Polyclonal antibody preparation, tissue localization and activity of serine protease inhibitor serpin of Cysticercus pisiformis

doi:10.12140/j.issn.1000-7423.2020.05.011

Abstract

Abstract:

Objective To study the tissue distribution of serine protease inhibitor of Cysticercus pisiformis (Cpserpin) and its inhibitory effect on serine proteases. Methods Prokaryotically expressed recombinant Cpserpin (rCpserpin) was used to immunize rabbits, and the titers of rabbit serum after immunization were determined by indirect ELISA. The polyclonal antibodies were purified and analyzed for the specificity by Western blotting. The tissue distributions of rCpserpin in C. pisiformis adult and larva were assayed by immunohistochemistry. The recombinant plasmid pPIC9K-Cpserpin was constructed, transformed into Pichia pastoris KM71 competent cells after linearization, induced for expression in high-copy strains obtained by G418 screening, and then the products were purified. The purified recombinant protein was tested using chromogenic substrate method for its inhibitory effects on the activity of three serine proteases including α-chymotrypsin, trypsin and elastase, at the concentrations of 6, 9, 18 and 21 μg/ml, respectively. Results The results of indirect ELISA showed that the titer of serum polyclonal antibody IgG reached 1 ∶ 51 200. Western blotting showed that the purified polyclonal antibody could specifically react with rCpserpin and the C. pisiformis worm soluble antigen. Immunohistochemistry showed that Cpserpin was expressed at a low level during the cysticercus stage, but was highly expressed in mature proglottid and gravid proglottid, with wide distribution in the worm body wall and eggs. The rCpserpin expressed by P. pastoris showed enhanced inhibitiory effect on the three n of serine proteases with increased concentration, having highest inhibition rate at 21 μg/ml for α-chymotrypsin (50.5 ± 2.5)%, trypsin (71.5 ± 1.5)% and elastase (77.4 ± 1.5)% (P < 0.05). Conclusion The polyclonal antibody produced displays specificity against rCpserpin, the Cpserpin is highly expressed in mature proglottid and gravid proglottid of C. pisiformis, and the recombinant Cpserpin exhibits strong inhibitory effect on α-chymotrypsin, trypsin, and elastase.

Key words: Cysticercus pisiformis, Serine proteinase inhibitor (serpin), Tissue localization, Enzyme activity

CLC Number:

R383.32

WANG Li-jie, WANG Li-qun, LIU Ting-li, ZHANG Shao-hua, LIU Guang-xue, LI Yan-ping, LIANG Pan-hong, LUO Xue-nong. Polyclonal antibody preparation, tissue localization and activity of serine protease inhibitor serpin of Cysticercus pisiformis[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2020, 38(5): 595-601.

Figures/Tables 6

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