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目的克隆、表达广州管圆线虫(Angiostrongylus cantonensis)半乳糖凝集素-1(galectin-1)基因并分析其凝集反应。方法用Swiss Model分析galectin-1的三维结构。提取广州管圆线虫雌性成虫总RNA,根据galectin-1基因编码序列(Gen Bank登录号为JN133961.1)设计引物,RT-PCR扩增目的基因,亚克隆至p ColdⅢ质粒,转化大肠埃希菌(Escherichia coli)BL21,取重组质粒进行双酶切、PCR鉴定和测序。阳性质粒经0.1 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,镍柱亲和层析法纯化galectin-1重组表达蛋白后,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析重组蛋白表达情况。以广州管圆线虫全虫免疫的小鼠血清为一抗,蛋白质印迹(Western blotting)验证目的蛋白。将重组蛋白10倍稀释为5.55×10~(-1)~5.55×10~(-5)ng/μl,显微镜观察其与新鲜的ICR小鼠血液中红细胞的凝集反应。结果 Swiss Model分析结果显示,galectin-1以非二聚体形式发挥作用。galectin-1基因RT-PCR扩增产物约为850 bp,与预期结果一致。经双酶切、PCR和测序结果显示,重组质粒p ColdⅢ-galectin-1构建成功。SDS-PAGE结果显示,经IPTG诱导获得相对分子质量约36 000的可溶性重组融合蛋白。Western blotting分析结果显示,galectin-1蛋白能被全虫免疫的小鼠血清识别。凝集反应结果显示,galectin-1蛋白与小鼠红细胞发生明显凝集反应的最低浓度为5.55×10~(-4)ng/μl。结论克隆的galectin-1基因能在p ColdⅢ原核表达系统中呈可溶性表达,并与小鼠红细胞发生凝集反应。
Objective To clone and express galectin - 1 gene of Angiostrongylus cantonensis and analyze its agglutination. Methods The three-dimensional structure of galectin-1 was analyzed by Swiss Model. According to the coding sequence of galectin-1 gene (Gen Bank accession number JN133961.1), primers were designed and the target gene was amplified by RT-PCR and subcloned into p ColdⅢ plasmid to transform into E.coli (Escherichia coli BL21, take the recombinant plasmid for double enzyme digestion, PCR identification and sequencing. Positive plasmids were induced by 0.1 mmol / L isopropyl-β-D-thiogalactopyranoside (IPTG). After recombinant protein galectin-1 was purified by nickel column affinity chromatography, sodium dodecyl sulfate-poly Acrylamide gel electrophoresis (SDS-PAGE) analysis of recombinant protein expression. Serum of mice immunized with C. elegans were used as primary antibodies, and the target protein was verified by Western blotting. The recombinant protein was diluted 10-fold to 5.55 × 10-5.55 × 10-5 ng / μl. The agglutination of erythrocytes in fresh ICR mice was observed by microscope. Results Swiss Model analysis showed that galectin-1 functions as a non-dimer. The galectin-1 gene RT-PCR product was approximately 850 bp, consistent with the expected results. Double enzyme digestion, PCR and sequencing results showed that the recombinant plasmid p ColdⅢ-galectin-1 was successfully constructed. The result of SDS-PAGE showed that soluble recombinant fusion protein of about 36 000 in molecular weight was induced by IPTG. The result of Western blotting showed that galectin-1 protein could be recognized by serum of mice immunized with all-insect. The result of agglutination showed that the lowest concentration of galectin-1 protein and mouse erythrocyte agglutination was 5.55 × 10 ~ (-4) ng / μl. Conclusion The cloned galectin-1 gene can be expressed in p Cold Ⅲ prokaryotic expression system and agglutinated with mouse erythrocytes.