The Molecular Cloning Of One Protein Antigen Gene Of Cysticercus Cellulosae

Object: In order to study the cysticercus cellulosae gene structure in molecular level, a cDNA library of cysticercus cellulosae was constructed and one protein antigen gene was immunoscreened.Methods: A cDNA library of cysticercus cellulosae was constructed after cDNA was synthesized using total RNA, extracted from the cysticercus cellulosae by the single step of acid guanidium thiocyanate-phenol-chloroform, and then linked with the Uni-ZAP XR vector. The library was immunoscreened using rabbit anti-cysticercus cellulosae proteinase polyclone antibody. One positive cDNA clone, named pTS, was isolated. The insert of the clone was sub-cloned to pBluescript SK plasmid with helper phage and the nucleotide sequence of the cDNA was determined by universal primers and dideoxynucleotide chain termination method using a Taq DyeDeoxy Terminator Cycle Sequencing Kit. The amino acid sequence was deduced from nucleotide sequence using GENETYX software. The homological search of the nucleotide and amino acid sequences was done using BLASTN in GenBank.Results: A cDNA clone (pTS) of 1358 bp was isolated The clone contained one opening reading frame (ORF) of 1302 bp encoding 434 amino acid (MW=50785Da). The initiation codon of5 ' end and poly(A) signal of 3 ' end were not found, but polyA of 3 ' end was found in the nucleotide sequence. The termination codon was composed of TAA. The percentage of the hydrophobic, neutral and hydrophilic residues in the amino acids sequences were 40.09% (174/434), 17.74% (17/434) and 41.24% (179/434), respectively. Two potential glycosylation sites were found in the amino acid sequence. The homology search found that the homogeneity of the partial nucleotide sequences was 100% with erythrocyte surface antigen gene of Plasmodium falciparum and 97% with the elastin-like protein gene of Drosophila melanogaster. The amino acid homology was 49%, 58%, 47%, 49% when compared with the a-spectrin of human, Drosophila melanogaster, Caenorhabditis elegans, mouse respectively, and was 30% with the human p-spectrin.Conclusion: The nucleotide sequence of pTS cDNA fragment had highly homogeneity with the elastin-like protein gene of Drosophila melanogaster and correlation with erythrocyte surface antigen gene of Plasmodiianfalcipanon. However, the amino acids sequence had higher homology with the a-spectrin of human, Drosophila melanogaster, Caenorhabditis elegans, mouse but lower with the human p-spectrin.