[广州管圆线虫半乳糖凝集素-1基因的克隆及该半乳糖凝集素-1蛋白凝集特性的检测]

[Cloning of galectin-1 gene of Angiostrongylus cantonensis and testing the agglutination property of the galectin-1 protein].

作者信息

Li Xing-pan, Zhao Meng-jing, Shi Xiao-meng, Yan Lan-zhu, Yan Bao-long, Huang Hui-cong

出版信息

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2017 Feb;35(1):48-52.

Abstract

OBJECTIVE

To clone and express the galectin-1 gene of Angiostrongylus cantonensis, and test the agglutination property of its protein.

METHODS

The three-dimensional structure of galectin-1 was analyzed with Swiss Model. Total RNA was extracted from male worms of A. cantonensis. Primers were designed for galectin-1 based on its coding region (GenBank Accession No. JN133961.1). RT-PCR was performed, and the PCR products were subcloned to pCold Ⅲ plasmid and transduced into Escherichia coli BL21 strain. The recombinant plasmid was extracted from positive clones on LB plate containing 100 μg/ml Kanamycin, and validated with double digestion, PCR identification and sequencing. The confirmed positive clones of E. coli BL21 with the recombinant plasmid were grown in LB medium containing ampicillin (100 μg/ml, 100 μl). IPTG was added to induce expression of the plasmid. The galectin-1 recombinant protein was purified with Ni-NTA beads, and analyzed with SDS-PAGE and Western blotting using anti-serum of mouse immunized with whole worms of A. cantonensis. The agglutination reaction with red blood cells in fresh blood of ICR mouse was observed for the 10-fold serial dilutions of recombinant proteins (5.55 × 10(-1)-5.55 × 10(-5) ng/μl).

RESULTS

The Swiss Model analysis showed that the functional galectin-1 had a non-dimeric form. As was expected, the RT-PCR products had a size of 850 bp. Results of double digestion, PCR and sequencing showed successful construction of the pCold Ⅲ-galectin-1 plasmid. SDS-PAGE revealed expression of soluble recombinant fusion protein with molecular weight of ~36 000. Western blotting showed that the galectin-1 protein was recognized by mouse anti-serum. In addition, the minimun concentration of galectin-1 that showed significant agglutination reactions with mouse red blood cells was 5.55 × 10(-4) ng/μl.

CONCLUSION

The galectin-1 clone can be expressed in the pCold Ⅲ plasmid, and its protein product has agglutination property.

摘要

目的

克隆并表达广州管圆线虫半乳糖凝集素-1基因，并检测其蛋白的凝集特性。

方法

用瑞士模型分析半乳糖凝集素-1的三维结构。从广州管圆线虫雄虫中提取总RNA。根据其编码区（GenBank登录号：JN133961.1）设计半乳糖凝集素-1的引物。进行逆转录聚合酶链反应（RT-PCR），将PCR产物亚克隆到pColdⅢ质粒中，并转化到大肠杆菌BL21菌株中。从含100μg/ml卡那霉素的LB平板上的阳性克隆中提取重组质粒，通过双酶切、PCR鉴定和测序进行验证。将含有重组质粒的经确认的大肠杆菌BL21阳性克隆在含氨苄青霉素（100μg/ml，100μl）的LB培养基中培养。加入异丙基-β-D-硫代半乳糖苷（IPTG）诱导质粒表达。用镍-亚氨基二乙酸（Ni-NTA）磁珠纯化半乳糖凝集素-1重组蛋白，并用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和用经广州管圆线虫全虫免疫的小鼠抗血清进行蛋白质免疫印迹法分析。对重组蛋白进行10倍系列稀释（5.55×10⁻¹-5.55×10⁻⁵ng/μl），观察其与ICR小鼠新鲜血液中红细胞的凝集反应。

结果

瑞士模型分析表明，具有功能的半乳糖凝集素-1呈非二聚体形式。正如预期的那样，RT-PCR产物大小为850bp。双酶切、PCR和测序结果表明成功构建了pColdⅢ-半乳糖凝集素-1质粒。SDS-PAGE显示表达了分子量约为36000的可溶性重组融合蛋白。蛋白质免疫印迹法表明半乳糖凝集素-1蛋白能被小鼠抗血清识别。此外，与小鼠红细胞呈现明显凝集反应的半乳糖凝集素-1的最低浓度为5.55×10⁻⁴ng/μl。