永生化猪视网膜色素上皮细胞的建立及初步应用

Establishment and application of immortalized porcine retinal pigment epithelial cells

【目的】建立永生化猪视网膜色素上皮细胞系,为病毒学研究提供一种新的细胞材料。【方法】合成猪腺病毒3型E1基因,将其克隆至真核表达质粒pCI-neo中,构建E1基因真核表达质粒pCI-neo-E1,进行PCR和测序鉴定。提取pCI-neo-E1质粒,转染原代猪视网膜色素上皮细胞,用新霉素G418筛选猪永生化视网膜色素上皮细胞系(RPECs)。检测RPECs的角蛋白18和19,通过细胞计数测定其生长曲线,流式细胞仪检测细胞周期,并对其进行染色体核型分析。分别将猪流行性腹泻病毒(PEDV)CV77毒株、猪圆环病毒2型(PCV2)DBN-SX07株和猪伪狂犬病毒(PRV)Bartha-K61株接种RPECs,同时将PEDV CV77毒株接种绿猴肾细胞(Vero细胞),将PCV2 DBN-SX07株接种猪肾细胞(PK15细胞),将PRV Bartha-K61株接种仓鼠肾成纤维细胞(BHK细胞),观察细胞病变效应(CPE),并采用间接免疫荧光法测定病毒的滴度。【结果】PCR和测序结果显示,真核表达质粒pCI-neo-E1构建成功。将pCI-neo-E1转染原代猪视网膜色素上皮细胞,经G418筛选得到永生化细胞株,该细胞株表达角蛋白18和19,传代50代仍保持上皮样细胞形态,增殖活性良好,细胞周期及染色体核型特征与正常的二倍体细胞一致。PEDV CV77毒株感染猪RPECs 24 h后,CPE明显,滴度为10^(5.25)TCID_(50)/mL,低于其在Vero细胞中的滴度(10^(8.125) TCID_(50)/mL)。PCV2 DBN-SX07毒株感染猪RPECs后可产生明显CPE,病毒滴度为10^(6.125)TCID_(50)/mL,略高于其在PK15细胞上的滴度(106.0 TCID_(50)/mL)。PRV Bartha-K61毒株感染猪RPECs后,CPE明显,病毒滴度为10^(8.75)TCID_(50)/mL,略低于其在BHK上的滴度(108.875 TCID_(50)/mL)。【结论】成功构建了永生化猪视网膜细胞系,可用于猪源病毒的培养,尤其是培养PCV2可产生明显CPE,易于观察,为后续开展疫苗工艺的提升提供了一种新的细胞材料。

【Objective】This study established an immortalized porcine retinal pigment epithelial cell line to provide a new cell material for virological research.【Method】The E1 gene of porcine adenovirus type 3 was synthesized and cloned into the eukaryotic expression plasmid pCI-neo.The eukaryotic expression plasmid pCI-neo-E1 was constructed and identified by PCR and sequencing.The pCI-neo-E1 plasmid was extracted and transfected into primary porcine retinal pigment epithelial cells.Porcine immortalized retinal pigment epithelial cell lines(RPECs)were selected by neomycin G418.Keratins 18 and 19 were detected in RPECs.Cell growth curve was determined by cell counting,cell cycle was detected by flow cytometry,and chromosome karyotype analysis was performed.RPECs were inoculated with porcine epidemic diarrhea virus(PEDV)CV77 strain,porcine circovirus type 2(PCV2)DBN-SX07 strain and porcine pseudorabies virus(PRV)Bartha-K61 strain,respectively.PEDV CV77 strain was inoculated into green monkey kidney cells(Vero cells),PCV2 DBN-SX07 strain was inoculated into pig kidney cells(PK15 cells),and PRV Bartha-K61 strain was inoculated into hamster kidney fibroblast cells(BHK cells)to observe the cytopathic effects(CPE).Indirect immunofluorescence assay was also used to measure virus titers.【Result】PCR and sequencing results showed that the eukaryotic plasmid pCI-neo-E1 was successfully constructed.It was transfected into primary porcine retinal pigment epithelial cells,and the immortalized cell line was obtained by G418 selection.The cell line expressed keratins 18 and 19.The cell line maintained the epithelioid cell morphology and proliferation activity for 50 passages.CPE was observed in RPECs 24 h after infe-ction with PEDV CV77 strain.The titer of PEDV CV77 strain was 10^(5.25) TCID_(50)/mL,which was lower than that in Vero cells(10^(8.125) TCID_(50)/mL).The PCV2 DBN-SX07 strain infected porcine RPECs and produced CPE.The virus titer was 10^(6.125)TCID_(50)/mL,which was slightly higher than that in PK15 cells(106.0 TCID_(50)/mL).CPE was observed in RPECs infected with PRV Bartha-K61 strain.The titer of PRV Bartha-K61 strain was 10^(8.75)TCID_(50)/mL,which was slightly lower than that of PRV Bartha-K61 strain in BHK(108.875 TCID_(50)/mL).【Conclusion】The immortalized porcine retinal cell line was successfully constructed,which can be used for the culture of porcine viruses.PCV2 could produce CPE and it is easy to observe,providing a new cell material for subsequent improvement of vaccine technology.