文献类型：期刊文献

中文题名：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

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

作者：孙娜[1] 丁伟峰[1] 刘志刚[1] 张欣[1] 李娴[1] 冯颖[1]

第一作者：孙娜

机构：[1]中国林业科学研究院资源昆虫研究所国家林业局资源昆虫培育与利用重点实验室

年份：2019

卷号：0

期号：3

起止页码：304-311

中文期刊名：昆虫学报

外文期刊名：Acta Entomologica Sinica

收录：CSTPCD;;Scopus;北大核心:【北大核心2017】；CSCD:【CSCD2019_2020】；

基金：中央级公益性科研院所基本科研业务费专项资金(CAFYBB2016ZD005)

语种：中文

中文关键词：柑橘凤蝶;单细胞克隆;重组杆状病毒;重组蛋白;简单重复序列区间

外文关键词：Papilio xuthus;single cell cloning;recombinant baculovirus;recombinant protein;inter simple sequence repeat(ISSR)

分类号：Q962

摘要：【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。
【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.