关键词: 达摩凤蝶, 新孵幼虫, 昆虫细胞系, 分泌型碱性磷酸酶, 重组杆状病毒

Abstract: 【Aim】 This study aims to establish and characterize cell lines developed from the neonate larvae of Papilio demoleus Linnaeus. 【Methods】 Modified Grace’s medium supplemented with 20% fetal bovine serum was used to culture tissues from neonate larvae of P. demoleus. The methods of cell morphology analysis, growth analysis, chromosome analysis, and molecular identification of the cell lines were used to obtain the biological characteristics of new cell lines. In order to study the viral susceptibility of four cell lines derived from P. demoleus, recombinant baculovirus carrying secreted alkaline phosphatase (AcMNPVSEAP) was constructed using Bac-to-Bac baculovirus expression system to infect the cell lines. The median tissue culture infectious dose (TCID₅₀) of each cell line was measured using limiting dilution method by measuring the expression of recombinant SEAP. 【Results】 Four new cell lines developed from the neonate larvae of P. demoleus were established on modified Grace’s medium and named RIRI-PaDe-1, RIRI-PaDe-2, RIRI-PaDe-3 and RIRI-PaDe-4, respectively. These cells were subcultured for approximately 60 passages. There were significant differences in biological characteristics between different cell lines. According to morphology, RIRI-PaDe-1 and RIRI-PaDe-4 were composed of round, spindle and polygonal cells, RIRI-PaDe-2 was mainly composed of round cells, and RIRI-PaDe-4 was mainly composed of epidermal-like and fibrous cells. The cell population doubling time (PDT) of IRI-PaDe-1, RIRI-PaDe-2, RIRI-PaDe-3 and RIRI-PaDe-4 was 69.77, 67.42, 81.48 and 65.43 h, respectively. Karyotype analysis showed that the number of chromosome of each cell line fitted the normal distribution. The chromosome numbers of RIRI-PaDe-2, RIRI-PaDe-3 and RIRI-PaDe-4 varied widely from 45 to 97, while that of RIRI-PaDe-1 varied from 36 to 90. All of the four cell lines could be infected with the AcMNPV-SEAP, but RIRI-PaDe-3 was more susceptible to AcMNPV-SEAP. 【Conclusion】 Although the four new cell lines developed from the neonate larvae of P. demoleus have the same genetic background analyzed by DNA amplification fingerprinting (DAF) using mitochondrial cytochrome c oxidase subunit I (COI) gene, there are still significant differences in the basic biological characteristics among them. It is worthy to be further researched.

Key words: Papilio demoleus, neonate larvae, insect cell line, secreted alkaline phosphatase, recombinant baculovirus