| Cystic echinococcosis (CE) caused by infection with larval stage of Echinococcus granulosus(E.g), affects both humans and domestic animals, which has been considered as one of the worldwide major zoonoses. In some highly prevalent regions in China, such of Xinjiang, Qinghai, Gansu, Ninxia, etc, there were more than 5%-10% peoples have been infected by this parasite. Development and application of effective vaccine have been being considered as the most efficient way to control this sort of transmission-infected diseases. Eg95 protein was regarded as a vaccine candidate to prevent from hydatid transmission for use in the parasite's natural animal intermediate hosts.Part 1: Cloning and sequence analysis of Eg95/XJThere is genetic variability within the E.g species and antigen variability in the Eg95 protein has the potential to limit the effectiveness genes that encode the Eg95-based vaccine. No information is available as yet concerning genetic variability in the gene encoding Eg95 between E.g strains from New Zealand sheep origin and E.g isolates from Xinjiang sheep origin. Here, have investigated the Eg95 gene of Xinjiang and its cDNA encoding Eg95 protein.E.g specimens used in this study, were protoscoleces from either individual hydatid cyst or pooled hydatid cysts collected from naturally infected Xinjiang sheep. The primers were designed according to the parent Eg95 sequence (GenBank accession No.X90928). Genomic DNA was extracted from protoscoleces using Trizol reagent. The GenomicDNA was used as template in PCR amplification with the primers. The PCR amplified DNA fragments were purified after separation by agarose gel electrophoresis and then ligated to the pUCm-T Easy plasmid. The positive clones of pUCm-Eg95/XJ were screened by blue/white screening, identified by EcoR I/Xho I restrication endonuclease and by PCR amplification.The result showed that genomic Eg95 PCR fragment was about 1200 bp DNA. The sequencing record revealed Eg95/XJ of genomic DNA was 1191bp, named as Eg95-XJ(GenBank accession No.AF465599). By homology comparison, the gene had 86%-97% identity with other members of Eg95 accessed in GenBank. analysis of its DNA sequence revealed that Eg95/XJ contained the gene with 100% identity to the Eg95 cDNA(GenBank accession No.X90928). The multiple nucleotide differences, which represented in Eg95/XJ gene in comparison with that of the New Zealand strain, occurred predominantly in the non-coding regions of gene. The result indicated that Eg95/XJ was a new sequence and belong to the Eg95 gene family members.To study expression and sequence differences of Eg95 antigen cDNA from different E.g stages in Xinjiang, such of protocolex, oncosphere and adult worm, Eg95 antigen cDNAs were amplified by E.g PCR from protocolex, oncosphere and adult worm cDNA libraries in Xinjiang, and cloned into pUCm-T plasmid, and then sequenced. The sequences were analyzed by DNAman and GenBank/BLAST biosoftware. PCR results showed that Eg95 antigen cDNA could be amplified from three E.g stages of cDNA libraries. Sequencing analysis indicated that the Eg95 cDNA length was 402bp wich are prving gene record the same as the reported data in GenBank. The result indicated Eg95 antigen cDNA could be expressed in the different E.g life-cycle stages in Xinjiang and there was no nuclear acid sequence difference of Eg95 antigen among the E.g protocolex, oncosphere and adult worm.Part 2:Construction of pcDNA3-Eg95/XJ and immune response to Eg95/XJ vaccineTo construct the Eg95/XJ DNA vaccine for further study, the PCR product of Eg95/XJ cDNA was purified and digested by double restriction enzymes EcoR I/Xho I. The digested PCR product was recovered using DNA recover kit and was Hgated to pcDNA3 backbone that was digested by EcoR I/Xho I to construct the recombinant pcDNA3-Eg95/XJ. After identifying them by EcoR I/Xho I digestion and PCR, the pcDNA3- Eg95/XJ consrtructs were transferred into HeLa cells using Lipofectamine-Plus reagent according to the supplied protocol. Then serum-free supernata...
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