Cloning And Serum-free Culture Of Cell Line RIRI-PX1 Developed From Papilio Xuthus(Lepidoptera:Papilionidae)

In this study,the cell lines RIRI-PX1 developed from Papilio xuthus was used as the material.Semi-solid culture method was used to clone the Papilio xuthus cell lines.The protein expression characteristics of cell line RIRI-PX1 and its clones were studied and compared,and the cloned strains RIRI-PX1-C24 and RIRI-PX1-C31 with significantly higher protein expression levels than the parent cell line were screened,then study their biological characteristics.Simultaneously,the clones RIRI-PX1-C24 and RIRI-PX1-C31 were adapted to serum-free medium.The cell population doubling time and recombinant protein production with baculovirus expression vectors of serum-free medium were compared with that of serum-containing cultures.The main results were summarized as follows:1.Isolation of clones from RIRI-PX1cell clonesIn this study,semi-solid culture method was used to clone the cell lines RIRI-PX1,in which the optimum inoculation density of cells 2.5x10~5/ml,14 clones were obtained:RIRI-PX1-C2,RIRI-PX1-C7,RIRI-PX1-C10,RIRI-PX1-C14,RIRI-PX1-C15,RIRI-PX1-C18,RIRI-PX1-C19,RIRI-PX1-C23,RIRI-PX1-C24,RIRI-PX1-C26,RIRI-PX1-C29,RIRI-PX1-C30,RIRI-PX1-C31 and RIRI-PX1-C32.2.Screening of highly expressed clones of RIRI-PX1We used recombinant baculovirus carrying three reporter genes,green fluorescent protein(GFP),β-galactosidase(β-Gal),and secreted alkaline phosphatase(SEAP),to infect the cell lines RIRI-PX1 and its clones,and to detect the expression levels of recombinant protein.The cloned strain RIRI-PX1-C24 with significantly higher expression level of recombinant GFP than the parent cell line RIRI-PX1(P<0.05)and the expression level ofβ-Gal in clone cell line RIRI-PX1-C31 was significantly higher than that in parent cell line RIRI-PX1(P<0.05).3.Biology characteristics of RIRI-PX1-C24 and RIRI-PX1-C31In morphology,the parent cell line RIRI-PX1 and its clones RIRI-PX1-C24 and RIRI-PX1-C31 were consisted of spindle-shaped cells and spherical cells.The percent of spherical cells in clonal cells was larger than that in parent cell line.The morphology of RIRI-PX1-C24 and RIRI-PX1-C31,became a little bigger than the parent cell line RIRI-PX1.The results of cell proliferation kinetics analysis showed that the cell population doubling time time of RIRI-PX1 and its cloned strains RIRI-PX1-C24 and RIRI-PX1-C31 was 47.86 h,40.79h and 62.51 h,respectively.The cell population doubling time of cloned strain RIRI-PX1-C24was shortened compared with that of parent cell line.Karyotype analysis showed that the number of chromosomes of the parent cell line RIRI-PX1 and its clonal cell line RIRI-PX1-C24 and RIRI-PX1-C31 fitted the normal distribution.The average chromosome numbers of RIRI-PX1-C24 cell line(107.65±33.71)and RIRI-PX1-C31 cell line(104.53±31.57)were significantly different from those of RIRI-PX1(119.75±32.79).The COI sequences from RIRI-PX1 cell lines and its clones RIRI-PX1-C24 and RIRI-PX1-C31confirmed that the clones were derived from RIRI-PX1.The fingerprint analysis of three cell lines using 10 ISSR primers generated 7 differential markers,indicating the difference in genotype.Viral susceptibility:Both of the parent cell line RIRI-PX1 and its clones could be infected by wt-Ac MNPV,but RIRI-PX1-C24 and RIRI-PX1-C31 were significantly more susceptible to wt-Ac MNPV than RIRI-PX1(P<0.05).4.Serum-free medium adaptation of RIRI-PX1-C24 and RIRI-PX1-C31The clonal cell RIRI-PX1-C24 and RIRI-PX1-C31 were cultured in serum-free medium Express Five SFM、EX-CELL 405、Sf-900 III SFM、Sf-900 II SFM、Hy Clone Serum-Free Media.At the same time,the serum content in the original medium(Grace)was gradually reduced,and the minimum serum content that the cell line could tolerate was determined.The cloned strain RIRI-PX1-C24 were not properly passed out in any of the five serum-free medium.The minimum serum content that can be tolerated by cloned strains RIRI-PX1-C24 in Grace medium is 5%.The cell population doubling time was extended from56.55 h to 88 h,and the expression level 3 recombinant protein was not significantly increased(P>0.05).The cloned strain RIRI-PX1-C31 was stably passaged in Express Five SFM,in the other four serum-free mediums,the cells gradually ceased to proliferate with the decrease of serum content.The proliferation rate of RIRI-PX1-C31 in serun-free mediums was significantly lower than that in serum-containing medium,but the expression levels of recombinantβ-Gal and SEAP in Express Five SFM medium was significantly higher than in Grace+20%FBS medium(P<0.05).The minimum serum content that can be tolerated by cloned strains RIRI-PX1-C31 in Grace medium is 5%.The cell population doubling time was extended from 62 h to 69 h,and the expression level 3 recombinant protein was not significantly increased(P>0.05).In summary,the high-efficiency expression cell line screening technology was used to screening single-cell clones of RIRI-PX1,and 14 clones were obtained.The expression level of GFP in RIRI-PX1-C24 was significantly higher than that of the parent cell line RIRI-PX1;the expression level ofβ-galactosidase protein in RIRI-PX1-C31 was significantly increased.In addition,RIRI-PX1-C31 was stably passaged in Express Five SFM,and the expression levels of recombinantβ-Gal and SEAP are significantly increased(P<0.05),which deserves further study.