virus (MDV), and HVT is not pathogenic to chickens. Therefore, HVT FC-126 strain is widely used Prevention of chicken Marek's disease (Marek's disease, MD). [0003] HVT is extremely safe for chickens and does not produce any side effects; it can be inoculated in embryo or immunized early at the age of 1 day, which can not only avoid the interference of maternal antibodies but also start immunity quickly; the immunity period is long, and the virus can last for up to 72 weeks of viremia. The HVT virus particle size is about 160nm, and the ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/66A61K39/245A61P31/20C12R1/19
CPCC12N15/70A61K39/12A61K2039/53C07K14/005C07K14/245C12N15/66C12N2710/16022C12N2710/16034C12N2800/101
Inventor 王继春王志胜许梦微刘芳乔永峰刘娅梅侯继波
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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