论文部分内容阅读
目的利用生物信息学方法分析华支睾吸虫LAP2全长基因的结构和功能,并将该基因克隆至原核表达载体进行表达、纯化,为进一步研究LAP2功能奠定基础。方法利用生物信息学相关软件,分析华支睾吸虫LAP2基因及其蛋白的结构、生物学和免疫学功能特征。针对LAP2的EST序列的编码区设计引物,从华支睾吸虫囊蚴cDNA质粒中扩增目的基因,克隆到原核表达质粒pET-28a(+)中,经PCR、双酶切及DNA测序鉴定的阳性克隆诱导目的蛋白表达、亲和层析纯化、免疫印迹鉴定及组化定位。结果华支睾吸虫LAP2基因全长为1761bp,其编码序列长度为1662bp,编码553个氨基酸,理论分子量是59696.5Da,与人的该基因氨基酸序列相似性为26.7%,一致性仅为15.4%。该基因在大肠杆菌中可被高效表达,纯化的重组蛋白能被感染华支睾吸虫的大鼠血清识别,免疫组化结果表明该重组蛋白定位于华支睾吸虫成虫的肠支及表膜。结论华支睾吸虫LAP2在大肠杆菌中呈高效的可溶性表达,具有良好的抗原活性,可能为华支睾吸虫成虫分泌排泄抗原的组份之一。
OBJECTIVE: To analyze the structure and function of full-length LAP2 gene of Clonorchis sinensis using bioinformatics method. The gene was cloned into prokaryotic expression vector for expression and purification, which laid the foundation for further study of LAP2 function. Methods The structural, biological and immunological functional characteristics of LAP2 gene and its protein in Clonorchis sinensis were analyzed by bioinformatics software. According to the coding region of LAP2 ESTs, primers were designed and cloned into the prokaryotic expression plasmid pET-28a (+). After PCR, double enzyme digestion and DNA sequencing Positive clones induced expression of the target protein, purified by affinity chromatography, identified by immunoblotting and histone localization. Results The full - length cDNA of LAP2 gene from Clonorchis sinensis was 1761bp in length and encoded a protein of 553 amino acids with a length of 1662bp. The theoretical molecular weight of LAP2 gene was 59696.5Da. The amino acid sequence of LAP2 gene shared 26.7% identity and 15.4% identity with human. This gene can be highly expressed in E. coli. The purified recombinant protein can be identified by serum of rats infected with Clonorchis sinensis, and immunohistochemistry results show that the recombinant protein is located in the intestinal branch and epidermis of adult Clonorchis sinensis. Conclusion Clonorchis sinensis LAP2 is highly soluble in E. coli and has good antigenic activity. It may be one of the components of Clonorchis sinensis secreting antigen.