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[Cloning of Taenia pisiformis Actin Gene and Assessment of Its Use as An Internal Control].

Objective
To clone the full-length cDNA of actin gene of Taenia pisiformis (Tp-actin), and analyze the gene structure, phylogenetic evolution and its use as an internal control.


Methods
Tp-actin was amplified by RT-PCR and the cDNA of 3′ and 5′ ends were obtained through RACE-PCR. After sequencing, these segments were linked to produce full-length cDNA of Tp-actin. The gene structure and phylogenetic evolution were analyzed using bioinformatics software. Primers for Tp-actin and cysteine peptidase (TpCP) were designed using Primer Express software. Primer specificity and amplification efficiency were analyzed with real-time fluorescence quantitative PCR (qRT-PCR). In addition, by using Tp-actin as an internal control, the expression of TpCP in T. pisiformis at various developmental stages was analyzed.


Results
As expected, sequencing results showed that the Tp-actin fragment was 1 048 bp in length, and the 3′ and 5′ ends were 428 bp and 945 bp, respectively. The full-length cDNA of Tp-actin generated from the 3 segments (submitted to GenBank with accession No. JX624787) was 1 279 bp, containing a 30-bp 5′-untranslated region（5′-UTR）, a 118-bp 3′-UTR, and a 1 131-bp open reading frame (ORF). Bioinformatics analysis showed that the Tp-actin encoded a protein of 356 amino acids, with a predicted relative molecular weight of 41 749 and a PI value of 5.29. This protein was predicted to contain 6 functional sites and 3 typical signatures of the actin family. Phylogenetic analysis showed that the Tp-actin was 100% and 99.7% homologous in amino acid sequence to those of Taenia solium and Diphyllobothrium dendriticum. qRT-PCR resulted in specific products of 82 bp and 108 bp from Tp-actin and TpCP, respectively, melting curves of which both showed a single signal peak, verifying the high specificity of primers. The linear correlation coefficient（R2） in standard curve of Tp-actin was 0.999, showing high amplification efficiency. Using Tp-actin as the internal control, the relative expression ratio of TpCP gene in gravid proglottid of T. pisiformis（1.65） was significantly higher than that in oncospheres （1.00）, mature proglottids （0.87） and cysticercus (0.62) (P<0.05).


Conclusion
Tp-actin gene is highly conserved and can be used as a reliable internal control.