| Parasitic nematodes cause diseases of major socio-economic importance in animals and humans world-wide. Ascaris suum is one such nematode which causes major economic losses globally to the pig and pork industries and farming communities as a consequence of reduced feed conversion and weight gains, and the condemnation of affected organs after slaughter. Currently, the control of these parasites relies chiefly on the use of anthelmintic drugs. However, their excessive and uncontrolled use has resulted in serious problems with resistance in nematodes and risks of residue problems in animal products and the environment. Consequently, there has been substantial interest in the development of alternative means of parasite control. Suppression of parasite development at particular developmental stages could offer an alternative approach for nematode control. The control of larval development would substantially reduce disease and production losses, since they can be pathogenic in the process of the transition and migration in the host. However, the success of such a novel control approach depends on our understanding of the developmental biology of the parasitic nematodes and consequently the capacity to interfere with their development.In this study, the genes differentially expressed in infective larvae of A. suum using cDNA microarray analysis by construction of a specific cDNA library for A. suum infective larvae were identified by semi-quantitative RT-PCR. The full length sequence of the differentially expressed gene 28A02 which identified by semi-quantitative RT-PCR was obtained and the characteristics of its predicted protein was analyzed.Firstly, the infective larvae in the eggs were isolated using 7.5%NaClO to treat the infective eggs in 37℃overnight and shaken with glass-beads; the third (L3) in lungs, the fourth (L4) in intestines were isolated with a method which is a modification of conventional Baerman's technique by using 0.3%-0.4% agar-gel; Also, females and males were collected from abattoirs and eggs were acquired from females uterus. Then all total RNA from different stages worms were obtained.Secondly, semi-quantitative RT-PCR was used to assess changes in mRNA abundance of parasite as a function of genes expression level at different stage worms. RT-PCR primers were designed for each of the 8 representative EST sequences selected from the SSH library and the cDNA microarray profiling, and control primers were designed according to theβ- actin gene sequence of A. suum. Visualization of RT-PCR products ofβ- actin in all the stages under UV-light exposure was used to confirm equal template. Each RT-PCR was replicated three or more times. The results showed the expression level of these genes in the infective larvae is much more than other stages.Thirdly, the full-length of the gene 28A02 was cloned by rapid amplication of cDNA ends (RACE). The full-length cDNA was 864 bp in size and encoded an open reading frame (ORF) of 450 amino acids with a calculated molecular weight (MW) of 16.706 kDa and an isoelectric point (pI) of 9.499. There was a 387 bp 3'untranslated region (3'UTR) following the stop codon (TGA), and a 27 bp 5'UTR upstream from the start codon (ATG).Amino acid sequence alignment indicated that 28A02 shared the highest identity (47%) with CBR-ATP-4 protein from Caenorhabditis briggsae, followed by Caenorhabditis elegans (45%) and had highly homology with ATP synthase family member from other nematodes, fish, Drosophila, Aedes and Culex. The molecular phylogeny was reconstructed. The predicted protein structure of 28A02 showed that it was composed by 38.9% Helix, 2.8% Sheet, 19.4% Turn and 38.9% Coil and had identity (32.877%) with ATP synthase-coupling factor 6 of Bos taurus (pDB: 1vzsA). The successful cloning of the full length cDNA of 28A02 provides foundation for further studying the developmental processes and infectivity of A. suum. |
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