柑橘凤蝶细胞克隆株RIRI-PX1-C24的生物学及重组蛋白表达特性

【目的】研究柑橘凤蝶Papilio xuthus细胞系RIRI-PX1的单细胞克隆株RIRI-PX1-C24的生物学和重组蛋白表达特性。【方法】用野生型苜蓿银纹夜蛾核型多角体病毒(wild-type Autographa californica multiple nucleopolyhedrosis virus, wt-AcMNPV)侵染RIRI-PX1-C24与RIRI-PX1，检测细胞对野生型病毒的敏感性；使用重组绿色荧光蛋白杆状病毒(AcMNPV-GFP)、重组β-半乳糖苷酶杆状病毒(AcMNPV-Gal)以及重组分泌型碱性磷酸酶杆状病毒(AcMNPV-SEAP)分别侵染细胞系RIRI-PX1-C24和RIRI-PX1，在之后的24, 48, 72, 96, 120, 144和168 h检测3种重组蛋白的表达量；通过简单重复序列区间(inter simple sequence repeat, ISSR)标记比较RIRI-PX1-C24与RIRI PX1的遗传相似性。【结果】亲本细胞系 RIRI-PX1和克隆株RIRI-PX1-C24均能被wt-AcMNPV侵染，其中RIRI-PX-C24对wt-AcMNPV的敏感性较RIRI-PX1有显著提高。3种重组蛋白均能在2个细胞系中表达，其中重组绿色荧光蛋白在RIRI-PX1-C24中的表达水平远高于在亲本细胞系RIRI-PX1中的表达水平，但重组β-半乳糖苷酶蛋白和重组分泌型碱性磷酸酶蛋白在RIRI-PX1-C24中的表达水平较在RIRI-PX1中的无显著提高。用10条ISSR引物进行的RIRI-PX1-C24和RIRI-PX1 2个细胞系的指纹图谱分析中，4条引物扩增条带在这2个细胞系间存在差异；2个细胞系的遗传相似性范围在0~83.33%之间，说明克隆株及其亲本细胞系在基因型上存在差异。【结论】通过对柑橘凤蝶细胞系RIRI-PX1进行单细胞克隆，确实获得了使重组绿色荧光蛋白表达水平有显著提高的克隆株RIRI-PX1-C24，其利用价值仍需进一步的研究。

Characterization and recombinant protein expression in the clonal strain RIRI-PX1-C24 derived from Papilio xuthus (Lepidoptera: Papilionidae)

【Aim】 This study aims to explore the biological characteristics and the expression of recombinant proteins in the cell line RIRI-PX1-C24 cloned from RIRI-PX1 cell line derived from Papilio xuthus. 【Methods】 The wild-type Autographa californica multiple nucleopolyhedrosis virus (wt-AcMNPV) was used to infect the clonal strain RIRI-PX1-C24 and the parent cell line RIRI-PX1, and the viral susceptibility of the two cell lines were detected. The recombinant baculoviruses carrying three reporter genes, i.e., green fluorescent protein (GFP) gene, β-galactosidase (Gal) gene, and secreted alkaline phosphatase (SEAP) gene, were used to infect RIRI-PX1-C24 and RIRI-PX1. The expression levels of the three recombinant proteins between the two cell lines were detected at 24, 48, 72, 96, 120, 144, and 168 h after viral infection. The genetic similarity between RIRI-PX1-C24 and RIRI-PX1 were compared by using inter simple sequence repeat (ISSR) markers. 【Results】 Both of the parent cell line RIRI-PX1 and the clonal strain RIRI-PX1-C24 could be infected by wt-AcMNPV, but RIRI-PX1-C24 was significantly more susceptible to wt-AcMNPV than RIRI-PX1. The recombinant GFP had significantly higher expression levels in RIRI-PX1-C24 than in RIRI-PX1. However, there were no significant differences in the expression levels of recombinant Gal protein and recombinant SEAP protein between RIRI-PX1-C24 and RIRI-PX1. The fingerprint analysis of RIRI-PX1-C24 and RIRI-PX1 using 10 ISSR primers generated four differential markers. The two cell lines had the genetic similarity levels ranging from 0 to 83.33%, indicating the difference in genotype. 【Conclusion】 This study has produced the clonal strain RIRI-PX1-C24 from the parent cell line RIRI-PX1 of P. xuthus by single cell cloning, which has a significantly enhanced expression level of recombinant GFP. The application value of RIRI-PX1-C24 needs further research.