重组抗原p29免疫保护小鼠细粒棘球蚴感染的差异长链非编码RNA的筛选研究

Study on screening of differential expression of long non-coding RNA in mice with Echinococcus granulosus infection after immunization with recombinant antigen p29

目的研究在筛选重组抗原p29诱导的免疫保护下细粒棘球蚴感染宿主后长链非编码RNA(lncRNA)差异分子的意义。方法将45只BALB/c小鼠随机分为空白对照组、感染组和p29免疫+感染组。p29免疫+感染组小鼠重组抗原p29免疫3次,末次免疫7周后,采用细粒棘球蚴原头蚴腹腔感染小鼠(包括感染组、p29+感染组);感染24周后,采集小鼠血液,密度梯度离心法获得外周血单个核细胞(PBMCs)。经Trizol提取总RNA后,采用Magnetic Core试剂盒构建lncRNA文库,通过HiSeq 2 000高通量测序与生物信息学方法筛选差异表达的lncRNA。最后,采用qRT-PCR验证候选lncRNA分子。结果高通量测序及生物信息学分析结果表明,与空白对照组相比,感染组小鼠PBMCs中有14个lncRNA显著差异分子(P<0.05);与感染组相比,p29+感染组外周血PBMCs细胞中有30个差异表达的lncRNA(P<0.05);进一步经qRT-PCR验证,确定XLOC-012763、INTE-015204与INTE-028446是p29+感染组的候选差异lncRNA。结论此研究筛选出p29免疫保护细粒棘球蚴感染小鼠PBMCs中的3个lncRNA差异分子,丰富了p29免疫保护理论机制,为其后续作为疫苗应用开发奠定了基础。

Objective To investigate the effects of immunization with recombinant antigen p29 on differential expression of long non-coding RNA(lncRNA) in mice with Echinococcus granulosus infection.Methods 45 female BALB/c mice were randomly divided into control group,infection group and p29+infection group. Mice in the p29+infection group were vaccinated with the recombinant antigen p29 for 3 times. After 7 weeks of immunization,mice in infection and p29+infection groups were infected with protoscoleces which were isolated from clinical operation. After 24 weeks of infection,blood was isolated from mice and then peripheral blood monocytes(PBMCs) were separated using density gradient centrifugation. The total RNA were obtained by Trizol method. Magnetic Core kit was used to construct lncRNA library. The differential lncRNAs were screened by HiSeq 2 000 high-throughput sequencing and bioinformatics analysis. Finally,lncRNAs candidates were identified by qRT-PCR.Results High-throughput sequencing and bioinformatics analysis showed that 14 lncRNAs of infection group were significantly different from those in control group(P<0.05);30 lncRNA molecules were significantly different between infection group and p29+infection group(P<0.05). Three lncRNA candidates including XLOC-012763、INTE-015204 and INTE-028446 were identified by qRT-PCR assay.Conclusion In the present study,3 differential lncRNA candidates were obtained after p29 immunization in mice with Echinococcus granulosus infection,which may potentially contribute to the foundation and application of p29 vaccine against the infection.