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Cloning, Expression and Detection of the gD Gene of Porcine Pseudorabies Virus Ja Strain

Author: LiuXiaoHui
Tutor: YangRunDe
School: Agricultural University of Hebei
Course: Preventive Veterinary Medicine
Keywords: Swine pseudorabies virus (PRV) Ji A strain gD gene Sequence analysis Clone Expression
CLC: S852.65
Type: Master's thesis
Year: 2006
Downloads: 212
Quote: 1
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Abstract


In this study, three different methods to extract swine pseudorabies virus Ji A strains of the nucleic acid, and according to the literature, designed a pair of primers, the the PRV 冀 A strains gD gene was amplified by PCR. Detected by agarose gel electrophoresis, purification purposes fragment with pUC18 cloning vector connection, and transformed into E. coli JM109 competent cells, blue-white screening, transformants were identified by PCR and restriction enzyme analysis of positive clones. The results showed that, of positive recombinants (pUC1.2) containing gD gene that is inserted correctly, and positive clones plasmid send Takara sequencing. Nucleotide sequencing of the gene length 1197bp, contains an open reading frame encoding 398 amino acids, compared with five different domestic and foreign sources of strain, found Ji A strain gD gene exist 13 point mutations and at deletion mutants, and the PRV gD 816nt-831nt of a C (A) GGCCC repeated hypervariable region, corresponding to gD268 bit -275 Arg-Pro repeat hypervariable region. Using the DNA Star software Hebei A strain gD gene nucleotide sequence with the above strain-Yangsan EA, Fa, Ka, Min A (min-A) strain of the gD gene sequence comparison, the homology of 98.8% to between 99.7%. -Ji A strain gD gene deduced amino acid sequence of the five strains of PRV gD gene amino acid sequence, the homology between 77.9% to 79.2%. Seen from different sources the PRV strains gD gene high homology at the nucleotide level, but with some differences in the amino acid level homology. And draw a phylogenetic tree, based on the phylogenetic tree analysis, A strain of Hebei and Fujian A (Min-A) strains showed obvious kinship. Author will correct recombinant cloning plasmid pUC1.2 of the EcoRI, BamHI double digestion, electrophoresis recovery target fragment, the objective fragment insert by EcoRI and BamHI double digestion process, pGEX-4T-3, and transformed into E. coli JM109 competent cells get transformed by restriction analysis of recombinants screened reading frame, constructed recombinant plasmid pGgD and successfully expressed in E. coli BL21 (DE3) host bacteria containing the target protein fusion protein, the fusion protein has a molecular weight of approximately 53kD, Add IPTG (1mmol / L) after 6h induction, protein expression reached the highest level. Western-blotting hybridization experiments, the target protein and the antibody response of the rabbit anti the PRV 冀 A strains, indicating that the expression product gD protein A strain of PRV Hebei, reactogenicity and has laid the foundation for the preparation of mono-specific antibodies.

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