Construction And Immunoprotection Efficacy Of Nucleic Acid Vaccine Of Trichinella Spiralis 49ku ES Antigen Gene

A pair of primers were designed and synthesized according to sequence of Trichinella spiralis published in GenBank (M64242). The total RNA of the pre-encysted Trichinella spiralis larva (PELA) of six Trichinella isolates (Swine isolate; Dog isolate; Cat isolate; American Swine isolate; Trichinella spiralis; Trichinella nativa) and different growth period of Swine isolate (pre-encysted larvae, muscle larvae and adult worm) was extracted with the SDS spallation liquid matching Protease K. The genes encoding 49ku of ES with Kozak sequence were amplified by RT-PCR. The target genes were directly cloned into the pMD18-T Vector, and then transferred into Escherichia coli strain TG1.The recombinant plasmid pMD18-T-S/EL, pMD18-T-D/EL, pMD18-T-C/EL, pMD18-T-AS/EL, pMD18-T-T1/EL, pMD18-T-T2/EL, pMD18-T-S/ML and pMD18-T-S/AM were constructed. The recombinant plasmids were identified by EcoR I and BamH I restriction enzyme digestion and PCR. The positive plasmids were subjected to sequencing.The gene-49ku of ES in six Trichinella isolates of pre-encysted larvae and three different growth period of Swine isolate were cloned successfully. The sequence analysis indicated that the 49ku ES antigen gene consists of 951bp contaning initiation codon, termination codon, and the ORF encoding a protein of 316 amino acid residues.The sequence analysis showed that the homology of the nucleotide sequence of 49ku ES gene between six Trichinella isolates and Trichinella spiralis published in GenBank was 97.0%~97.5%, and the homology of their deduced amino acid sequence is 93.4%～94.6%. The homology among the six Trichinella isolates was 99.3%～99.9%. The results revealed that the 49ku ES gene was very conservative, the diversity of the nucleotide sequence was very little among six Trichinella isolates, and the antigenicity of their encoding protein was similar.The sequence analysis showed that the homology of the nucleotide sequence of 49ku ES gene among pre-encysted larva, muscle larvae and adult worm of Swine isolate and Trichinella spiralis published in GenBank was 97.5%, 97.3% and 97.3% respectively, The homology between pre-encysted larva and muscle larvae was 99.8%, there were mutations in 535 T→C and 665 T→C. The homology between the muscle larvae and adult worm was 99.8%, and mutations were in 665 C→T and 848A→G. The homology between pre-encysted larvae and adult worm was 99.8%, mutations were in 535T→C and 848 A→G. Although the deduced amino acid was changed, the...