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Construction of Recombiant Marek’s Disease Virus Expressing HA and NA Gene of Avian Influenza Virus

Author: LiuShuiQing
Tutor: ZhangQiJin；LiMingYi；ShenZhiYi；GuanPingYuan
School: Inner Mongolia Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Avian influenza virus Recombinant Marek virus General transfer vector Hemagglutinin Neuraminidase
CLC: S852.5
Type: Master's thesis
Year: 2007
Downloads: 134
Quote: 1
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Abstract


Hemagglutinin (HA) is a major avian influenza virus protective antigen, neuraminidase (NA) surface after AIV HA is an important antigen for the analysis of HA, NA gene phylogenetic relationships, and explore strains differences in the molecular basis of virulence, this study isolates of avian influenza virus hemagglutinin (HA), neuraminidase (NA) genes were amplified by RT-PCR, and its cloned and sequenced. Sequencing results showed that the cloned HA gene containing the complete open reading frame gene is 1707bp, encoding 568 amino acids, wherein the signal peptide of 1-16 amino acids, HA1 330 amino acids (containing 5-6 cleavage basic amino acids), HA2 222 amino acids, and contains the start codon and stop codon; NA gene is 1410bp, encoding 469 amino acids. Extracts published in Genbank reference strains of avian influenza H5N1 sequences, sequence analysis and homology comparison and found that, HA nucleotide sequence homology between the 91.0% -98.7%, varied greatly, indicating that avian influenza virus over time with continuous genetic recombination, mutation. NA gene is relatively conserved in evolution, the sequence homology of 95.2% or more. For the development of novel recombinant vaccines, recombinant viral Marek live viruses as vectors genetically engineered vaccine has become a research focus. Construction of recombinant transfer vector is an important part of building Marek virus. The analysis of Marek virus genome, based on a US2 gene insertion site designed primers to amplify 2.6 kb of homology arms, the amplified connected in vitro homologous arms, inserted into pUC18, and the order of constructed based on the transfer of green fluorescent protein expression common carrier. The transfer vector containing the eGFP transfected Marek embryo fibroblasts infected cells, the reporter gene was successfully expressed viral vectors which will Marek basis for further development. This study is to overcome the current avian influenza inactivated vaccine cross-protection power is low vulnerability, to be constructed expression avian influenza virus HA and NA recombinant Marek virus, in order to develop new types of cross protection with recombinant virus vaccine. The AIV hemagglutinin protein (HA) gene was inserted into MDV universal transfer vector construct expressing HA protein alone can rMDV transfer vector; simultaneously turn AIV HA and NA genes cloned into MDV universal transfer vector construct containing HA- NA two genes transfer vector. By liposome-mediated transfection method will be transferred from the parent carrier Marek virus infection CVI988 chicken embryo fibroblasts. Based on green fluorescent protein (GFP) marker gene is expressed in cells, screening recombinant virus, after several rounds of plaque purification, PCR identification results obtained prove that the recombinant virus rMDV-HA-NA stable genetic traits, and be able to restructure its one or two high-level expression of foreign gene, and expression in cultured cells and AIV protein product has similar biological properties and immunological activity. The experiment for the next reorganization Marek vaccine provides the necessary experimental basis. In short, through this study, recombinant Marek Virus Transfer plasmid is a recombinant vaccine provides a material basis. Meanwhile, the initial gained a simultaneous expression of avian influenza HA, NA recombinant Marek virus, which will develop new genetically engineered vaccine for bird flu prevention and control to bring new technologies.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Serological
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