Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (11): 2253-2264.doi: 10.3864/j.issn.0578-1752.2025.11.013

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles     Next Articles

Construction and Evaluation of a PK15 Cell Line Stably Expressing Porcine CD163 Protein

ZHAO QingYang(), ZHANG XiaoXiao, GUO ChunHe()   

  1. College of Veterinary Medicine, South China Agricultural University/Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou 510640
  • Received:2025-01-07 Accepted:2025-04-05 Online:2025-06-01 Published:2025-06-09
  • Contact: GUO ChunHe

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Abstract:

【Background】 CD163 is an important transmembrane protein that plays a key role in host immune regulation, inflammatory response, and the infection process of various pathogens. Studies have shown that CD163, as a host receptor, not only influences cell signaling pathways but also regulates host susceptibility to foreign pathogens. Since the receptor-pathogen interaction mechanisms involving CD163 are not yet fully elucidated, establishing a cell model stably expressing CD163 is of great significance for related research. Porcine reproductive and respiratory syndrome virus (PRRSV) is an important model for studying the CD163-mediated viral entry mechanism. However, the primary target cells of PRRSV—porcine alveolar macrophages (PAMs)—cannot proliferate continuously in vitro, which limits their application in pathogenetic research. Therefore, the monkey-derived cell line MARC-145 is commonly used for PRRSV in vitro culture, but its non-porcine origin may affect the biological relevance of experimental results. 【Objective】 This study aimed to construct a porcine cell line stably expressing porcine CD163 protein and to evaluate its susceptibility to PRRSV infection.【Method】 This study was conducted at the Guangdong Provincial Laboratory of Lingnan Modern Agricultural Science and Technology from June 2022 to November 2023. The porcine CD163 gene was cloned, and two strategies were employed to express this gene in the porcine kidney cell line (PK15). Firstly, recombinant plasmid transient transfection was attempted, but due to the low transient transfection efficiency of PK15 cells, a stably expressing cell line could not be obtained. To optimize the expression strategy, lentiviral vector-mediated gene transduction was further applied, followed by screening for stable cell lines. Lentiviral packaging was performed using 293T cells, and the resulting virus was transduced into PK15 cells. Positive clones were screened using puromycin to ensure stable integration and long-term expression of the exogenous gene. CD163 mRNA and protein expression levels were detected by RT-qPCR and Western blot, and passage experiments were further conducted to evaluate the stability of CD163 expression, ensuring the long-term application potential of the cell line. 【Result】 A recombinant lentiviral vector carrying the CD163 gene was successfully constructed, and a stably expressing PK15 cell line, named PK15-CD163, was obtained through lentiviral transduction. RT-qPCR and Western blot results showed that the expression levels of CD163 mRNA and protein in PK15-CD163 cells were significantly higher than those in normal PK15 cells and remained stable after multiple passages. However, PRRSV infection experiments indicated that PRRSV N protein expression could not be detected by either RT-qPCR or Western blot, suggesting that PRRSV replication might be restricted in this cell line.【Conclusion】 This study successfully established a PK15-CD163 cell line, providing an important experimental tool for further investigation of CD163’s biological functions in host cells. Although PRRSV infection in PK15-CD163 cells did not yield significant results, this cell line could still be used to explore CD163-mediated immune regulation, receptor function, and other pathogen infection mechanisms. Additionally, it provided a potential platform for antiviral drug screening.

Key words: porcine reproductive and respiratory syndrome virus, CD163, lentiviral vector, PK15

Table 1

Reverse transcription system (10 μL)"

组分 Component 体积 Volume (μL)
RNA 2.5 (1000 ng)
StarScript III All-in-one RT Mix 1
5×StarScript III All-in-one RT Buffer 4
Nuclease-free Water（DEPC-treated） 补足至10μL Fill up to 10 μL

Table 2

Primer sequence"

基因 Gene 引物序列 Primer sequence 切口 Restriction site
CD163 F: 5'-ggatcgggtttaaacggatccATGGACAAACTCAGAATGGTGCT-3'
R:5'-gtagggccctctagactcgagTTGTACTTCAGAGTGGTCTCCTGAGG-3'		 BamH I
Xho I

Table 3

PCR amplification system (10 μL)"

组分 Component 体积 Volume (μL)
模板DNA Template DNA 1
引物 F Primer F 0.5
引物 R Primer R 0.5
2 × Taq PCR Mix 5
ddH2O 补足至10 μL Fill up to 10 μL

Table 4

Vector double digestion reaction system (50 μL)"

组分 Component 体积 Volume (μL)
载体 Carrier 0.5 (100 ng)
BamH I 1
XhoI 1
10 × NE Buffer 5
ddH2O 补足至50 μL Fill up to 50 μL

Table 5

Homologous recombination system (20 μL)"

组分 Component 体积 Volume (μL)
线性化载体 Linearized vector DNA 0.85 (170 ng)
CD163片段 CD163 fragment 1.3 (130 ng)
2 × ClonExpress Mix 5
ddH2O 补足至20 μL Fill up to 20 μL

Table 6

Primer sequences"

基因
Gene		 引物序列
Primer sequence		 产物长度
Product length
CD163 F: 5'-AGGAGCCCTCATGATAAATC-3'
R: 5'-ACCATTCCAGAAGAAAGTGC-3'		 3348 bp

Table 7

RT-qPCR reaction system（10 μL）"

组分 Component 体积 Volume (µL)
cDNA 模板 cDNA template 2
SYBR Green 2 × qPCR Mix 5
Primer F 0.2
Primer R 0.2
ddH2O 2.6

Fig. 1

PCR Amplification of porcine CD163 molecule A: PCR amplification of porcine CD163 gene from PAMs cDNA; B: Large-scale PCR amplification of CD163 gene"

Fig. 2

Construction of the pcDNA3.1-CD163 recombinant plasmid A: Electrophoresis verification of double digestion products of CD163 gel recovery product and pcDNA3.1 vector; B: PCR amplification of CD163 gene from 10 bacterial colonies"

Fig. 3

Fluorescence microscopy observation of pcDNA3.1-CD163 transfected 293T cells"

Fig. 4

Western blot validation of normal expression of porcine CD163 in 293T cells"

Fig. 5

Schematic map of the vector plasmid phage-mcs-CD163-3×HA"

Fig. 6

RT-qPCR detection of CD163 expression in PK15-CD163 cells"

Fig. 7

Western blot detection of CD163 expression in PK15- CD163 cells"

Fig. 8

RT-qPCR detection of PRRSV N expression in PK15- CD163 cells cafter PRRSV infection"

Fig. 9

WB validation of PRRSV N expression in PK15 cells"

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