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【目的】建立达摩凤蝶Papilio demoleus Linnaeus新孵幼虫细胞系,并对其生物学特性进行研究。【方法】使用改良配方的Grace培养基并辅以20%胎牛血清,通过原代和传代培养建立达摩凤蝶新孵幼虫细胞系。通过显微观察、细胞活力分析、核型分析和分子鉴定,获得4个新建细胞系的生物学特性数据。使用Bac-To-Bac杆状病毒表达系统构建重组分泌型碱性磷酸酶杆状病毒(Ac MNPV-SEAP)。使用有限稀释法测定达摩凤蝶细胞系对Ac MNPVSEAP的半数组织培养感染剂量(TCID50),比较达摩凤蝶细胞系对Ac MNPV-SEAP的敏感性。【结果】建立了4个贴壁生长的达摩凤蝶新孵幼虫细胞系(RIRI-Pa De-1,RIRI-Pa De-2,RIRI-Pa De-3及RIRI-Pa De-4),且均传至60代以上。形态学方面,细胞系RIRI-Pa De-1和RIRI-Pa De-4较为相近,均由圆形、梭形以及多边形细胞组成,细胞系RIRIPa De-2主要为圆形细胞,而RIRI-Pa De-3主要为类似表皮细胞和纤维状细胞。细胞增殖动力学方面,4个细胞系的群体倍增时间分别为69.77,67.42,81.48及65.43 h。核型分析显示4个细胞系染色体数量均呈正态分布,其中RIRI-Pa De-2,RIRI-Pa De-3以及RIRI-Pa De-4的染色体数目分布区间比较接近,在45~97条之间,RIRI-Pa De-1的染色体条数相比偏少,在36~90条之间。通过比对4个达摩凤蝶细胞系和虫卵的细胞色素c氧化酶亚基I(COI)基因序列,证明4个新建细胞系来源于达摩凤蝶组织。比较4个细胞系对Ac MNPV-SEAP敏感性发现RIRI-Pa De-3对此病毒较为敏感,可作为重组杆状病毒表达的宿主。【结论】虽然4个新建达摩凤蝶细胞系的来源相同,具有相同的遗传背景,但其生物学特征仍有明显差异,具有进一步研究的价值。
【Objective】 To establish a newly hatched larval cell line of Papilio demoleus Linnaeus and to study its biological characteristics. 【Method】 Newborn larvae cell lines of Papilio Papyrifera were established by using modified Grace’s medium supplemented with 20% fetal calf serum and primary and subculture. The biological characteristics of four newly established cell lines were obtained by microscopic observation, cell viability analysis, karyotype analysis and molecular identification. Recombinant secreted alkaline phosphatase baculovirus (Ac MNPV-SEAP) was constructed using the Bac-To-Bac baculovirus expression system. The limiting dilution method was used to determine the median tissue culture infective dose (TCID50) of Ac MNPVSEAP in the Papaver bracteosperm cell line, and to compare the sensitivity of Acantham & apos; s daphnida cell line to Ac MNPV-SEAP. 【Result】 Four new cell lines (RIRI-Pa De-1, RIRI-Pa De-2, RIRI-Pa De-3 and RIRI-Pa De-4) And are passed to more than 60 generations. Morphologically, the cell lines RIRI-Pa De-1 and RIRI-Pa De-4 are relatively similar and consist of round, fusiform and polygonal cells. The cell line RIRIPa De-2 is mainly round cells, whereas the RIRI-Pa De-3 is mainly similar to epidermal cells and fibroblasts. In terms of cell proliferation kinetics, the population doubling times of the four cell lines were 69.77, 67.42, 81.48 and 65.43 h, respectively. Karyotype analysis showed that the number of chromosomes in four cell lines showed a normal distribution. The distributions of chromosome numbers of RIRI-Pa De-2, RIRI-Pa De-3 and RIRI-Pa De-4 were close to each other. Between 45 and 97 RIRI-Pa De-1 compared to the number of chromosomes is less than the number of 36 to 90 between. By comparing the sequence of the cytochrome c oxidase subunit I (COI) gene in four Papaver somniferum cell lines and eggs, we found that four of the newly established cell lines originated from the Papaveraceae tissue. Comparing the sensitivity of four cell lines to Ac MNPV-SEAP, RIRI-Pa De-3 was found to be sensitive to this virus and could be used as a host for recombinant baculovirus expression. 【Conclusion】 Although the four new Papilio Papilioma cell lines share the same source and have the same genetic background, their biological characteristics are still significantly different and have the value of further research.