Cloning, expression and bioinformatics analysis of 27 kDa cysteine protease gene of Spirometra erinacei plerocercoid / 中国人兽共患病学报

To clone and express 27 kDa cysteine protease (CP) gene of Spirometra erinacei plerocercoid,and analyze the biology characteristics,a total of RNA was extracted from the plerocercoids and reversely transcribed into cDNA.The 27kDaCP gene was amplified by PCR and cloned into pM-19T vector for sequencing.The accurate sequence was subcloned into the expression vector pET-28a (+).The recombinant plasmid was transformed into Transetta (DE3) and the expression protein induced by IPTG.The recombinant protein was purified by Ni2 + affinity chromatography,and analyzed by SDS-PAGE and Western blotting.The 27 kDa CP gene and its expression protein were predicted and analyzed by bioinformatics analysis tools such as NCBI and ExPASy.Results showed that the ORF length of 27 kDa CP gene sequence was 1 011 bp,and the removed signal peptide sequence was 954 bp with the submission number of ANA52569 in GenBank.The whole sequence of 27 kDa CP (Mr 35 669.9,pI 5.92) was 317 amino acids conferred from cDNA,which belongs to the Peptidase_C39_like superfamily.The pET-28a (+)-27kDa-CP was expressed under the induction of IPTG.Western blotting analysis showed that the purified protein reach expectancy,and had better response with positive serum of Spirometra erinacei plerocercoid infection.In conclusion,the 27 kDa CP gene of Spirometra erinacei plerocercoid is successfully cloned and expressed and knowing coded sequences and bioinformatic.

Diagnostic Efficacy of a Recombinant Cysteine Protease of Spirometra erinacei Larvae for Serodiagnosis of Sparganosis

The mature domain of a cysteine protease of Spirometra erinacei plerocercoid larva (i.e., sparganum) was expressed in Escherichia coli, and its value as an antigen for the serodiagnosis of sparganosis was investigated. The recombinant protein (rSepCp-1) has the molecular weight of 23.4 kDa, and strongly reacted with the sparganum positive human or mice sera but not with negative sera by immunoblotting. ELISA with rSepCp-1 protein or sparganum crude antigen (SeC) was evaluated for the serodiagnosis of sparganosis using patient's sera. The sensitivity and specificity of ELISA using rSepCp-1 protein were 95.0% (19/20) and 99.1% (111/112), respectively. In contrast, the sensitivity and specificity of ELISA with SeC were 100% (20/20) and 96.4% (108/112), respectively. Moreover, in experimentally infected mice, the sensitivity and specificity of both ELISA assays were 100% for the detection of anti-sparganum IgG. It is suggested that the rSepCp-1 protein-based ELISA could provide a highly sensitive and specific assay for the diagnosis of sparganosis.

Recurred Sparganosis 1 Year after Surgical Removal of a Sparganum in a Korean Woman

Sparganosis, an infection due to the plerocercoid of Spirometra erinacei, are found worldwide but the majority of cases occur in East Asia including Korea. This report is on a recurred case of sparganosis in the subcutaneous tissue of the right lower leg 1 year after a surgical removal of a worm from a similar region. At admission, ultrasonography (USG) of the lesion strongly suggested sparganosis, and a worm was successfully removed which turned out to be a sparganum with scolex. Since sparganum has a variable life span, and may develop into a life-threatening severe case, a patient once diagnosed as sparganosis should be properly followed-up for a certain period of time. Although imaging modalities were useful for the diagnosis of sparganosis as seen in this case, serological test such as ELISA should also be accompanied so as to support the preoperative diagnosis.

Spargana in a Weasel, Mustela sibirica manchurica, and a Wild Boar, Sus scrofa, from Gangwon-do, Korea

To know the status of sparganum (plerocercoid of Spirometra erinacei) infection in the Korean wild life, several species of wild animals were captured in Gangwon-do and examined for their status of infection with spargana. From February to December 2011, a total of 62 wild boars, 5 badgers, 1 weasel, 1 Siberian chipmunk, and 53 wild rodents were captured, and their whole muscles were examined with naked eyes for the presence of spargana worms. From the weasel and 1 wild boar, a total of 5 spargana specimens were extracted. The weasel was for the first time recorded as an intermediate or paratenic/transport host of S. erinacei in Korea, and both the weasel (Mustela sibirica manchurica) and wild boar (Sus scrofa) were added to the list of wild animals carrying spargana.

Seroprevalence of Tissue Invading Parasitic Infections Diagnosed by ELISA in Korea

Seroprevalence of the IgG antibodies for Clonorchis sinensis, Paragonimus westermani, Taenia solium metacestode (cysticercus), and Spirometra erinacei plerocercoid (sparganum) was measured using enzyme-linked immunosorbent assay (ELISA) in sera of patients in Korea from 1993 to 2006. A total of 74,448 specimens referred nationwide from 121 hospitals revealed an IgG positive rate of 7.6% for the 4 parasites. The IgG positive rate (18.7%) for the 4 parasites in 1993 decreased gradually to 6.6% in 2006. Individual positive rate decreased from 5.2% (1993) to 1.6% (2006) for C. sinensis, from 2.8% (1993) to 1.1% (2006) for P. westermani, from 8.3% (1993) to 2.2% (2006) for cysticercus, and from 2.6% (1993) to 1.6% (2006) for sparganum. The positive rate was highest (21.2%) in the group of patients who ranged in age from 50-59 yr old, and in the group that was referred from the Seoul area (55.9%). In conclusion, our results suggest that tissue invading parasitic infections should always be included in differential diagnosis for patients with eosinophilia associated lesions of the central nervous system, liver, and lungs in Korea.

A Case of Vesical and Scrotal Sparganosis Presenting as a Scrotal Mass

A 59-year-old Korean man complained of a painless scrotal hard nodule and weak urine stream. The ultrasound scan revealed a 2.2-cm sized round heteroechogenic nodule located in the extratesticular area. Microscopic hematuria was detected in routine laboratory examinations. On scrotal exploration, multiple spargana were incidentally found in the mass and along the left spermatic cord. On cystoscopy, a 10-mm sized mucosal elevation was found in the right side of the bladder dome. After transurethral resection of the covered mucosa, larval tapeworms were removed from inside of the nodule by forceps. Plerocercoids of Spirometra erinacei was confirmed morphologically and also by PCR-sequencing analysis from the extracted tissue of the urinary bladder. So far as the literature is concerned, this is the first worm (PCR)-proven case of sparganosis in the urinary bladder.

Cloning of the novel putative apoptosis-related gene of Spirometra erinacei (Order Pseudophyllidae)

We postulated that apolysis was processed in accordance with apoptotic changes occurring in a cestode, Spirometra erinacei (Pseudophyllidea). We cloned the novel putative apoptosis-associated gene from S. erinacei via screening of a S. erinacei cDNA library with a ced-3 gene (activator of apoptosis) probe from Caenorhabditis elegans. We identified a 261-bp cDNA sequence, which encodes for an 86-amino acid protein. The cloned gene expression was observed in the neck and gravid proglottids via Northern blotting, using cloned cDNA inserts as probes, but the clone was not expressed in any of other tissues. We suggest that this gene may be involved in the apolysis of S. erinacei during normal tissue development and differentiation in cestode parasites.

A new method for concentration of proteins in the calcareous corpuscles separated from the spargana of Spirometra erinacei

Calcareous corpuscles are a characteristic structure found in larval and adult stage cestodes. These corpuscles are known to contain several protein components and to possess protein-binding activity. However, the proteins bound to calcareous corpuscles in situ have not been studied. The present study was undertaken to identify the proteins on calcareous corpuscles. Calcareous corpuscles were purified from the plerocercoids (= spargana) of Spirometra erinacei, and serially dissolved using 0.1 M sulfamic acid solution. Collected supernatants were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. The results showed that only the fraction remaining after the 19th dissolved fraction contained proteins. A total of 20 protein molecules were detected in gel, with major bands at 56, 53, 46, 40, 35, 29, 28, 24.5, 21, 19, 16, 13, 10 and 8 kDa. In particular, the proteins corresponding to the 21 and 16 kDa bands were most abundant. Our results demonstrated for the first time the protein contents of the calcareous corpuscles of spargana. Further studies on the functions of these proteins are required.

Screening of Stage-Specific Expression Genes of Spirometra erinacei-europaei Plerocercoid by mRNA Differential Display Technique / 中国寄生虫学与寄生虫病杂志

Objective To screen stage\|specific expression genes of plerocercoid of Spirometra erinacei\|europaei. Methods RNA was extracted by the acid guanidinium thiocyanate\|phenol\|chloroform from plerocercoid and adult worm of Spirometra erinacei\|europaei. Contaminated DNA in the RNA was digested by RNase\|free DNase. cDNA was synthesized by using T\-\{12\}MA, T\-\{12\}MC, T\-\{12\}MG and T\-\{12\}MT primers, and PCR was then done using the same T\-\{12\}MN and one random primers. The PCR products were fractionated on 8% denatured polyacrylamide gel, differential bands of plerocercoid found in the gel were cut out, amplified by PCR and sequenced after the gel was dried out and autoradiographed. Northern hybridization was conducted to identify the stage\|specific expression genes. Results Eleven differential bands were selected from the gel and classified into 3 kinds of gene fragments by hybridization after they were amplified by PCR. The fragments 1 and 2 were confirmed to express specifically in plerocercoid by Northern hybridization, but the fragment 3 was expressed in both plerocercoid and adult worm. When the 3 gene fragments were homologically analyzed in GenBank, the sequence which was homologous with the fragments 1 and 2 was not found, but the fragment 3 had high homology with many kinds of 28S rRNA. Conclusion The gene expression of plerocercoid was different from that of adult worm probably because they live in different hosts. Two kinds of different gene fragments in plerocercoid were identified by mRNA differential display technique.