title: Characterisation of a high-frequency gene encoding a strongly antigenic cystatin-like protein from Trichinella spiralis at its early invasion stage.
abstract: BACKGROUND
  The intestinal phase is the early invasion stage of Trichinella spiralis (T. spiralis), in which muscle larvae invade intestine epithelial cells and then develop into adult worms to breed newborn larvae. Thus, intestinal infective larvae are first exposed to the immune system of the host, and antigens from the worms may be the earliest marker in the diagnosis of trichinellosis and may contribute to vaccine development to prevent Trichinella infections in pigs.
  
  
  METHODS
  A cDNA library of intestinal infective larvae of T. spiralis at 6 hours post infection (p.i.) was constructed and immunoscreened using serum collected from pigs that were infected with T. spiralis at 26 days p.i. T. spiralis cystatin-like protein (Ts-CLP) gene encoding a 45.9 kDa protein was cloned and expressed in Escherichia coli. The rabbit antisera were generated and used to determine the location of Ts-CLP in the parasite. Transcription levels of Ts-CLP in different developmental stages of T. spiralis were observed by RT-PCR. The potential application of recombinant Ts-CLP in diagnosis against T. spiralis infection was tested by ELISA. The immune protection of recombinant Ts-CLP protein against T. spiralis infection was evaluated in mice.
  
  
  RESULTS
  Thirty-three positive clones were selected from cDNA library, among which 20 clones encoded the same novel cystatin-like protein (Ts-CLP). Immunolocalisation and real-time quantitative PCR revealed that native Ts-CLP was localised primarily to β-stichocytes and that the Ts-clp gene was transcribed and expressed in all developmental stages of T. spiralis. The recombinant protein rTs-CLP was recognised by pig antiserum as early as 15 days p.i., and could induce protective immunity in mice, with a 61.21% reduction in the number of muscle larvae.
  
  
  CONCLUSIONS
  These data preliminarily suggested that Ts-CLP may play an important role in the early infection of T. spiralis and that recombinant Ts-CLP protein is a candidate antigen for diagnosis and vaccine development in Trichinella infections.
authors: National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, Key Laboratory for Zoonoses Research, Ministry of Education, Institute of Zoonoses, Jilin University, Changchun, P. R. China. tangbin0912@gmail.com.
authors: Bin
authors: B
authors: Tang
authors: National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, Key Laboratory for Zoonoses Research, Ministry of Education, Institute of Zoonoses, Jilin University, Changchun, P. R. China. liumy@jlu.edu.cn.|Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, P. R. China. liumy@jlu.edu.cn.
authors: Mingyuan
authors: M
authors: Liu
authors: Yunnan Institute of Parasitic Diseases, 6 Xiyuan Road, Puer, Yunnan, P. R. China. ynwanglibo@126.com.
authors: Libo
authors: L
authors: Wang
authors: National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, Key Laboratory for Zoonoses Research, Ministry of Education, Institute of Zoonoses, Jilin University, Changchun, P. R. China. yushenye@163.com.
authors: Shenye
authors: S
authors: Yu
authors: Immunology Laboratory, Pediatric Gastroenterology Unit, Massachusetts General Hospital East, Charlestown, MA, 02129, USA. shiha@helix.mgh.harvard.edu.
authors: Haining
authors: H
authors: Shi
authors: ANSES, Laboratory for Animal Health, Maisons Alfort, France. pboireau@vet-alfort.fr.
authors: Pascal
authors: P
authors: Boireau
authors: University of Agricultural Science and Veterinary Medicine, Cluj-Napoca, Romania. cozmavasile@yahoo.com.
authors: Vasile
authors: V
authors: Cozma
authors: National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, Key Laboratory for Zoonoses Research, Ministry of Education, Institute of Zoonoses, Jilin University, Changchun, P. R. China. lvy.2001@163.com.
authors: Xiuping
authors: X
authors: Wu
authors: National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, Key Laboratory for Zoonoses Research, Ministry of Education, Institute of Zoonoses, Jilin University, Changchun, P. R. China. liuxlei@163.com.
authors: Xiaolei
authors: X
authors: Liu
mesh_terms: D000818:Animals; D000947:Antigens, Helminth / Q000235:genetics / Q000276:immunology*; D015891:Cystatins / Q000235:genetics / Q000276:immunology*; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D013552:Swine; D013553:Swine Diseases / Q000276:immunology / Q000469:parasitology*; D017160:Trichinella spiralis / Q000235:genetics / Q000254:growth & development / Q000276:immunology* / Q000472:pathogenicity*; D014235:Trichinellosis / Q000276:immunology / Q000469:parasitology / Q000662:veterinary*; D014774:Virulence
chemical_list: D000947:Antigens, Helminth; D015891:Cystatins
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