Construction Of Pam Cell Lines With Stable-Expression Of Porcine CD163Proteins

CD163is a member of the SRCR protein superfamily. As a type I transmembrane protein, CD163consists of9extracellular consecutive SRCR domains. Some researches showed that CD163is a key factor in the initiation of PRRSV infection, CD163paly a role in viral uncoating and genomic RNA releasing. Porcine alveolar macrophages (PAM) is the target cells of PRRSV, transfected SV40Large T antigen into primary PAM cells, we get three cell lines, but these cell lines do not support PRRSV infection becuse of no CD163expression. This study was designed to transfected PAM with eukaryotic expression plasmid pcDNA4.0-CD163, in order to obtain cell lines which is stable expression of CD163, supporting PRRSV infection.1. The construction and identification of recombinant eukaryotic expression plasmid for porcine CD163full length cDNAThe3348bp cDNA encoding open reading frame of porcine cDNA was then cloned by RT-PCR from primary porcine alveolar macrophages saved by the laboratory with sepecific primes based on the published GenBank sequence of porcine CD163gene, inserted into pMD19-T plasmid, then identifieded by PCR, enzyme digestion, and sequence identification.In view of CD163cDNA in the amplified process having five mutation sites which can cause an corresponding change in amino acids, the involved mutation were consistent with the record GenBank sequence by the site-directed mutagenesis kit from TaKaRa company. And then the correct sequencing gene fragment was cloned into the eukaryotic expression vector pcDNA4.0and pIRES vector, determining the recombinant expression plasmid successfully constructed by restriction enzyme digestion.2. The function of porcine CD163as a PRRSV receptorBy RT-PCR method and indirect immunofluorescence technique verified the eukaryotic expression plasmid pIRES-CD163transiently transfected PRRSV non-sensitive cell line BHK-21and PK-15. By PRRSV proliferation assay, compared to the changes of the cells transfected with CD163before and after to the sensitivity of PRRSV. To verify the porcine CD163as a PRRSV receptor, only expression of CD163will be able to make non-sensitive cell lines sensitive to PRRSV.3. The establishment of PAM which can stably express CD163The recombinant plasmid pcDNA4.0-CD163was transfected into PAM cell line3D4/21(CRL-2843), the expression of CD163was detected in the cells by RT-PCR and IFA. Cells were selected by culture adding zeocin. Using the limiting dilution assay, we got the PAM cell line stable expression CD163protein after three times of screening. RT-PCR and IFA were used to determine the recombinant PAM cell line successful construction. I got eight positive cell clones, named for the3G10-C2,3G10-D2,3G10-F2,3G10-B3,3G10-D4,3G10-B6,3G10-A8,3G10-H8. The PRRSV reproduction was detected in the recombinant PAM cell line, the CD163have a catalytic effect on influenza virus reproduction. The stability of recombinant cell lines was detected by IFA after ten generations of continuous passage.The experiment building a stable expression of CD163PAM cell lines can be used to proliferate PRRSV in vitro and evaluate the different PRRSV vaccine quality by the neutralizing antibody experiments.