目的:克隆、表达、定位日本血吸虫腺苷酸激酶-1(Schistosoma Japonicum Adenylate Kinase-1,SjAK1)及日本血吸虫程序性细胞死亡蛋白-10(Schistosoma Japonicum Programmed Cell Death-10,SjPCD10),为进一步研究其基因在生长发育中的功能奠定基础。方法:据SjAK1、SjPCD10基因完整开放阅读框(长度分别为594bp、651bp)分别设计并合成特异性引物,提取日本血吸虫成虫总RNA,RT-PCR获得cDNA,以cDNA为模板分别扩增SjAK1、SjPCD10目的基因片段,与载体pET28a质粒共同进行双酶切,连接、转化大肠杆菌DH5α,筛选阳性克隆并测序鉴定后,抽提重组质粒,转化表达宿主菌BL21。PCR鉴定阳性重组克隆,IPTG诱导表达,SDS-PAGE、Western Bloting检测表达结果,镍柱亲和层析纯化目的蛋白,BCA法定量目的蛋白;ATP荧光检测试剂盒检测重组SjAK1的活性。将目的蛋白分别免疫新西兰兔制备多抗,ELISA鉴定多抗效价。免疫组化法检测SjAK1和SjPCD10基因在日本血吸虫皮肤型童虫(30min)、肺型童虫(3天)、肝门型童虫(10天、14天、18天、22天)以及成虫(26天)中表达的变化。结果:成功构建重组表达质粒pET28-SjAK1和pET28-SjPCD10,原核表达的目的蛋白rSjAK1为可溶性、rSjPCD10呈包涵体形式;SDS-PAGE鉴定目的蛋白rSjAK1约26kDa、rSjPCD10约28kDa,与理论分子量一致;经镍柱亲和层析纯化获得高纯度目的蛋白,rSjAK1的比酶活为135.2U/mg。将目的蛋白免疫新西兰兔后血清效价均高于1∶1 280。免疫组化结果显示,SjAK1在各型童虫均有表达,皮肤型和肺型童虫中表达较少,肝门型童虫感染10天开始增加,但成虫期表达又减少;SjPCD10从18天开始表达增多,在雄虫和虫卵表达最为明显。结论:本实验成功克隆表达及纯化SjAK1、SjPCD10,其基因在日本血吸虫发育过程中均有表达,为深入研究其基因功能奠定了基础。 Objective: To clone, express and locate Schistosoma Japonicum Adenylate Kinase-1 (SjAK1) and Schistosoma Japonicum Programmed Cell Death-10 (SjPCD10) for further study Its gene in the growth and development of the function laid the foundation. Methods: According to the complete open reading frame of SjAK1 and SjPCD10 genes (594 bp in length and 651 bp in length), specific primers were designed and synthesized respectively. Total RNA was extracted from adult Schistosoma japonicum, cDNA was amplified by RT-PCR. CDNAs were used as template to amplify SjAK1 and SjPCD10 The target gene fragment was digested with restriction endonuclease and ligated with the vector pET28a, and then transformed into E. coli DH5α. The positive clones were screened and identified by sequencing. The recombinant plasmids were extracted and transformed into host strain BL21. The positive recombinant clones were identified by PCR, induced by IPTG, expressed by SDS-PAGE and Western Bloting, purified by nickel affinity chromatography and quantified by BCA method. The activity of recombinant SjAK1 was detected by ATP fluorescence detection kit. The target protein were immunized New Zealand rabbit polyclonal antibody, ELISA identification of multi-titers. Immunohistochemistry was used to detect the expression of SjAK1 and SjPCD10 genes in Schistosoma japonicum skin-type schistosomiasis (30 min), lung-type schistosomiasis (3 days), portal-type schistosomiasis (10 days, 14 days, 18 days and 22 days) 26 days). Results: The recombinant plasmids pET28-SjAK1 and pET28-SjPCD10 were constructed successfully. The prokaryotic expression vector rSjAK1 was soluble and rSjPCD10 was in the form of inclusion body. SDS-PAGE showed that the target protein rSjAK1 was about 26kDa and rSjPCD10 was about 28kDa, which was consistent with the theoretical molecular weight. Purification of nickel column by affinity chromatography to obtain high purity of the target protein, rSjAK1 specific enzyme activity was 135.2U / mg. The target protein immunized New Zealand rabbit serum titer higher than 1: 280. The results of immunohistochemistry showed that SjAK1 was expressed in all species of schistosomiasis and was less expressed in skin and lung-type schistosomiasis. The onset of hepatic-type schistosomiasis increased at 10 days, but decreased at adult stages. The expression of SjPCD10 increased from 18 days Increased expression began, the most obvious expression in the male and insect eggs. Conclusion: The successful cloning and expression of SjAK1 and SjPCD10 in this experiment were all expressed in the development of Schistosoma japonicum, which lays the foundation for further study of gene function.