| Dunaliella salina, a unicellular eukaryotic green alga without cell well, can live in a wide range of NaCl concentration from0.5M-5M, and has flagella of equal-length of～13μm. The modle of the flagellum of D. salina has a typical structure deemed as“9+2”, which can be observed by using optical microscope. Flagellar resorption and regeneration take place after treated with machinery and some chemical agents such as colchicine, so it is much suited for a model organism for reaserching the resorption and regeneration and diseases related to flagella.The flagellum/cilium is an organelle on the surface of cells and plays vital roles in cell communication, division, motion and signal transduction. The dysfunction of flagellum/cilium is bound up with several human diseases, such as left-right asymmetry in the organization, Kartagener syndrome, polycystic kidney disease, Multi-fingered or polydactyly and so on. Hence, the regulation mechanism of flagellum/cilium plays important roles in occurrence and pathogenesis of these diseases. But at present the exact regulation mechanism of flagellum/cilium is poorly understood. Flagellar/cilia assembly is a bidirectional process, which means that both anterograde and retrograde exist in the flagellum during the same period. Intraflagellar transport (IFT) particles return along the axonemal of microtubles from the tip of the flagella back to the cell body by dynein with the microtubule depolymerization macterials. When the anterograde process is predominant, the flagella will regenerate; on the contrary, flagella will demonstrate resorption. There is a link between flagella regeneration, resorption and cell devision, abnormality of flagellar length is also observed in many human diseases.Based on the sequence in GenBank, the Open Reading Frame of FLA8was cloned by RT-PCR. The pET28a-FLA8of prokaryotic expressing vector was constructed and then transfected into E. coli BL21. The high-purity proteins of FLA8were purified by IPTG induction at its optimal concentration. The specific polyclonal antibodies were obtained by immunizing New Zealand rabbits for further study about FLA8.This study also detected the expression of FLA8during the resorption and regeneration using polyclonal antibody. To further study the relationship between FLA8and his homology gene KIF3B, a fluorescent orientation vector pEGFP-N1-FLA8was constructed, then transfected into esophageal squamous cell carcinoma (ESCC) EC9706cells with pEGFP-N1-FLA8and found out where is the fusion proteins in the position of the cell.Methods1Preparation of FLA8polyclonal antibody1.1Cloning and sequence analysis of FLA8geneAccording to the sequence on Genbank, a pair of primers was designed to amplify the FLA8gene using RT-PCR. The resulting products of PCR were cloned into pMD-T vector and analyzed after sequencing.1.2Construction of FLA8prokaryotic expression vectorThe plasmid of pMD-FLA8and empty pET28a (+) were digested by restriction enzymes Nde Ⅰ and HindⅢ, the recombinant plasmid pET28a-FLA8was constructed and identified by DNA sequencing.1.3Expression of FLA8in E.coli BL21and its purificationAfter the pET28a-FLA8was transferred into E.coli BL21cultured in LK medium,IPTG was add to the medium to induce the expression of FLA8proteins. Subsequently, the sample of proteins was loaded to SDS-PAGE to see what is the best condition to induce the proteins. In order to get the most quantity of precipitate inclusion proteins, the precipitate inclusion body was collected by repeated washing, dissolved, and then purified by Ni2+-NTA-agarose purification column.1.4Preparation of FLA8polyclonal antibodySpecific antibodies with a high titer were obtained after separation of antiserum and Western blots was also conducted for verifying the specificity of the antibody.2Expression of FLA8during the flagellar regenerationThe expression of FLA8in the total protein is low under the normal. But the total protein of FLA8is apparently improved during the regeneration by Western blots.3Localization of FLA8in ESCC EC97063.1Construction of fluorescence vectorThe ORF of FLA8was amplified, digested with proper restriction enzyme, and then inserted into the polyclonal sites of fluorescent vector pEGFP-N1.3.2TransfectionThe ESCC EC9706cells were seeded in6-well plates, and fluorescent vector pEGFP-N1-FLA8was transfected by LipofectamineTM2000according to the explanatory memorandum. Meanwhile the cells transfected with empty vector pEGFP-N1were used as negative control.3.3Cellular localization of the fusion proteinsThe localization of pEGFP-N1-FLA8fusion proteins in the EC9706cells was observed using fluorescence microscopy camera system24-48h after transfected by pEGFP-N1-FLA8.Results1Prokaryotic expression and preparation FLA8polyclonal antibody1.1Cloning of FLA8and ORF sequence analysis The total RNA extracted from D. salina using Trizol reagent and the results were clear and distinct for the two bands of28S and18S. The FLA8gene ORF was amplified by RT-PCR, there are about2355bp bases which encoded784amino acids. Homologous analysis suggested that the sequence was the same as the FLA8on GenBank.1.2Construction prokaryotic expression vectorThe results of restriction enzymes digestion show two brands about2500bp and5OOObp, respectively.The DNA sequencing was identify with the sequence of GenBank.it is revealed that the prokaryotic expression vector was constructed successfully.1.3Expression of FLA8in E.coli BL21and purificationThe result of SDS-PAGE showed that a distinct band approximate94kDa appeared when the fusion FLA8proteins were expressed at optimum levels after the induction of1.1mM IPTG at37℃in E.coli BL21, which was estimated to be the recombinant FLA8. The proteins were purified by Ni2+-NTA-agarose purification column that accounts for about95%of the total proteins.1.4Preparation of FLA8polyclonal antibodyProkaryotic expression vector pET28a-FLA8was successfully constructed with a size of about94kDa The fusion protein which was expressed in E. coli BL21successfully. Inclusion bodies fusion protein was purified using the Ni-NTA-agarose purification column. The SDS-PAGE shows its purity up to95%and the titer of anti-serum of FLA8was about1:512000and proved that the antibody has a high specificity.2Expression of FLA8following the flagellar regenerationSDS-PAGE analysis demonstrated that the FLA8expressed was relatively rare in the D. salina total proein expression. Western blots showed that the expression of FLA8protein was increased significantly during the flagellar regeneration.3Localization of FLA8in ESCC EC9706cells3.1Construction of fluorescence localization vectorThe results of restriction enzyme digestion and the DNA sequencing showed the fluorescence localization vector pEGFP-N1-FLA8was successfully constructed.3.2Cellular localization of pEGFP-N1-FLA8fusion proteinsESCC EC9706cells were transfected by pEGFP-N1, pEGFP-N1-FLA8respectively, and then observed by the fluorescence microscope for24-48h. Green fluorescence distributed throughout the cells when transfected by empty vector pEGFP-N1, while green fluorescence is located in the nucleus when transfected with pEGFP-N1-FLA8.ConclusionIn this study, It is proved that FLA8genes involved in the flagellar regeneration using the fusion protein as the antibody and observed FLA8gene located in the centrosome when it has been transfected into esophageal squamous cell carcinoma. |
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