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目的克隆编码日本血吸虫视黄酸X受体2(SjRXR2)蛋白的全长cDNA,并对其进行初步研究。方法利用cDNA末端快速扩增技术(RACE)获得SjRXR2蛋白全长编码cDNA。利用生物信息学技术,对基因结构进行初步分析。利用实时荧光定量(Real time)PCR技术对该基因在日本血吸虫不同时期虫体中的转录情况进行分析。应用在线抗体表位预测软件获得SjRXR2配体结合区抗原性较强的一个多肽序列,合成该多肽片段,并免疫小鼠制备抗血清。利用Western blot技术分析该蛋白在日本血吸虫中的表达。结果采用RACE技术成功获得了SjRXR2蛋白全长编码cDNA,总长度为5 960bp,其完整开放阅读框为4 308 bp,编码1 435个氨基酸,预测分子量为159 kDa。生物信息学分析表明该基因编码的蛋白质序列具有核受体家族2的典型结构域特征,且与曼氏血吸虫RXR2有较高的相似性。Real time PCR分析表明,该基因在21、42 d龄日本血吸虫虫体内有较高的转录水平。Western blot分析表明,小鼠SjRXR2多肽免疫血清可特异性识别日本血吸虫虫体150 kDa蛋白。结论成功获得了编码SjRXR2蛋白的全长cDNA,并制备了针对该蛋白的特异性多克隆抗体,为进一步研究该蛋白的功能奠定了基础。
Objective To clone the full-length cDNA encoding the SjRXR2 protein of Schistosoma japonicum, and to conduct a preliminary study. Methods Full-length cDNA encoding SjRXR2 protein was obtained by rapid amplification of cDNA ends (RACE). The use of bioinformatics technology, preliminary analysis of gene structure. The transcription of this gene in different stages of Schistosoma japonicum was analyzed by real time PCR. An on-line epitope prediction software was used to obtain a polypeptide sequence with high antigenicity of SjRXR2 ligand binding domain. The polypeptide fragment was synthesized and immunized to prepare antiserum. Western blot analysis of the protein expression in Schistosoma japonicum. Results The full-length cDNA encoding SjRXR2 was successfully obtained by RACE technique. The total length of the cDNA was 5 960 bp. The complete open reading frame was 4 308 bp encoding a protein of 1 435 amino acids with a predicted molecular mass of 159 kDa. Bioinformatics analysis showed that the protein sequence encoded by this gene possesses typical domain characteristics of nuclear receptor family 2 and has high similarity with RXR2 of Schistosoma mansoni. Real time PCR analysis showed that the gene had higher transcriptional levels in Schistosoma japonicum at 21 and 42 days old. Western blot analysis showed that the mouse SjRXR2 polypeptide serum could specifically recognize 150 kDa protein of Schistosoma japonicum. Conclusion The full-length cDNA encoding SjRXR2 protein was successfully obtained and a specific polyclonal antibody against this protein was prepared, which laid the foundation for further study on the function of this protein.