Establishment And Characterization Of Clones Developed From Papilio Demoleus Linnaeus (Lepidoptera:Papilionidae) Cell Lines

The insect cell-baculovirus expression vector system has been in recent years the rapidly developing eukaryotic cells expression system,and has significant value in genetic engineering,protein engineering,vaccine and pharmaceutical research.In this study,four cell lines developed from Papilio demoleus Linnaeus were used as the material for single-cell cloning.Different cloning methods were compared that aims to design a new cloning procedure suiting for Papilio demoleus cell.The expression characteristic between parental cell lines and their clones was analysed to screen out high-expression clone cells,then biological characteristics of which were study in detail.Establishment of Papilio demoleus cell clones not only provided a new cloning method,but laid a foundation for utilization of clone cells of high expression level.The main results were summarized as follows:1.Isolation of clones from Papilio demoleus cell clonesIn this study,four single-cell cloning methods,limiting dilution,micromanipulation,feeder layer and semi-solid culture,were used to clone four Papilio demoleus cell lines.The results showed that only semi-solid culture method could be used to obtain subclones.when the seeding density of cells was in the range of 3.05×105~6.10×105cells/m L,single cells could grow to form cell clusters,and the clusters were distributed evenly with an appropriate distance,which allowed the micromanipulator system to easily pick cell clusters.After the cell clusters were transferred to new environmemt from semi-solid mudium,the cells continued to divide,and to form a stable cell colonies finally.The whole process of single-cell cloning needed approxiately to cost 41~64 days.Semi-solid culture method was used to clone the Papilio demoleus cell lines,and 61 clones were obtained,including 6 clones from RIRI-Pa De-1,11 clones from RIRI-Pa De-2,40 clones from RIRI-Pa De-3,and 4 clones from RIRI-Pa De-4.2.Expression characteristic of clonal population of Papilio demoleus cell linesWe used recombinant baculovirus carrying three reporter genes,green fluorescent protein(GFP),?-galactosidase(?-Gal),and secreted alkaline phosphatase(SEAP),to infect four of Papilio demoleus cell lines(RIRI-Pa De-1?RIRI-Pa De-2?RIRI-Pa De-3?RIRI-Pa De-4)and their clones,and to detect the expression levels of recombinant protein.(1)Expression of GFP in Papilio demoleus cell lines and their clonesCell line RIRI-Pa De-1,RIRI-Pa De-3 and their clones could express GFP,and the expression level in parental cell line was higher than that in clones.After inoculation with Ac MNPV for 96 h,the number of emitting-fluorescent cells increased to the maximum in RIRI-Pa De-3 cell line,but the expression level was much lower than in that in Sf9.Green fluorescent cells in RIRI-Pa De-2,RIRI-Pa De-4,and their clones.were not detected.(2)Expression of ?-gal in Papilio demoleus cell lines and their clonesThree Papilio demoleus cell lines and their clones could be infected with Ac MNPV-Gal,expression level of ?-Gal in different cell lines was varied.Expression level of ?-Gal in clone cell line RIRI-Pa De-1-C1 was significantly higher than that in parental cell line RIRI-Pa De-1.Expression level of ?-Gal in RIRI-Pa De-C6 was twice as that in parental cell line RIRI-Pa De-2.Expression level of ?-Gal in cell line RIRI-Pa De-3-C52(0.270±0.009 milliunits/?L)was at most,but have no significant differences from that in RIRI-Pa De-3 cell line,and the expression level was much lower than in that in Sf9(4.132±0.941 milliunits/?L).(3)Expression level of SEAP in Papilio demoleus cell lines and their clonesThree Papilio demoleus cell lines and their clones could be infected with Ac MNPV-SEAP,expression level of SEAP in different cell lines was varied widely.There were no remarkable difference between RIRI-Pa De-1 cell line and their clones at expression level of SEAP(P>0.05);Expression level of clones of RIRI-Pa De-2 didn't surpass that of parental cell line;Expression level of SEAP in clones of RIRI-Pa De-3 were significantly less than that in parental cell line(P<0.05).3.Biology characteristics of RIRI-Pa De-2-C6Studying the expression characteristic of four Papilio demoleus cell lines and their clones showed that expression level of ?-Gal in RIRI-Pa De-2-C6 was significantly higher than that in RIRI-Pa De-2.Biological characteristics of RIRI-Pa De-2-C6 has been studied.All cells in RIRI-Pa De-2-C6 were spindle-shaped,which was more homogeneous than RIRI-Pa De-2 in cell morphology;The cell population doubling time of RIRI-Pa De-2-C6 was 94.94 h which was longer than those of RIRI-Pa De-2(67.42 h);The averages chromosome numbers of RIRI-Pa De-2-C6 was 52.26±30.48,which was significant difference from those of RIRI-Pa De-2(73.19±24.274);The virus was used to test the viral susceptibility of RIRI-Pa De-2-C6 and RIRI-Pa De-2,the result showed that RIRI-Pa De-2-C6 cell line was easier to be infected.In this dissertation,four single-cell cloning methods were used to isolate subclones from four Papilio demoleus cell lines.The semi-solid culture method could generate well-dispersed clone cell plaques,and plaques could be picked by a micromanipulator system and cultured to produce 61 individual cell clones.Expression level of ?-Gal in RIRI-Pa De-C6 was nearly twice as that in RIRI-Pa De-2.It was valuable to study biological characteristic of RIRI-Pa De-2-C6,which could lay a foundation for further development.