关键词: 长角血蜱, 人兽共患病, 田鼠巴贝虫, 天冬氨酸蛋白水解酶8, RNA干扰

Abstract:

Objective To identify the Haemaphysalis longicornis caspase 8 (cas8) gene, and to investigate its role in resisting Babesia microti infection, to lay the foundation for development of vaccine interrupting transmission of B. microti. Methods The RNA of the H. longicornis was extracted and reverse transcribed into cDNA, the cas8 was amplified by PCR and sequenced, and sequence alignment was performed in GenBank with BLAST. Using MEGA software, a phylogenetic tree based on the cas gene was constructed with the neighbor-joining method. The H. longicornis nymph that had bitten B. microti infected-mice were assigned as infection group, while the same batch of ticks that had bitten normal mice as the control group. Nine ticks from each group were collected for extraction of RNA to reverse transcribe into cDNA. Quantitative PCR (qPCR) was used to analyze the relative transcriptional differences of cas8 between the infection group and the control group ticks. The total protein of H. longicornis nymph from the infection group and the control group were extracted for analysis of relative expression level of by Western blotting. Double stranded RNA (dsRNA) of cas8 gene and luciferase gene were synthesized and microinjected into the tick nymphs for RNA interference (RNAi) (RNAi group and control group, respectively). Upon 12-24 hours post microinjection, the ticks with good activity were picked and fed with B. microti infected-mice by biting. Immediately after fully fed of blood, the ticks were collected to detect relative transcriptional level of cas8 gene with qPCR, and to detect the 18S rRNA of B. microti in H. longicornis by probing with real-time PCR. The comparison of relative transcription levels and protein expression levels of cas8 gene between the infection group and the control group, and content of B. microti 18S rRNA in the RNAi group and control group were performed using t-test. Results The cas8 gene of H. longicornis is 1 377 bp in length, and the accession number obtained by submitting the sequence to GenBank is PP407944. The predicted amino acid sequence is 21.26% and 61.24% in consistence with the amino acid sequences of H. longicornis CAS8 (GenBank: ABG48761) and Rhipicephalus haemaphysaloides CAS8 (GenBank: ALQ43547.1) from GenBank, respectively. The phylogenetic tree analysis showed that the cas8 were not at the same branch with the H. longicornis cas sequences (ABG48665.1, ABG48761.1) from GenBank, indicating a distant genetic relationship; while the cas8 was at the same branch with the cas8 of R. haemaphysaloides (GenBank: ALQ43547.1), indicating a relatively closer genetic relationship. The qPCR analysis results showed that the relative transcription level of the cas8 in infection group was 0.562 ± 0.036, which was higher than that in the control group (0.198 ± 0.071) (t = 7.910, P < 0.01). The Western blotting results showed that the relative expression level of CAS8 protein in the H. longicornis in infection group was 0.460 ± 0.013, which was higher than that in the control group (0.346 ± 0.007) (t = 9.368, P < 0.01). After silencing cas8 by RNAi, the relative transcription level of the cas8 in RNAi group was 0.036 ± 0.003, which was lower than that in the control group (0.081 ± 0.006) (t = 10.680, P < 0.01). The content of B. microti 18S rRNA in the ticks in RNAi group was 3.35 × 10⁶ copies/μl, which was higher than that in the control group (1.35 × 10⁶ copies/μl) (t = 4.570, P < 0.05). Conclusion CAS8 is an apoptosis related molecule, which can resist the infection of B. microti to H. longicornis. It may be used as a candidate target molecule for the transmission blocking vaccine of B. microti.

Key words: Haemaphysalis longicornis, Zoonosis, Babesia microti, Caspase 8, RNA interference