Acta Entomologica Sinica ›› 2019, Vol. 62 ›› Issue (3): 304-311.doi: 10.16380/j.kcxb.2019.03.004

• RESEARCH PAPERS • Previous Articles     Next Articles

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

SUN Na, DING Wei-Feng, LIU Zhi-Gang, ZHANG Xin, LI Xian, FENG Ying*   

  1. (Key Laboratory of Cultivating and Utilization of Resource Insects of State Forestry Administration, Research Institute of Resource Insects, Chinese Academy of Forestry, Kunming 650233, China)
  • Online:2019-03-20 Published:2019-03-14


【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.

Key words: Papilio xuthus; single cell cloning, recombinant baculovirus, recombinant protein, inter simple sequence repeat (ISSR)