Identification and characterization of a full-length cDNA encoding paramyosin of Trichinella spiralis

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Abstract

A full-length cDNA encoding Trichinella spiralis paramyosin (Ts-Pmy) was cloned by immunoscreening a cDNA library of the adult T. spiralis worm. Ts-Pmy cDNA consists of 2655 bp that encode 885 amino acids. The recombinant protein (rTs-Pmy) was expressed and purified by Ni-affinity chromatography. Western blot analysis showed that rTs-Pmy could be recognized by sera from T. spiralis-infected humans, swine, rabbits, and mice. Immunolocalization demonstrated that Ts-Pmy was abundant on the surface of T. spiralis larvae. BALB/c mice vaccinated with rTs-Pmy demonstrated 36.2% reduction in muscle larvae burden following T. spiralis larvae challenge. Vaccination of the mice with rTs-Pmy resulted in a high level of specific anti-Ts-Pmy IgG antibodies and generated a Th1/Th2 mixed type of immune response, with Th2 predominant. These studies showed that rTs-Pmy induced protective immunity in mice and could be considered as a potential vaccine candidate for trichinellosis.

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Materials and methods

Parasites and antigen preparation. T. spiralis (ISS533) was maintained in female ICR mice. Adult worm and muscle larvae were recovered from infected mice by the standard pepsin digestion method as described [11] and infection sera were collected. Crude somatic extracts of adult worms, muscle larvae, and excretory–secretory (ES) products were prepared by conventional methods [12].

Mice. Female BALB/c mice aged 6–8 weeks were obtained from the Laboratory Animal Center of the Academy of Military

Molecular characterization of the cDNA encoding paramyosin

After immunoscreening of a cDNA expression library (200,000 plaques) with rabbit antisera against T. spiralis adult extracts and T. spiralis-infected rabbit sera, the longest cDNA fragment was sequenced and its amino acid sequence was deduced. The full length of cDNA was 2996 bp. The ORF of the cDNA consisted of 2655 bp encoding 885 amino acids with a predicted molecular mass of 102 kDa and an isoelectric point of 5.4. The cDNA consisted of a 5′-untranslated region of 74 bp and a 3′-untranslated

Acknowledgments

We thank Fengyun Wang and Jin Pan for their technical assistance. This work was supported by grants from the National Natural Science Foundation of China (30571626), the Natural Science Foundation of Beijing (5062005), the Beijing Municipal project for Developing Advanced Human Resources for Education (BAHED), and the PhD Programs Foundation of the Ministry of Education of China (20060025003).

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