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The Molecular Basis Study of Infection to Target Cells of Equine Infectious Anemia Virus, a Single Receptor Utilizing Lentivirus

Author: HaoFeiFei
Tutor: TianJinHong；ZhouJianHua
School: Southwestern University
Course: Preventive Veterinary Medicine
Keywords: EIAV Acceptor Protein expression Purification Crystallization
CLC: S852.65
Type: Master's thesis
Year: 2010
Downloads: 42
Quote: 0
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Abstract


Horse lentiviral receptor 1 (Equine Lentivirus Receptor-1, ELR1) Zhang, 2005 Cloning and Expression of technology found in equine infectious anemia virus (Equine Infectious Anemia Virus, EIAV) cell receptor, based on its sequence features attributable to the tumor necrosis factor receptor (Tumour necrosis Factor Receptor, TNFR) family of proteins. Cat, monkey, and human immunodeficiency virus with the same as lentivirus by using two different co-receptor to infect cells, ELR1 EIAV infection of target cells with a single functional receptor. To further elaborate the pathogenic mechanism of EIAV main application of protein crystallography method vitro prokaryotic expression of eukaryotic expression of the virus binds ELR1 extracellular region (ELR1-T) and crystallization trials, this receptor main function X-ray diffraction of protein crystals, protein structural analysis and structural and functional studies have laid a good foundation. The main results of this study are as follows: 1, ELR1 gene sequence analysis: analysis by the secondary structure of the software, the ELR1 gene sequence into a signal peptide, extracellular domain, a transmembrane region, and intracellular area. 2, ELR1 expression vector construct: In this study, the gene extracellular region ELR1 the (Truncated ELR1, ELR-T) was cloned into the expression vector pET-30 on the His-MBP recombinant plasmid of pET30-ELR1-T. After sequencing, the pET30-ELRl-T plasmid was transformed into E.coli Rosetta competent cells induced protein expressing recombinant ELR1-T. 3, the choice of expression system: First trial prokaryotic expression system, ELR1-T and His-MBP fusion form of double-tag fusion expression of the recombinant protein. His-MBP fusion ditag recombinant ELR1-T protein detected by SDS-PAGE electrophoresis, although the detected expression, but after the excision of the fusion protein His-MBP ditag, protein precipitation occurs. After the baculovirus expression system in eukaryotic expression systems attempt to express the target protein, intracellular expression and secretion of extracellular expression. 4, the purification of the recombinant protein ELR1-T: The baculovirus expression system secretory expression of the protein supernatant by Amicon Stirred Cell stirring type ultrafiltration apparatus system sample pre-enrichment process in the supernatant after the processed high affinity and Ni-NTA Resin preliminary purification by gel filtration chromatography methods for purification. By SDS-PAGE electrophoresis, the protein was purified ELR1-T to a single band, already highly purified, can be used for crystallization trials. Reorganization ELR1-T protein crystal growth research: vapor diffusion method, crystal screening, Hampton Research Crystal Screen2 31 # 39 # 52 # 44 # and Index under the conditions of growth of micro-crystals for the ultimate high-quality crystal, the three-dimensional conformation of ELR1 protein application of X-ray diffraction analysis, and laid an important foundation.

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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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