蓝氏贾第鞭毛虫α-19贾第素的单克隆抗体制备与亚细胞定位研究

Preparation of monoclonal antibody and subcellular localization of a-19 giardinin Giardia lamblia trophozoites

目的制备蓝氏贾第鞭毛虫(Giardia lamblia,简称贾第虫)α-19贾第素的特异性抗体,并通过免疫荧光对该贾第素的亚细胞定位进行鉴定。方法经PCR获得α-19贾第素编码区片段,双酶切将目的片段连入原核表达载体pET-28a(+)-α-19,重组载体转化E.coli Rosetta(DE3),IPTG诱导表达,SDS-PAGE及Western blot验证表达情况;采用设计的合成抗原肽免疫小鼠经细胞融合和筛选制备特异性单克隆抗体,分别用贾第虫滋养体裂解物和α-19贾第素重组蛋白Western blot验证抗体特异性和结合力,选择特异性最好的单克隆抗体通过免疫荧光技术对α-19贾第素的亚细胞定位进行观察。结果成功构建了原核表达载体pET-28a(+)-α-19,经SDS-PAGE及Westernblot分析显示重组载体在大肠杆菌中表达出相对分子量约49 kD的重组蛋白,与理论值相符;Western blot显示单克隆杂交瘤细胞株α-19-3-7-3-5分泌的抗体结合力和特异性俱佳,可用于定位研究;免疫荧光定位结果表明α-19贾第素主要位于贾第虫滋养体的腹鞭毛。结论制备了α-19贾第素的特异性单克隆抗体,免疫荧光显示α-19贾第素定位于贾第虫滋养体的腹鞭毛。

The coding region ofα-19 was obtained by PCR.Transforming the recombinant vector into E.coli Rosetta(DE3)and IPTG induced expression.Immunized mice with the designed synthetic peptide by cell fusion and preparation of specific monoclonal antibody.The monoclonal antibodies(mAb)againstα-19 giardin with the advantages of high affinity and specificity were pre-pared by using hybridoma technology.Western blot was used to verify the specificity and affinity of the antibodies.Subcellular localization ofα-19-giardin by immunofluorescence with the most specific monoclonal antibody.The recombinant plasmid pET28a(+)-α-19 was successfully constructed by gene sequencing.The relative molecular weight of recombinantα-19 Giardia protein was 49 kDa by SDS-PAGE.Western blot results showed that the mAb secreted by a monoclonal hybridoma cell lineα-19-3-7-3-5 with the highest specificity with nativeα-19 giardin protein and used for immunofluorescence analysis.The results of confocal microscopy showed thatα-19 giardin was located in the ventral flagella of the trophozoites of Giardialamblia.