| The technique of insect cell culture, as one of the bases of cell engineering, is a valuable method of modern experimental biology. It is applied widely to various fields of biology, medicine and agriculture. The insect cells- baculovirus expression victor system(BEVS), which has the advantages of the extremely high expression of foreign genes and the ability to translationally modify the newly synthesis preteins, is being increasingly used in gene engineering. In this study, the neonate larvae of Papilio demoleus Linnaeus were used as the material for establishment of cell lines. After a long period, the cells continued to multiply in some cultures, and finally 4 continuous cell lines were established. This thesis describes the establishing process of the cell lines, biological characteristics and virus susceptibility of the cell lines. The expression of recombinant protein of the established cell lines are also provided. The results were summarized as follows.1. The cell culture of neonate larvae of P. demoleus L.Modified Grace’s medium supplemented with 20% fetal bovine serum was used culturing tissues frome neonate larvae of P. demoleus L. The primary cell lines were cultured for over 7 months before any observable replication in cell numbers. After that time, the cells continued to multiply in some cultures. Finally continuous 4 cell lines were established, named RIRI-Pa De-1, RIRI-Pa De-2, RIRI-Pa De-3 and RIRI-Pa De-4 respectively.2. The basic biological characteristics of new cell lines2.1 Identification of the cell lines: The COI(mitochondrial cytochrome c oxidase subunit I gene) sequences from RIRI-Pa De cell lines and the eggs of P. demoleus L. exhibited 100% identity. These results confirmed that the new established cell lines were derived from P. demoleus.2.2 Cell morphology: Four cell lines mostly attached to the flask. At the early culture phase, the various types of cells that migrated from tissues. After subculture of 4 cell lines was stably, the cell type of each cell lines appeared morphological differences.The RIRI-Pa De-1 were mainly round(accounted for 61.73±1.33%, average diameter ranged from 7.06μm to 35.112μm), short spindle-like, and epithelial-like cells. The average diameter of cells were 6.79±0.27μm under suspension.The RIRI-Pa De-2 were mainly round(average diameter ranged from 7.14μm to 27.66μm) cells. The average diameter of cells were 6.87±0.27μm under suspension.The RIRI-Pa De-3 were mainly epithelical-like or fibroblast-like cells. The average diameter of cells were 6.66±0.56μm under suspension.The RIRI-Pa De-4 were mainly round(accounted for 58.35±4.22%, average diameter was 13.813±0.421μm), short spindle-like and epithelial-like cells. The average diameter of cells were 6.71±0.47μm under suspension.2.3 Growth analysis: The growth rate of 4 cell line cultures were expressed by growth curves. The population doubling time(PDT) of each cell lines was measured with a growing cell population during the exponential phase and calculated to be 69.77 h for RIRI-Pa De-1, 67.42 h for RIRI-Pa De-2, 81.48 h for RIRI-Pa De3, 65.43 h for RIRI-Pa De-4.2.4 Chromosome analysis: The suitable concentration of hypotonic KCl solution was 0.60% for RIRI-Pa De cell lines. Under this concentration of hypotonic solution, the cells membrane were broken, cytoplasm scattered cytoplasm of the cells at mitotic metaphase. after 61 st passages, the chromosome number distribution of RIRI-Pa De-1 varied widely from 36 to 90 with a average mode of 63; RIRI-Pa De-2(60th passages) varied widely from 49 to 97 with a average mode of 73; RIRI-Pa De-3(52nd passages) varied widely from 46 to 90 with a average mode of 68; RIRIPa De-4(52nd passages) varied widely from 45 to 91 with a average mode of 68.2.5 Cryopreservation and thawing: The experimental results showed that the post-thaw viability of 4 cell lines decreased with the time of cryopreservation in liquid nitrogen expended. All 4 cell lines could recovery in 10 days after thawing. That means cell lines derived from P. demoleus could be stored frozen in a liquid nitrogen using routine operation.3. Viral susceptibilityThe following viruses were used to test the viral susceptibility of 4 cell lines: Autographa californica multicapsid nucleopolyhedrovirus(Ac MNPV), Bombyx mori nucleopolyhedrovirus(Bm NPV), Apocheima cinerarius nucleopolyhedrovirus(Aci NPV), Hyphantria cunea nucleopolyhedrovirus(Hycu NPV), Lymantria dispar nucleopolyhedrovirus(Ld NPV), and Antheraea pernyi nucleopolyhedrovirus(Anpe NPV).The result showed that Ac MNPV could infect all 4 cell lines derived from P. demoleus; Aci NPV and Hycu NPV could infect cell line RIRI-Pa De-1, RIRI-Pa De-2, and RIRI-Pa De-4. All of 4 cell lines were found to be insusceptible to Bm NPV, Ld NPV, and Anpe NPV.4. Characteristics of expressing foreign genesIn order to study the characteristics of expressing foreign genes of 4 cell lines derived from P. demoleus, 3 recombinant baculovirus were constructed using Bac-to-Bac® Baculovirus Expression System. That were recombinant baculovirus carring with green fluorescent protein(GFP) named Ac MNPV-GFP, recombinant baculovirus carring with β-galactosidase named Ac MNPV-Lac Z, recombinant baculovirus carring with secreted alkaline phosphatase(SEAP) named Ac MNPV-SEAP. The 6 cell lines(RIRI-Pa De cell lines, Sf9 and High Five as ontrols) would be infected with 3 rocombinant baculovirus separately. The expression level of foreign proteins would be tested.4.1 The sensitivity of Ac MNPV-GFPGFP could be observed with fluorescent microscope directly. If 6 insect cell lines were infected by Ac MNPV-GFP, the median tissue culture infectious dose(TCID50) of each cell lines could be measured using limiting dilution method. The smaller the TCID50, the more sensitive to Ac MNPV-GFP. The test results have shown that GFP could not be detected with microscope which means that GFP was faintly expressed in cell line RIRI-Pa De-2 and RIRI-Pa De-4. The TCID50 of High Five was 10-6.88, 10-6.55 for RIRI-Pa De-1, 10-6.5 for Sf9, and 10-5.45 for RIRIPa De-3.4.2 Expression level of GFPIn order to measure the expression of recombinant GFP, emission spectrum data of each cell lines infected Ac MNPV-GFP after 5 days were collected with an emission wavelength of 533 nm using microplate spectrophotometer. Although the sensibility of RIRI-Pa De-1 was higher than Sf9 to Ac MNPV-GFP, the expression level of GFP in RIRI-Pa De-1 was almost 1/10 in Sf9. The expression level of GFP in RIRI-Pa De-3 was 1/18 in Sf9. The expression level of GFP in RIRIPa De-2 and RIRI-Pa De-4 were 1/27 in Sf9. The expression level of GFP in High Five was 1/2 in Sf9.4.3 Expression level of recombinant β-galactosidaseIn order to measure the expression of recombinant β-galactosidase, enzyme activity of β-galactosidase was measured everyday when each cell lines infected Ac MNPV-Lac Z. The results showed that the expression level of recombinant β-galactosidase in cell lines derived from P. demoleus was much lower than Sf9 and High Five. After 48 h when cells were inoculated with Ac MNPV-Lac Z, recombinant β-galactosidase was prominently expressed in Sf9 and High Five. At the same time, the expression level of recombinant β-galactosidase in cell lines derived from P. demoleus was very low. After 96 h when cells were inoculated with Ac MNPV-Lac Z, the expression level of recombinant β-galactosidase in RIRI-Pa De-3 was significant increased, but it was the equivalent of 1/11 in Sf9. After 216 h, the expression level of recombinant β-galactosidase in each cell lines reached highest. The expression level of recombinant β-galactosidase in High Five was highest. The Sf9 was followed by High Five. The expression level of recombinant β-galactosidase in RIRI-Pa De-3 was 1/8 in High Five. RIRI-Pa De-4 ranked fourth in all 6 cell lines, and the the expression level of recombinant β-galactosidase in RIRIPa De-4 was 1/18 in High Five. The the expression level of recombinant β-galactosidase in RIRIPa De-2 was 1/24 in High Five. The the expression level of recombinant β-galactosidase in RIRIPa De-1 was the lowest.4.4 Expression level of recombinant SEAPIn order to measure the expression of recombinant SEAP, enzyme activity of SEAP was measured everyday when each cell lines infected Ac MNPV-SEAP. The results showed that the expression level of recombinant SEAP in cell lines derived from P. demoleus was comparable to Sf9 and High Five, even higher at one time. After 48 h when cells were inoculated with Ac MNPVLac Z, recombinant SEAP was prominently expressed in All cell lines. After 144 h when cells were inoculated with Ac MNPV-SEAP, the expression level of recombinant SEAP in RIRI-Pa De-1 and RIRI-Pa De-2 was increased significantly. After 169 h, the expression level of recombinant SEAP in all cell lines had reached peaks. After 216 h, the expression level of recombinant SEAP in all cell lines, except RIRI-Pa De-3, was gradually decreased. After 240 h, the expression of recombinant SEAP in RIRI-Pa De-3 was maintaining high level. The research suggests that the expression level of recombinant SEAP in cell lines derived from P. demoleus was comparable to Sf9 and High Five, even higher in some respects. The expression level of recombinant SEAP in RIRI-Pa De-3 was significant higher than Sf9 and High Five. It had potential to develop and utilize the cell line RIRI-Pa De-3 as host of baculovirus expression system.In this dissertation, four continuous cell lines were established successfully applying the neonate larvae of P. demoleus as the material for establishment of cell lines. The research results showed that differences among 4 cell lines in characteristics of basic biological characteristics, virus susceptibility, and expression level of recombinant protein. Four cell lines were sensitive to Ac MNPV. The expression levels of recombinant GFP and β-galactosidase were lower than Sf9 and High Five, but significant higher than Sf9 and High Five. |
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