Establishment And Application Of One MARC-145 Cell Line Stably And Highly Expressing Porcine CD163

Porcine reproductive and respiratory syndrome(PRRS) was characterized by the productive failure and stillbirth in sows, and respiratory problems in piglet, which was caused by porcine reproductive and respiratory syndrome virus(PRRSV). PRRSV could damage immune system, causing serious infections and secondary infections. In China, PRRSV causes major economic losses in the pig industry. Nowadays, vaccination is the major protective measure to control PRRS.Isolation and identification of PRRSV is very important for PRRSV research. However,PRRSV has a strong restricted tropism for infected cells both in vivo and in vitro. In vitro,PRRSV only replicates in primary pig macrophages and African green monkey kidney derived cells, such as CL2621 and MARC-145. Porcine alveolar macrophages(PAMs) are susceptive to PRRSV, however, they are primary cells and hard to harvest. Nowadays,MARC-145 cell line is frequently-used for proliferation of PRRSV, but it has some limitations such as low virus titer and low growth rate. Therefore, it is very important to establish one cell line for efficient and rapid proliferation of PRRSV.Binding to the receptors on the host cells is the first step for PRRSV infection. Studies had shown that CD163 was functioned as a receptor-mediated virus stripping its shell and releasing its viral nucleic in the cytoplasm, which was closely related to PRRSV infection.What is more, Patton et al had shown that the expression of CD163 receptor was directly related to PRRSV replication. So this study was attempted to generate one MARC-145 cell line stably and highly expressing porcine CD163 to facilitate virus propagation and set the foundation for PRRSV isolation and vaccine production.The CD163 gene was amplified by RT-PCR from PAMs and cloned into the eukaryotic expression vector pCI-neo, then the positive plasmid pCI-CD163 was transfected into MARC-145 cells. After selecting with G418 and subcloning for 3 times, MARC-145 cell line expressing porcine CD163 was established. IFA results indicated that the fluorescence of pCD163-MARC cells was significantly brighter than MARC-145 cells. Western blot results indicated that the pCD163-MARC cells could express higher levels of CD163 and the expression level was 8.7 times higher than MARC-145 cells. The pCD163-MARC cell line could be stably passaged for 20 passages and the expression level of CD163 was similar with different passages. The pCD163-MARC and MARC-145 cells were simultaneously infectedwith 1MOI of classic SD-1 strain and highly pathogenic SD-JN strain. Growth curves showed that virus proliferated by pCD163-MARC cell line has a high virus titer. The result of RTPCR assays showed that virus proliferated by pCD163-MARC cell line has a larger amount of RNA.pCD163-MARC cells and MARC-145 cells wre simultaneously inoculated with 120 clinical PRRSV positive tissue samples which were positive by RT-PCR and kept in our laboratory from June 2012 to June 2014. After passaged for 5 times, the rate of PRRSV isolation for pCD163-MARC cells and MARC-145 cells was 100% and 80%, respectively. It was indicated that pCD163-MARC cell line was more suitable for clinical PRRSV isolation.In addition, pCD163-MARC cells and MARC-145 cells were cultuered in roller bottles and infected with 1MOI PRRSV JXA1-R vaccine strain, which was produced by Guangdong Dahuanong Animal Health Products Co., Ltd. After cultured for 96 h, PRRSV titer produced by pCD163-MARC cells and MARC-145 cells was 1×108.5TCID50/ml and 1×107.6TCID50/ml,indicating that pCD163-MARC cell line had stronger proliferation of PRRSV and could be used in vaccine production.