目的 对长角血蜱天冬氨酸蛋白水解酶8（cas8）基因进行鉴定，探究其在长角血蜱抵抗田鼠巴贝虫感染中的作用，为田鼠巴贝虫传播阻断疫苗的研究奠定基础。 方法 提取长角血蜱RNA，逆转录为cDNA，PCR扩增cas8，测序后在GenBank中进行BLAST比对。用MEGA软件，采取邻接法构建基于cas基因的系统进化树。以叮咬田鼠巴贝虫感染小鼠的长角血蜱若蜱为感染组，以叮咬正常小鼠的同批次若蜱为对照组。感染组与对照组各取9只蜱，提取RNA并逆转录为cDNA，采用定量PCR（qPCR）分析感染组与对照组长角血蜱cas8基因相对转录水平差异；提取感染组和对照组长角血蜱若蜱总蛋白，蛋白质免疫印迹（Western blotting）检测CAS8的相对表达量。合成cas8基因和荧光素酶基因的双链RNA（dsRNA），分别显微注射至长角血蜱若蜱体内进行RNA干扰（RNAi）（RNAi组和对照组），12~24 h后取活性良好的蜱叮咬田鼠巴贝虫感染小鼠，饱血后立即收集蜱，qPCR检测cas8基因的相对转录水平，实时PCR（探针法）检测长角血蜱体内田鼠巴贝虫的18S rRNA基因。感染组和对照组cas8基因相对转录水平和蛋白相对表达水平、RNA干扰组和干扰对照组田鼠巴贝虫18S rRNA基因含量的比较采用t检验。 结果 长角血蜱cas8基因长1 377 bp，序列提交GenBank获得的登录号为PP407944，其预测的氨基酸序列与GenBank中的长角血蜱CAS8（GenBank：ABG48761.1）、镰形扇头蜱CAS8（GenBank：ALQ43547.1）的氨基酸序列一致性分别为21.26%和61.24%。系统进化树分析结果显示，cas8基因与GenBank中的长角血蜱cas基因（ABG48665.1、ABG48761.1）均不在同一分支上，且亲缘关系较远；与镰形扇头蜱cas8基因（GenBank：ALQ43547.1）的亲缘关系较近，在同一分支上。qPCR分析结果显示，感染组cas8基因的相对转录水平为0.562 ± 0.036，高于对照组的0.198 ± 0.071（t = 7.910，P < 0.01）。Western blotting结果显示，感染组CAS8蛋白的相对表达水平为0.460 ± 0.013，高于对照组的0.346 ± 0.007（t = 9.368，P < 0.01）。沉默cas8后，RNAi组cas8基因的相对转录水平为0.036 ± 0.003，低于对照组的0.081 ± 0.006（t = 10.680，P < 0.01）；RNAi组田鼠巴贝虫18S rRNA基因为3.35 × 10⁶ 拷贝/μl，高于对照组的1.35 × 10⁶ 拷贝/μl（t = 4.570，P < 0.05）。 结论 CAS8是长角血蜱凋亡相关分子，能够抵御田鼠巴贝虫感染长角血蜱，可以作为田鼠巴贝虫传播阻断疫苗的候选靶向分子。

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.