华支睾吸虫LAP2基因的生物信息学分析及克隆表达、组织定位

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。

Bioinformatics analysis,cloning,expression and immunolocalization of the LAP2 gene from Clonorchis sinensis

Objective To characterize the structure and function of Clonorchis sinensis LAP2（CsLAP2） gene by bioinformatics,and express and purify the recombinant CsLAP2 in prokaryotic expression system for the further studies of its functions.Methods The characteristics of the structure,biology and immunity of CsLAP2 were analyzed using several bioinformatic softwares.Specific primers were designed according to the coding region of EST sequence of LAP2 to amplify the target gene from the cDNA plasmid of Clonorchis sinensis metacercaria.PCR products were subcloned into the prokaryotic expression plasmid pET-28a（＋）.After digestion with restriction enzymes,the recombinant plasmid was confirmed by DNA sequencing.The selected clone was expressed in E.coli BL21 with IPTG induction.The total protein was subjected to affinity chromatography and identified by immunoblotting.The recombinant protein was used to raise antibodies for the immunolocalization.Results CsLAP2 is a conserved gene with a full length of 1 761 bp.The open reading frame（ORF） of this gene is 1,662 bp,coding for 553 amino acids.Theoretical molecular mass of CsLAP2 is 59 696.5 Da.The sequence share 26.7% homology with that of Homo sapiens,with the identity of 15.4%.The CsLAP2 gene was highly expressed in E.coli BL21.The purified recombinant protein could react with the Clonorchis sinensis infected rat serum.The result of immunolocalization showed that CsLAP2 located at the intestine and tegument of Clonorchis sinensis adult worm.Conclusion CsLAP2 is highly expressed in E.coli BL21 as a soluble protein,retaining its antigenicity and may be one of the components of ESA of Clonorchis sinensis adult worm.