Abstract:

Objective To obtain the gene for proteasome α5 subunit (pas-5) of Angiostrongylus cantonensis, construct the recombinant plasmid, and investigate the effect of PAS-5 recombinant protein on the apoptosis of human THP-1 macrophages. Methods The pas-5 gene was cloned from the cDNA of A. cantonensis, inserted into the prokaryotic expression vector pcoldⅢ to construct the recombinant plasmid pcoldⅢ-pas-5, and transformed into Escherichia coli DH5α competent cells. The cultures underwent PCR, double digestion, and sequencing to confirm the construction of recombinant plasmid. Expression of the recombinant plasmid was induced by IPTG, and the expressed recombinant proteins were analyzed by SDS-PAGE and Western blotting, and purified with Nickel affinity chromatography using Ni-NTA beads. The phorbol myristate acetate-induced THP-1 macrophages were incubated with PAS-5 for 18 h, and then flow cytometry was performed to evaluate the apoptosis of macrophages. The level of cytokine IL-10 in the culture supernatant was assessed by ELISA. Negative control and blank control groups were set with bovine serum albumin(BSA) and RPMI 1640, respectively, instead of PAS-5. Results The PCR amplification product of pas-5 was about 800 bp, consistent with expectation. The pcoldⅢ-pas-5 recombinant plasmid was verified by PCR, double digestion and sequencing results. SDS-PAGE results showed that the recombinant protein PAS-5 was expressed mainly in the form of inclusion body, with a relative molecular weight of 28 000. Western blotting showed that the recombinant protein could specifically bind to rabbit anti-mouse His monoclonal antibody. A single strip was obtained from Nickel affinity chromatography. The flow cytometry showed that the THP-1 macrophages apoptosis rate was(0.380 ± 0.194)% in the BSA group, and (0.052 ± 0.036)% in the PAS-5 group (P < 0.05). ELISA results showed a significantly higher level of supernatant IL-10 in the PAS-5 group than in the BSA group［(77.606 ± 1.766) pg/ml versus(45.652 ± 5.975) pg/ml, P < 0.05］.  Conclusion The recombinant plasmid pcoldⅢ-pas-5 was constructed, and the recombinant protein PAS-5 can inhibit the apoptosis of THP-1 macrophages, which may be related to the increased level of IL-10.
  

Key words: Angiostrongylus cantonensis, Proteasome, Recombinant protein, Human THP-1 macrophages, Apoptosis