Establishment And Identification Of HEK-293 Cell Lines Stably Expressing Porcine CD163 Protein

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important destructive disease of swine. The disease has caused heavy economic impacts of the pig industry all over the world. The causative agent of PRRS, the PRRS virus, has a complicated immune mechanism, it can accomplish immune evasion through variety pathways. We have not found a feasible and effective measures to prevent and cure this disease until now. Virus receptor is a type of molecular complex on the surface of host cells which interact with the viral proteins and assist virus infection. Study of virus receptor will reveal the mode of action of virus-cell further detail, accordingly displays new solving ideas for PRRS. In the natural host, porcine alveolar macrophages (PAM) are predominant target cell of PRRS V, three receptors for PRRSV have been identified on PAM cell surface, including heparin sulfate (HS), sialoadhesin (Sn) and CD 163. HS helps PRRSV binding to the cell membrane, Sn mediates the virus attachment and internalization, while CD 163 has been described to function as a receptor for PRRSV replication. The expression of CD 163 was shown to allowed non-permissive cells to become permissive to PRRSV replication with production of progeny infectious virus. Consequently, this study, based on this incredible research procedure, cloned porcine CD 163 gene from PAM cell, constructed porcine CD 163 expressing plasmid pCDNA3.1/V5-pCD163, and established stable HEK-293 cell lines expressing porcine CD163(pCD163). The main research contents include:1. The cloning, sequence analysis of porcine CD163 and construction of the p CD163 expressing plasmidAccording to a porcine CD 163 mRNA sequence on GeneBank (EU016226.1), a pair of primers pCD163-F/R was designed, and the cDNA of porcine CD 163 complete coding sequence was amplified via RT-PCR from total celluar RNA of PAM cells. The sequence was cloned into pGEM-T easy vector and sequencing result showed that the cloned sequence was 3348bp in length and encoded 1115 amino acid. The sequence had a homology as high as 99.7% with the reference sequence on GenBank database, especially the critical SRCR 5 domain shared a homology of 100%, on the level of amino acid sequence. The pCD163 cDNA fragment obtained from pGEM-T easy then subcloned into pCDNA3.1/V5-His B vector using BamHI and Xhol restriction site to construct the pCD163 expressing plasmid pCDNA3.1/V5-pCD163. 2. Generation of HEK-293 cells stably expressing porcine CD163The eukaryotic expressing plasmid pCDNA3.1/V5-pCD163 was constructed and transfected into HEK-293 cells. Under the selection of G418, the HEK-293 cell lines stable expressing porcine CD 163 was constructed. RT-PCR, IFA and Western Blot were performed to verify the pCD163 expressing in the constructed cell lines on mRNA level and protein level respectively.3. Identification of PRRSV infection in HEK-293-pCD163 cell linesThe HEK-293-pC163 cells were inoculated with PRRSV WUH3 strain, obvious CPE was viewable, RT-PCR, IFA and Western Blot were performed, confirming PRRSV infection in HEK-293-pCD 163 cell lines.4. Growth kinetics of PRRSV on HEK-293-pCD163 cell lines and Marc-145 cel IsHEK-293-pCD163 cells and Marc-145 cells were inoculated with PRRSV WUH3 strain under the totally equal conditions, six time periods were planned to measure TCID50 for the PRRSV titeration, drafted the comparison diagram of Growth kinetics of PRRSV on HEK-293-pCD163 cell lines and Marc-145 cells.