›› 2014, Vol. 32 ›› Issue (2): 2-86-95.

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Prokaryotic Expression and Function Analysis of Schistosoma japonicum Calpain

DU Xiao-feng1，XU Bin2，LIU Jian1，WANG Ji-peng1，LIU Mu1，LIU Xiu-feng1，MA Xiao-lin1，HU Wei1，2，JU Chuan2 *   

  1. 1 Department of Microbiology and Microbial Engineering，School of Life Sciences，Fudan University，Shanghai 200433，China；2 National Institute of Parasitic Diseases，Chinese Center for Disease Control and Prevention；Key Laboratory of Parasite and Vector Biology，Ministry of Health；WHO Collaborating Centre for Malaria，Schistosomiasis and Filariasis，Shanghai 200025，China
  • Online:2014-04-30 Published:2014-07-03

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Abstract:  Objective  To clone and express recombinant calpain of Schistosoma japonicum（Sjcalpain），　observe the distribution of Sjcalpain in S. japonicum cercariae and analyze its role in skin invasion.  Methods  The primers were designed according to the full-length sequence of calpain（GenBank accession No.　AB016726）. The genes encoding catalytic domain and Ca2+ binding domain of Sjcalpain were amplified by PCR， and the target fragments were subcloned into pET-28a. The recombinant proteins were expressed in E. coli BL21（DE3）and purified by Ni-NTA resin. The rabbit polyclonal antibodies were prepared with the two purified recombinant proteins by immunizing New Zealand white rabbits. ELISA was used to detect the titer of rabbit antiserum. Immunolocalization was used to investigate the distribution of Sjcalpain in S. japonicum cercariae. Cercariae were incubated with specific inhibitor before infection of mice and the worm reduction rate was calculated.  Results  The recombinant expression vector Sjcalpain catalytic domain/pET28a and Sjcalpain Ca2+ binding domain/pET28a were constructed and the recombinant proteins were successfully expressed in E. coli BL21（DE3）（about Mr 43 000 and Mr 39 000， respectively）. The two target proteins were expressed as inclusion bodies. The purified target proteins were obtained through Ni-NTA affinity purification. ELISA result showed that the titer of prepared rabbit polyclonal antibodies was higher than 1 ∶ 80 000. Immunolocalization study demonstrated that Sjcalpain protein was mainly expressed in the head of cercariae. Inhibition assays suggested that the average number of adult worms in calpain inhibitor-incubation group and control group was 19 and 23， respectively, with a worm reduction rate of 17.4%.  Conclusion  Sjcalpain is mainly expressed in the head of S. japonicum cercariae. Inhibition of Sjcalpain could reduce the number of invading cercariae in infected mice， which suggest that Sjcalpain may play a role in skin invasion by cercariae.

Key words: Schistosoma japonicum, Cercaria, Calpain, Immunolocalization, Infection