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Objective: To conduct eukaryotic expression of the leucine-rich repeat containing 15 (LRRC15), a differentially expressed protein in excretory secretory antigens of Taenia solium cysticercus, and predict its antigen epitope., Methods: The molecular weight, stability, amino acid sequence composition, isoelectric point and T lymphocyte epitope of the LRRC15 protein were predicted using the bioinformatics online softwares ExPASy-PortParam and Protean. The full-length splicing primers were designed using PCR-based accurate synthesis, and the LRRC15 gene was synthesized. The recombinant pcDNA3.4-LRRC15 plasmid was constructed and transfected into HEK293 cells to express the LRRC15 protein. In addition, the LRRC15 protein was characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting., Results: The recombinant pcDNA3.4-LRRC15 plasmid was successfully constructed, which expressed the target LRRC15 protein with an approximately molecular weight of 70 kDa. Bioinformatics prediction with the ExPASy-PortParam software showed that LRRC15 was a hydrophilic protein, which was consisted of 644 amino acids and had a molecular weight of 69.89 kDa and an isoelectric point of 5.6. The molecular formula of the LRRC15 protein was C 3073 H 4942 N 846 O 953 S 28 and had an instability coefficient is 50.3, indicating that LRRC15 was an instable protein. Bioinformatics prediction with the Protean software showed that the dominant T-cell antigen epitopes were located in 292 to 295, 353 to 361, 521 to 526 and 555 to 564 amino acids of the LRRC15 protein, and the T-cell antigen epitopes with a high hydrophilicity, good flexibility, high surface accessibility and high antigenicity index were found in 122 to 131, 216 to 233, 249 to 254, 333 to 343, 358 to 361, 368 to 372, 384 to 386, 407 to 412, 445 to 450, 469 to 481, 553 to 564, 588 to 594, 607 to 617 and 624 to 639 amino acids. Following transfection of the recombinant pcDNA3.4-LRRC15 plasmid into HEK293 cells, SDS-PAGE and Western blotting identified LRRC15 proteins in cell secretory culture media, cell lysis supernatants and sediments. The LRRC15-His fusion protein was purified from the cell culture medium, and SDS-PAGE identified a remarkable band at approximately 70 kDa, while Western blotting successfully recognized the band of the recombinant LRRC15 protein., Conclusions: The eukaryotic expression and antigen epitope prediction of the LRRC15 protein in the excretory secretory antigens of T. solium cysticercus have been successfully performed, which provides insights into further understandings of its biological functions.

Objective: To discover a new gene of candidate molecule for the diagnosis of cysticercosis., Methods: Cysticercus cellulosae cDNA library was immunoscreened with pooled serum of cysticercosis patients. The encoded protein of a new gene, Ts3, was analyzed through biology software (EXPASY) on line. The new gene was cloned, the prokaryotic expression vector pET28a (+)-Ts3 was constructed, and the expressed result was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting., Results: The new gene Ts3 (GenBank accession No. EU338456) was obtained with 531 bp and an ORF of C. cellulosae. With relatively stable structure and physicochemical properties, the new gene was anon-transmembrane protein and contained more protein kinase C phosphorylation site. To induce its expression, the Ts3 gene was cloned into the vector pET28a (+), and a protein with a relative molecular mass Mr21000 was obtained which was recognized by the sera of cysticercosis cases., Conclusion: The Ts3 gene of C. cellulosae was expressed, and the protein shows adequate antigenicity.

To obtain the recombinant 18 kD protein with high purity and normal bioactivity of Cysticercus cellulosae (rCE18), E. coli cells with the rCE18 were disrupted ultra-sonically, and the inclusion bodies were washed with a solution containing 0.2% deoxycholic acid sodium (DOC)and 2% DOC, respectively. Then they were denatured with 0.9% sodium lauroyl sarcosine (SKL) followed by dialysis and gel filtration to refold and purify the target protein. At the same time, this method was compared with GST-FF affinity chromatography and recovering from SDS-PAGE gel. Biological activity of purified rCE18 was analyzed with indirect ELISA, and the purity of the products was identified using SDS-PAGE. The purity of refolded inclusion bodies exceeded 60% and the total recovery of activated protein rCE18 was about 41.3%. The specificity of rCE18 reached up to 97.2% using indirect ELISA. An effective way for purifying and refolding rCE18 expressed in E. coli as inclusion bodies was established, rCE18 with higher purity and activity was obtained, which has the potential for developing diagnosis methods of porcine cysticercosis.

Objective: To immunoscreen one protein antigen gene from a cDNA library of Cysticercus cellulosae., Methods: A eDNA library of C. cellulosae was constructed after cDNA was synthesized, and immunoscreened using rabbit anti-C. cellulosae polyclone antibody. The gene structure and its possible function were analyzed by comparing with the sequences available in the GenBank, after the insert of positive clone was subcloned to pBluescript SK plasmid and the nucleotide sequence of the cDNA was determined by dideoxynucleotide chain termination method using a Taq DyeDeoxy Terminator Cycle Sequencing Kit. The amino acid sequence was deduced from nucleotide sequence using GENETYX software. Homological search of the nucleotide sequences was done using BLAST in GenBank., Results and Conclusion: A cDNA clone of 1320 bp was isolated. The clone contained one open reading frame composed of 1260 bp encoding 420 amino acids, in which two potential glycosylation sites were found. The partial nucleotide sequence of the gene fragment showed high homology with the essential spectrin gene of Caenorhabditis elegans and the erythrocyte surface antigen gene of Plasmodium falciparum, when the gene fragment was homologically analyzed in GenBank.

Objective: To obtain related genes of Cysticercus cellulosae from spliced leader (SL) cDNA library., Methods: Spliced leader library of Cysticercus cellulosae was constructed using SL specific primer and oligo (dT) 15 with M13M4 primer, and positive clones were then screened randomly, identified with enzyme restriction, followed by sequencing and homologous analysis., Results: The amino acid sequence, encoded by the positive clone with a poly (A)22 tail and a complete open reading frame (ORF), was with homology of RNA polymerase subunit genes of human, B. napus, fission yeast, A. thaliana, C. elegans and fruit fly up to 71.6%., Conclusion: The protein, RNA polymerase subunit encoded putatively by the clone, is high conservative in different species.

Objective: To clone and characterize the NADH1 gene of Cysticercus cellulosae., Methods: A cDNA library was constructed from Cysticercus cellulosae and was immunoscreened by using rabbit anti-Cysticercus cellulosae polyclonal antibody. The gene structure and its possible function were analyzed by comparing with sequences available in the GenBank, after the insert of positive clone was subcloned and the nucleotide sequence of the insert was determined by dideoxynucleotide chain termination method., Results and Conclusion: A cDNA clone (named TS5) with a length of 1,082 bp was isolated. The 5' terminal of cloned gene contained one open reading frame of 1-578 bp encoding 192 amino acid residues of mitochondrial NADH dehydrogenase subunit 1 and the 3' terminal contained three kinds of tRNA genes.

Aim: To construct a fusion expression vector for DNA vaccine including porcine interleukin-4(IL-4) and antigen cC1 to enhance the protective immunity of Cysticercus cellulosae antigen cC1., Methods: The cDNA fragments encoding porcine IL-4 and cC1 were amplified respectively by PCR and the fused. The obtained chimeric gene IL-4cC1 contained a synthetic linker of ten amino acids and the sequence surrounding its 5' AUG initiatory codon was changed to optimized translational initiation., Results: Identified by restriction enzyme analysis, an insert fragment of 1.5 kb was demonstrated. It had the same sequence as reported and designed by DNA sequencing analysis., Conclusion: A fusion expression plasmid containing porcine IL-4 and cC1 was constructed.

Aim: To provide simple and useful method for the immunodiagnosis of cysticercosis., Methods: Fusion protein of beta-galactosidase-Cysticercus cellulosae cDNA was analysed with simplified Western blot., Results: The positive rate was 93.5% when detecting 124 cases of human cysticercosis sera with four clones (cC1, cC2, cP1 and cH1) and 100% when detecting 38 cases of porcine cysticercosis sera with three clones (cC1, cC2 and cP1), being higher than those using monoclone fusion protein alone. Moreover, the fusion proteins did not react with other parasitosis sera., Conclusion: Simplified Western blot analysis using recombinant fusion protein as antigen was highly sensitive and specific and easy to be operated.