游仆虫EoRab2a蛋白的表达、纯化及定位分析

EXPRESSION,PURIFICATION AND LOCALIZATION OF EORAB2A FROM EUPLOTES OCTOCARINATUS

Rab GTPase家族蛋白是真核细胞内膜系统转运途径中重要的调控因子,不同的Rab家族成员在细胞具有功能多样性。为了解Rab2的功能,八肋游仆虫EoRab2a基因连接入原核表达质粒pGEX-6P-1中,获得重组表达质粒pGEX-6P-1-EoRab2a。质粒pGEX-6P-1-EoRab2a转化大肠杆菌BL21(DE3),经IPTG诱导,大肠杆菌BL21(DE3)/pGEX-6P-1-EoRab2a高效表达了可溶性GST-EoRab2a蛋白。融合蛋白GST-EoRab2a经亲和层析获得电泳纯蛋白。纯化后的GST-EoRab2a免疫BALB/c小鼠制备多克隆抗体。ELISA和Western blotting检测显示制备的抗体效价1∶25600,特异性良好。免疫荧光定位表明EoRab2a在游仆虫细胞质中点状分布,推测参与内质网与高尔基体间膜泡转运。

Rab family proteins are key regulators of vesicular traffic in eukaryotic cells.As an important member of the Rab family,Rab2 protein has been shown to be involved in various vesicular trafficking processes,and showed func-tional diversity in different cell types and species.In the present study,EoRab2a,Rab2 homology,was obtained from Euplotes octocarinatu.The universal TGA stop codon appeared at position 66th in the EoRab2a,encoded cysteine in E.octocarinatum,was replaced by TGC by PCR-mediated site-directed mutagenesis.Mutated EoRab2a open reading frame（ORF） was subcloned into expression vector pGEX-6P-1,and the recombinant plasmid pGEX-6P-1-EoRab2a was transformed into E.coli BL21（DE3）.GST-EoRab2a protein was expressed in E.coli BL21（DE3） strain by induction with 0.1 mmol/L isopropyl β-D-thiogalactoside for 6 hours at 37℃.GST-EoRab2a was purified by affinity chromatog-raphy and analyzed by SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining.Two BALB/c mice were immunized with 80 μg purified GST-EoRab2a emulsified in complete Freund’s adjuvant and boosted two times with 50 μg purified GST-EoRab2a emulsified in incomplete Freund’s adjuvant.The titer of anti-EoRab2a polyclonal antibody（1∶25600） was detected by indirect ELISA assay and the specificity of the antibody was detected by Western blotting.The cellular localization of EoRab2a in E.octocarinatum was determined by immunofluorescence with polyclonal an-tibody raised against GST-Rab2a.Localization showed that Eorab2a displayed a dotted pattern in the E.octocarinatum cytoplasm.The results implied that EoRab2a could be involved in trafficking between the endoplasmic reticulum and the Golgi apparatus in E.octocarinatum.