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目的克隆表达一个新的旋毛虫组织蛋白酶基因CathepsinX,初步探讨其免疫学特性,为研究新的防治方法奠定科学基础。方法从GenBank旋毛虫核酸数据库中筛选出功能未知的CathepsinX基因部分EST序列,使用RACE-PCR方法得到其全长编码基因序列。半定量RT-PCR分析该基因的转录。构建该基因的原核表达载体pET30a-TsCPX,大肠杆菌中IPTG诱导表达,His-tag亲和层析纯化获重组蛋白TsCPX;将纯化的重组蛋白40μg/只免疫BALB/c小鼠,免疫前1周及每次免疫后2周收集免疫血清,间接ELISA检测血清总IgG、亚类IgG1、IgG2a抗体浓度。结果 RACE-PCR克隆出TsCPX全长基因序列,共1227bp;RT-PCR揭示TsCPX在旋毛虫三个发育时期均有380bp扩增条带,条带光密度分析显示最高转录水平出现在新生幼虫期。E.coli原核表达体系中成功克隆表达出重组蛋白TsCPX;重组蛋白免疫小鼠二次后,免疫血清总IgG迅速上升,三次免疫后达到峰值(P<0.001);IgG亚类IgG1抗体浓度随着免疫次数的增加迅速上升,亚类IgG2a无明显上升,两者间差异有统计学意义(P<0.001)。结论成功克隆的TsCPX基因在旋毛虫的三个生长发育时期均有转录表达,TsCPX重组蛋白具有较强免疫原性,能引起宿主(小鼠)Th2型为主的免疫应答,具有作为旋毛虫病免疫预防研究靶分子的潜在价值。
Objective Cloning and expression of a new Trichoplusia cathepsin gene CathepsinX preliminary study of its immunological properties and lay a scientific foundation for the study of new methods of prevention and treatment. Methods A partial EST sequence of CathepsinX gene with unknown function was screened from the GenBank database and the full-length coding sequence was obtained by RACE-PCR. Semi-quantitative RT-PCR analysis of the gene transcription. The prokaryotic expression vector pET30a-TsCPX was constructed and induced by IPTG in E. coli. The recombinant protein TsCPX was purified by His-tag affinity chromatography. BALB / c mice were immunized with purified recombinant protein 40μg / The immune serum was collected 2 weeks after each immunization, and the total IgG, subclass IgG1, IgG2a antibody concentrations were detected by indirect ELISA. Results The full-length cDNA sequence of TsCPX was cloned by RACE-PCR with a total length of 1227bp. RT-PCR revealed that 380 bp of TsCPX was amplified in all three developmental stages of Trichinella spiralis. The optical density analysis showed that the highest level of TsCPX appeared in the neonate larval stage. E.coli prokaryotic expression system successfully cloned and expressed the recombinant protein TsCPX; recombinant protein immunized mice twice, the total immune serum IgG increased rapidly after three immunizations peak (P <0.001); IgG subclass IgG1 antibody concentration with the Immunization times increased rapidly, subclass IgG2a no significant increase, the difference between the two was statistically significant (P <0.001). Conclusion The cloned TsCPX gene is transcribed in all Trichinella spiralis growth and development stages. TsCPX recombinant protein has strong immunogenicity and can cause host (mouse) Th2 type-based immune response. As a trichinosis The potential value of immune prevention research targets.