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

Abstract

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^-4ng/μl. Conclusion The galectin-1 clone can be expressed in the pCold Ⅲ plasmid, and its protein product has agglutination property.

Key words: Angiostrongylus cantonensis, Galectin-1, Prokaryotic expression, Agglutination

CLC Number:

R383.19

Xing-pan LI, Meng-jing ZHAO, Xiao-meng SHI, Lan-zhu YAN, Bao-long YAN, Hui-cong HUANG. Cloning of galectin-1 gene of Angiostrongylus cantonensis and testing the agglutination property of the galectin-1 protein[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2017, 35(1): 48-52.

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