Preparation Of Polyclonal Antibody To DsGPI Of Dunaliella Salina And Localization In MDCK Cells

GPI, which is short for the glucose-6-phosphate isomerase or the phosphoglucose isomerase, catalyzes the transformation of Glucose-6-phosphate and Fructose-6-phosphate in glycometabolism. GPI is universally distributed among eukaryotes, bacteria and archaea, which is an essential metabolic enzyme involved in both catabolic glycolysis and anabolic gluconeogenesis. In diverse organisms, the molecular weights of GPIs are different, but their functional regions are rather conservative and are usually homodimer molecules, and have high homology.In recent years, it has been demonstrated that GPI, as a highly conservative enzyme, not only involves in glucose metabolism, but also has some special functions, for example, as autocrine motility factors (AMF) in animal cells to promote proliferation and invasion of tumor cells. GPI is also one of neuroleukins (NLK) secreted by lectin stimulated T cells, which can help cultivated feeling and special embryonic spinal cord neuron survive. As a maturation factor (MF), GPI can also mediates human myeloid leukemia HL-60 cells differentiate to terminal monocytes. Although still controversial, in a number of literatures, the anti-GPI autoantibody is considered to have positive correlation with human arthritis. Genetic defect of GPI in human mainly affects red blood cells and causes hereditary non-spherical red blood cell hemolytic anemia (HNSHA).Dnualiella salina is a kind of unicellular green algae without cell wall,which belongs to Chlorophyta, Chlorophyceae and Volcocales. It is one of the most extremely halotolenrant eukaryotes and can live in a wide range of NaCl concentration from 0.05 M-5 M. However, there is still little researches on GPI in D. salina, in addition, there is no commercial anti-DsGPI antibody, making it impossible to detect the DsGPI expression at the protein level.Based on the sequence of DsGPI logged in GenBank, the ORF of it was cloned by RT-PCR. The prokaryotic expression vector, pET28a-DsGPI, was constructed and transfected into E. coli BL21 (DE3).High-purity DsGPI proteins were purified after induction of IPTG at its optimal concentration. By immunizing New Zealand rabbits, the specific polyclonal antibody to DsGPI were obtained, which provided experimental materials for further study.This study also detected the expression of DsGPI under salt stress and completed the first application of self-made DsGPI polyclonal antibody on biological function research of DsGPI.To further study the relationship between DsGPI and human AMF, this study also transfected the recombinated fluorescent orientation vector pEGFP-C1-DsGPI and pEGFP-C1-AMF to MDCK, and was observed under fluorescence microscope.Methods1 Cloning and sequence analysis of the ORF of DsGPI geneAccording to the cDNA sequence of DsGPI on GenBank, a pair of primers was designed to amplify the DsGPI gene using RT-PCR. The PCR product was inserted into pMD18-T vector and then sequenced. Homology analyze was performed after sequencing.2 Construction of DsGPI prokaryotic expression vectorAfter the digestion of pMD18-DsGPI and pET28a(+) by restriction enzymes EcoR I and Nde I, pET28a-DsGPI was constructed using the purified fragment at the presence of T4 DNA ligase. This is followed by identification by enzymes digestion and DNA sequencing.3 Expression and purification of DsGPI in in.coli BL21 (DE3)After the constructed expression vector pET28a-GPI was transformed to E.coli BL21(DE3),IPTG was added into the medium to induce the expression of DsGPI proteins. Following that the sample was loaded to 12% SDS-PAGE to see the result of expression.The inclusion body was collected by repeated washing and dissolved in dissolving buffer, then purified by Ni2+-NTA purification column after being filtered by the 0.45μm filter membrane.4 Preparation of DsGPI polyclonal antibodyThe purified DsGPI fusion proteins were emulsified using ultrasonic method with Freund's adjuvant, and then immunized New Zealand rabbits for the preparation of polyclonal antibody. After 7 times immunization, the anti-serum was collected and detected by ELISIA and Western blots, respectively.5 Detection of the expression of DsGPI under salt stressPut the D.salina, growing in normal medium(1.5 M NaCl) UTEX into the culture medium under salt stress with different concentration of NaCl(1.5 M-3.5 M). The cells were collected after 16 h and the total proteins of the algae were extracted, and then the DsGPI proteins were detected by Western blots using self-made polyclonal antibody.6 The localization of DsGPI and human AMF in MDCK cellsAccording to the GenBank, the ORF of DsGPI gene with BamHⅠand EcoRⅠrestriction sites were amplified using RT-PCR, and then inserted into the multiple cloning sites of fluorescent vector pEGFP-C1.MDCK (dog kidney epithelial cell line) cells were seeded in 6 well plates and transfected by pEGFP-C1-DsGPI and pEGFP-C1-AMF at the presence of LipofectamineTM2000 according to the instruction, meanwhile transfected by empty vector pEGFP-C1 as negative control. The localization of EGFP-DsGPI and EGFP-AMF fusion proteins were observed in MDCK cells using fluorescence microscopy system after 24 h.Results1 Cloning and sequence analysis of the ORF of DsGPI geneThe total RNA extracted from D. salina using TRIzol reagent were very perfect. The two bands of 28S and 18S were clear and distinct. Using RT-PCR, the ORF of DsGPI gene was amplified, which contained 1980 bp, encoding 559 amino acids. Homologous analysis suggested that the sequence was that exactly wanted to be.2 Construction of DsGPI prokaryotic expression vectorThe results of restriction enzymes digestion and DNA sequencing revealed that the prokaryotic expression vector was successfully constructed.3 Expression and purification of DsGPI in E.coli BL21 (DE3)The result of SDS-PAGE showed that the fusion DsGPI proteins were expressed at a high level after the induction of 3 mM IPTG at 37℃. A distinct band approximate 78 kDa was expressed in E.coli BL21 (DE3), which was estimated to be the recombinant DsGPI and accounted for about 41% of the total proteins. The proteins were purified by Ni2+-NTA purification column. After purification,the recombinant proteins account for about 96% of the total proteins.4 Preparation of DsGPI polyclonal antibodiesIndirect ELISA showed that the titers of anti-serum to DsGPI were about 1:512 000 after immunization. Western blots showed that anti-DsGPI polyclonal antibody was successfully prepared in that an obvious band about 78 kDa could be seen clearly.5 Detection of the expression of DsGPI under salt stressSDS-PAGE analysis showed that the proteins expressed by D. salina under salt stress were relatively rare in the DsGPI expression district (70 kDa-80 kDa). Western blots showed that the expression of DsGPI increased significantly when the concentration of NaCl increased.6 The localization of DsGPI and human AMF in MDCK cellsThe results of restriction enzymes digestion and DNA sequencing revealed that the fluorescence localization vector pEGFP-C1-DsGPI was successfully constructed. MDCK cells were transfected by pEGFP-C1,pEGFP-C1-DsGPI and pEGFP-C1-AMF respectively, and then observed by the fluorescence microscope after 24 h. Green fluorescence distributed throughout the cells when transfected by empty vector pEGFP-C1,while green fluorescence is located in the nucleus and its nearby when transfected with pEGFP-C1-DsGPI and pEGFP-C1-AMF.Conclusion1 This study has successfully cloned the ORF of DsGPI and constructed DsGPI prokaryotic expression vector then successfully expressed in E. coli BL21 (DE3). High-pure DsGPI fusion proteins were obtained and used as antigens. Polyclonal antibody to DsGPI with a titer of 1:512 000 was successfully prepared and confirmed to be of good specificity.2 Using the prepared DsGPI polyclonal antibody to detect the expression of it in D. salina under salt stress completed the first application of polyclonal antibody to DsGPI in the molecular biology study of D.salina. 3 The green fluorescent showed that DsGPI located in the nucleus of MDCK cells and its periphery area, obviously similar to the cellular localization of human AMF, which provide groundwork for exploration of functional relevance between DsGPI and human AMF.