关键词: 细粒棘球绦虫, 丝氨酸蛋白酶, 原头节, 生发层

Abstract:

Objective To compare the expression levels of Echinococcus granulosus serine protease (EgSP) in protoscoleces, germinal layer (metacestode), and adult worms, and assay of the protease activity. Methods RNA was extracted from the protoscoleces (with pepsin digestion and without pepsin digestion), germinal layer (metacestode), and adult worms using TRIzol reagent, and EgSP was amplified by PCR. The PCR products were sequenced, sequence alignment was performed using the Clustal × 2.0 software, and the phylogenetic tree was constructed by the neighbor-joining method. A gene sequence with M_r of 38 000 containing the SP reactive motif was used for prokaryotic expression. cDNA with proper open reading frame was cloned into the expression vector pET-30a, and then transformed into the E. coli BL21 strain, followed by IPTG induction of recombinant protein (rEgSP) expression in the LB medium. The proteins were purified using the Ni-IDA affinity chromatography column. A total of 5 BALB/c mice were immunized with 25 μg rEgSP emulsified with the same amount of Freund’s complete adjuvant by subcutaneous multi-site injection. The same amount of incomplete Freund’s adjuvant was used from the second immunization. The fourth booster immunization was done by injecting intraperitoneally. There was an interval of one week between each immunization. After the end of immunization, blood samples were collected from the tail vein, and the serum was separated and the level of specific antibody in serum was detected by ELISA. The relative expression of EgSP mRNA in different developmental stages was measured by qRT-PCR. The expression of EgSP in different developmental stages of E. granulosus was analyzed by Western blotting, and the protein expression abundance was analyzed with the Image Lab 6.0 software. The distribution of EgSP in the protoscoleces and germinal layer (metacestode) of E. granulosus was detected by immunohistochemistry. The protease activities of rEgSP and hydatid cyst fluid (HCF) proteins were assayed using the substrates for trypsin like serine protease and cysteinase (Z-Phe-Arg-AMC) and the substrate for Cathepsin L (Z-Arg-AMC). Data were analyzed with one-factor ANOVA using the Graphpad Prism 7 software. Results PCR produced target bands from cDNA samples from protoscoleces, germinal layer (metacestode), and adult worms, with an expected size of 1 455 bp. Sequencing of PCR products showed that the EgSP open reading frame encoded 484 amino acids with 1 455 bp bases, with a relative molecular weight (M_r) 54 870. The EgSP nucleotide and amino acid sequence showed 97.73% and 96.69% identity to E. multilocularis serine protease (EmSP), respectively. The phylogenetic tree analysis showed that the amino acid sequence identity to other tapeworm genera was 33.10%-87.19%. In particular, the identity to Taenia asiaica (GenBank accession number: VDK43949.1) was 87.19% and that to Taenia solium (GenBank accession number: VDM30859.1) was 52.89%, respectively. qRT-PCR evidenced the relative transcription levels of EgSP mRNA in protoscoleces digested with pepsin, germinal layer (hydatid cyst) and adult worms were 2.1 ± 1.2, 9.1 ± 2.1, 5.8 ± 2.3 respectively, which were higher than protoscoleces without pepsin treatment (1.0 ± 0). Western blotting results showed that the gray values of EgSP in protoscoleces, germinal layer (hydatid cyst) and adult worm tissues were 1.22 ± 0.09, 3.60 ± 0.23, and 4.00 ± 0.19 respectively. The expression of EgSP in germinal layer (hydatid cyst) and adult worms was higher than that of protoscoleces (P < 0.01). The results of immunohistochemistry showed that EgSP polyclonal antibody could specifically recognize cyst germinal layer but not protoscoleces. Examining the rEgSP and HCF protease activity in 3 different pH buffer, highest serine protease activity was detected in the pH 7.2 buffer (t = 53.79, 63.82; P < 0.01). Conclusion EgSP is highly expressed in the germinal layer (hydatid cyst) and adult worms of E. granulosus, while the expression is lower in protoscoleces. Both rEgSP and HCF proteins have strong serine protease activity.

Key words: Echinococcus granulosus, Serine protease, Protoscolex, Germinal layer