The Molecular Basis Study Of Infection To Target Cells Of Equine Infectious Anemia Virus, A Single Receptor Utilizing Lentivirus

The receptor for equine infectious anemia virus (EIAV) was identified as equine lentiviral receptor 1 (ELR1) through an approach of cloning and expression by Zhang et al in 2005. EIR1 was classified as a member of the tumor necrosis factor receptor (TNFR) family according to its sequence characteristics. Differing from human, simian and feline immunodeficiency viruses (acronymized as HIV, SIV and FIV respectively), which need two co-receptors for efficient infection; EIAV completes its infection only by only a single receptor, i.e. the ELR1. To understand better the mechanism of EIAV pathogenesis, prokaryotic,eukaryotic expression and crystallization of out-membrane region of ELR1(ELR1-T) was employed in this study for further structural dissection of the protein by X-diffraction and functional analysis. The major objectives and results of this study are listed as follows:1. ELR1 sequence analysis:This receptor was composed of the signal peptide, out-membrane region, trans-membrane region and cytoplasmic region by using a secondary structure prediction software.2. Construction of ELR1 expression vectors:The gene encoding ELR1 out-membrane region was cloned and inserted into prokaryotic expression vectors pET-30 His-MBP. Resulting recombinant plasmids were selected by sequencing and transformed into E.coli Rosetta for producing recombinant ELR1-T.3. Selection on ELR-T expression system:Firstly, ELR1-T was expressed in the prokaryotic expression system by fussing to the His-MBP double tag. Though the His-MBP double tag fusion protein was detected from transformed E.coli by SDS-PAGE, this protein was insolublized when the tags were cut out. The GST and 6×His tagged soluble ELR1-T was eventually expressed in insect cells in forms of intracellular protein and secreted protein respectively by using the Bac to Bac Baculovirus Expression system. 4. Purification of ELR1-T:The secreted form of ELR1-T expressed by eukaryotic cells was concentrated from supernatant of cell culture by passing through the Amicon Stirred Cell concentration apparatus. Purification of this tagged receptor was performed by using Ni-NTA affinity chromatography and followed by gel-filtration. High purity of the recombinant ELR1-T was confirmed by SDS-PAGE.5. Crystal growth of ELR1-T:Vapor diffusion was adapted for crystal screening. The formation of microcrystals was observed in boxes 31#,39# and 44# of Crystal Screen2 system and 52# of Index system produced by Hampton Research. The successful growth of ELR1-T microcrystal provides essential information on crystallizing this specific protein and therefore is a key step for getting high quality crystals of the EIAV receptor. This study has set up an important base for the final three-dimensional conformation analysis of ELRl by X-Ray diffraction.