| Specific receptors used by viruses determine the cellular tropism and virulence of these viruses. Interactions between virus and receptor provide an important target for antibodies and drugs to block virus infection. Alternative splicing is a mechanism that increases transcriptome and proteome diversifications by generating multiple mRNA products from a single gene. Identifying different functions of alternative spliced species is a hot research area in biology.Recently, the receptor of equine infectious anemia virus (EIAV) was identified and termed as equine lentivirus receptor-1 (ELR1), which belongs to the super family of tumor necrosis factor receptor (TNFR). Reported here, are two novel species of ELR1 mRNA, which were identified from cultured primary equine macrophages by screening cDNA clones amplified by RT-PCR. One of the ELR1 species (termed as ELR1-IN) contains an insertion of 153bp between the 786-787nt of the published ELR1 cDNA sequence, and the another one (termed as ELR1-DE) has a deletion of 64bp between the sites of 415nt to 478nt. Experiments were performed to rule out the possible contamination of chromosomal DNA for the insertion form of receptor. Both of the insertion and the deletion shifted the ORF of ELR1 and resulted terminations of translation in front of the transmembrane domain.The SYBR Green real-time RT-PCR was established to measure the ratio and relative amount of these three isoforms of ELR1 mRNA. Three pairs of primers were designed to quantitate PCR products corresponding to ELR1+ELR1-IN+ELR1-DE, ELR1+ELR1-IN and ELR1-IN. The amount of each isoform was determined by arithmetic. In order to reduce experimental errors, a single plasmid (pMD-18T-ELR1-IN) was used to set up the standard line. By this approach, the copy number of each type of isoform was measured using its specific primes. The feasibility and accuracy of this method was tested by comparing the amplification rates of the standard plasmid using these three pairs of primers. The ratio of these three species of ELR1 mRNA was measured by this methods as: ELR1-IN:ELR1-DE:ELR1=3:1.7:1.To investigate the proteins expressed by these alternative splicing variants, cDNAs of these isoforms of receptor were inserted into an eukaryotic expression vector pcDNA3.1(+). Cell lines of 293 and HeLa were transiently transfected with these recombinant plasmids and expressions of these vectors were verified by Western-blot and indirect immunofluorescence assay. The membrane-bond receptor ELR1 was expressed as a protein of 49 ku, and was located on the cell membrane. The inserted receptor ELR1-IN was expressed as a soluble protein of 38 ku. The deleted receptor ELR1-DE cDNA primarily encoded a protein for the second open reading frame, with the mass of 36 ku.To study the function of the soluble form of receptor, cell lines that consistently express ELR1 and ELR1-IN were constructed and designated 293-ELR1 and 293-ELR1-IN, respectively. Results of real-time RT-PCR and reverse transcriptase assay showed that the EIAV vaccine strain FDDV was able to enter and replicate in the 293-ELR1 cell line. In contrast, although FDDV was able to enter in the 293-ELR1-IN cell line, no replication could be detected. When the 293-ELR1 cell line was transiently transfected with the expression vector of ELR1-IN, the viral replication, which was indicated by the viral reverse transcriptase activity, was significantly lower than that transfected with the empty vector. This result implicates that the soluble receptor inhibits EIAV infection, probably at the entry stage.The specifical antibody of the virus receptor are needed for research the mechanism of virus and its receptor. The completed ORF and intracellular domain of ELR1 was expressed in E.coli , the expression product were subjected to SDS-PAGE and Western-blot analysis. The result lay foundation for the development of the polyclonal antibody. |
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