Cloning, expression and identification of cysteine protease genes AcCBL1 and AcCBL2 of Angiostrongylus cantonensis

Abstract

Abstract:

Objective To clone and express the cysteine protease genes AcCBL1 and AcCBL2 of Angiostrongylus cantonensis, and analyze the immunoreactivity of the purified recombinant proteins. Methods Two cysteine protease genes AcCBL1 and AcCBL2 were identified from Angiostrongylus cantonensis fourth-stage larvae cDNA library, and cloned into pET-28a vector to generate recombinant plasmids pET-28a-AcCBL1 and pET-28a-AcCBL2, respectively. The constructed recombinant plasmids were transformed into（E. coli） BL21（DE3） respectively for expression under the induction of isopropyl β-D-1-thiogalactopyranoside(IPTG). The recombinant proteins rAcCBL1 and rAcCBL2 were purified by Ni-NTA affinity chromatography and identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). The immunoreactivity of rAcCBL1 and rAcCBL2 were analyzed by Western blotting with 6 × His tag antibody, as well as anti-sera from mice infected with A. cantonensis and patients. Results PCR generated a specific band at 1 000 bp and 1 100 bp for AcCBL1 and AcCBL2, respectively, consistent with the theoretical values. The constructed vector pET-28a-AcCBL1 and pET-28a-AcCBL2 were confirmed by gene sequencing. SDS-PAGE revealed that rAcCBL1 was soluble with relative molecular mass of 37 800, and rAcCBL2 was expressed as inclusion body in E. coli with relative molecular mass of 40 800. Western blotting showed that the two purified recombinant proteins can be recognized by 6 × His tag antibody, as well as anti-sera from mice infected with A. cantonensis and patients. Conclusion AcCBL1 and AcCBL2 genes have been expressed in prokaryotic expression system. The two recombinant proteins both show adequate immunoreactivity.

Key words: Angiostrongylus cantonensis, Cysteine protease, Expression, Identification

CLC Number:

R383.19

Hui-fang BAI, Qi-wen KE, Yun-qiu CHEN, Zhi-qing LIU, Ling-min ZHANG, Chun-yun WU, Gui-xiu YUAN, Xi-mei ZHAN, Mei CHENG. Cloning, expression and identification of cysteine protease genes AcCBL1 and AcCBL2 of Angiostrongylus cantonensis[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2018, 36(5): 469-473.

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