siRNA-757干扰旋毛虫Ts-ODC基因对其抗酸能力的影响

doi:10.11843/j.issn.0366-6964.2023.05.032

Abstract

Abstract: In order to explore the existence of ornithine-dependent anti-acid system in Trichinella spiralis(T. spiralis, Ts)muscle larvae, three specific siRNAs molecules were designed according to the complete genome sequence of Ts-ODC(Trichinella spiralis ornithine decarboxylase) in GenBank. Three siRNAs were transfected into T. spiralis muscle larvae by liposome transfection. The best interference siRNA molecules and the best interference conditions were determined by qPCR, Western blot and indirect immunofluorescence assays. The muscle larvae were cultured at pH 1.5, 2.5, 4.5 and 6.6 for 0.5, 1 and 2 h, respectively. The effects of Ts-ODC gene interference on the acid resistance of muscle larvae were evaluated from three aspects of the ODC transcription level, ornithine decarboxylase activity and larvae survival rate. Mice were infected with muscle larvae of T. spiralis after Ts-ODC gene interference. Adults and muscle larvae were collected at 7 d and 42 d, respectively, and the worm reduction rates were calculated. The first-generation muscle larvae were cultured under acidic conditions at pH 2.5 for 2 h, and the survival rate was calculated to analyze the heritability of Ts-ODC gene interference. The results showed that Ts-ODC siRNA-757 was the optimal interference molecule, and the best interference conditions were transfection for 12 h and 3 d culture at a concentration of 2 μmol·L^-1; the highest transcription level of Ts-ODC gene was reached at pH 2.5 for 2 h, and the survival rate was significantly lower than other culture conditions at pH 1.5 for 2 h (P<0.01). The ornithine decarboxylase activity was the highest at pH 4.5 for 0.5 h. After the mice were orally gavaged with muscle larvae transfected with Ts-ODC siRNA-757, the worm reduction rates were 58.95% and 65.91% at 7 d and 42 d, respectively. The survival rate of the first-generation muscle larvae was 49% when cultured in a pH 2.5 medium for 2 h. The above experiments concluded that T. spiralis muscle larvae has an ornithine-dependent acid-resistant system. In this study, the Ts-ODC gene of T. spiralis muscle larvae was interfered by siRNA technology for the first time, and it was confirmed that interfering with the Ts-ODC gene could reduce the acid resistance of muscle larvae, which provided a new idea for the prevention and treatment of Trichinellosis.

Key words: Trichinella spiralis, ornithine decarboxylase, RNA interference, anti-acid system

CLC Number:

S852.731

BO Luqi, WANG Xueying, HOU Jiaming, ZHANG Li, ZHANG Jianhui, RUAN Rulin, ZHANG Bohan, WANG Shuang, SONG Mingxin, ZHANG Ziqun. Interference of Ts-ODC Gene of Trichinella spiralis by siRNA-757 Effect on Its Acid Resistance[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2114-2125.

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Interference of Ts-ODC Gene of Trichinella spiralis by siRNA-757 Effect on Its Acid Resistance

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