Prokaryotic expression and preliminary functional identification of Echinococcus granulosus calreticulin

doi:10.12140/j.issn.1000-7423.2025.02.012

Abstract

Abstract:

Objective To express Echinococcus granulosus (Eg) calreticulin (CRT) in prokaryotic cells and preliminarily explore the function of EgCRT. Methods The cDNA of Eg protoscoleces was used as a template to amplify the target gene using PCR assay, which was ligated into the pET-28a (+) vector to construct the recombinant plasmid pET28a-CRT. The recombinant plasmid was transformed into the Escherichia coli BL21 strain. The rEgCRT was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, and the physicochemical properties and some biological functions of the EgCRT protein were predicted using online bioinformatics tools. Mice were immunized with rEgCRT to prepare the polyclonal antibody, and the antibody titer and reactogenicity were detected using indirect ELISA and Western blotting, respectively. The distribution of CRT in protoscoleces and adults and the binding of rEgCRT to canine intestinal epithelial cells were analyzed using indirect immunofluorescence. In addition, the relative transcription level of the crt gene at different developmental stages and the effect of rEgCRT (experimental group and PBS as a control group) on apoptosis and pyroptosis of canine intestinal epithelial cells were examined using real-time quantitative reverse transcription PCR (RT-qPCR) assay. Results The molecular formula of the CRT protein was C₂₀₃₃H₃₀₅₁N₅₁₁O₆₅₀S₉, and the EgCRT protein was predicted to contain α-helix (24.05%), irregular coils (60.00%) and extended chain structures (15.95%). EgCRT, along with the CRTs of E. multilocularis and Taenia solium, were clustered into a clade on the phylogenetic tree. The rEgCRT protein was successfully expressed and highly purified, with a relative molecular weight of approximately 50 000. The rEgCRT protein showed a high antigenic specificity, and indirect immunofluorescence assay showed that EgCRT was localized in the epidermis of protoscoleces and adults and some parenchyma tissues. RT-qPCR assay showed higher expression of EgCRT in the adult stage (24.83 ± 0) than in the protoscoleces stage (27.05 ± 0.008) (t = 45.76, P < 0.01), and indirect immunofluorescence assay showed that the canine small intestinal epithelial cells co-cultured with rEgCRT presented green fluorescence aggregation. RT-qPCR assay showed that the relative transcription levels of the Bax gene were (31.63 ± 0.001) and (24.17 ± 0.027) in the experimental group and the control group 24 hours post-culture (t = 7.94, P < 0.01), indicating up-regulated expression, and the relative transcription levels of Bcl-2 were (34.55 ± 0.070) and (19.65 ± 0.042) (t = 15.70, P < 0.01), indicating downregulated expression, while the relative transcription levels of IL-18 were (25.62 ± 0.067) and (20.93 ± 0.014) (t = 5.05, P < 0.01), indicating downregulated expression, and the relative transcription levels of IL-1β were (28.18 ± 0.001) and (28.71 ± 0.020) (t = 0.26, P > 0.05), indicating downregulated expression. Conclusion The rEgCRT protein has a high antigenicity, and EgCRT is expressed in different developmental stages of E. granulosus, and may play an important role in the fixation, colonization, growth or development of E. granulosus. In addition, rEgCRT adheres to canine small intestinal epithelial cells and regulates cell apoptosis and pyroptosis, which may be involved in the process of E. granulosus infections or host immune responses, and has a promise as potential vaccine candidates and drug targets.

Key words: Echinococcus granulosus, Calreticulin, Cloning expression, Immunofluorescence, Molecular function

CLC Number:

R383.3

PU Na, BO Xinwen, ZHAO Wenqing, CHEN Xuke, ZHANG Yuxia, ZHANG Yanyan, SUN Yan, WANG Zhengrong. Prokaryotic expression and preliminary functional identification of Echinococcus granulosus calreticulin[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2025, 43(2): 223-230.

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基因Gene	引物序列（5'→3'） Primer sequences (5'→3')	片段大小/bp Product length/bp
EgCRT	F：CCG GAATTCTACGACTATAATAATGATGC-CGAAC	1 108
	R：CCG CTCGAGCTTCTCTCCCAAAAGGGTCAT
qCRT	F：TCGCATTGTTATTCGTTGTT	150
	R：GGCATCTTGTGTTGTTCGT
GAPDH^a	F：CGCATCGGTCGTCTTGTGTT	190
	R：CGGTAATCCTGTGGCTGTCAAT
Bax	F：GATGAACTGGACAGTAACATGGA	158
	R：AGTTTGCTGGCAAAGTAGAAGAG
Bcl-2	F：GATTGTGGCCTTCTTTGAGTTC	168
	R：TACAGTTCCACAAAGGCATCC
IL-1β	F：TTGTAGCTTTGGAGAAGCTGAAG	179
	R：GTGGCTTATGTCCTGTAACTTGC
IL-18	F：AGGATATGCCCGATTCTGACT	179
	R：ATCCGGAGGACTCATTTTCTG
β-actin^b	F：CTGACCCTGAAGTACCCCATT	168
	R：GGTCATCTTCTCACGGTTGG