猪带绦虫乳酸脱氢酶基因的序列分析、克隆表达和免疫学分析

Sequence analysis,cloning expression and immunogenicity analysis of lactate dehydrogenase gene from Taenia solium

目的利用生物信息学方法从猪带绦虫成虫cDNA文库中识别乳酸脱氢酶A(lactate dehydrogenase A,LDH-A),分析和预测其编码蛋白的结构和功能,并对其进行克隆表达和免疫学特性研究。方法利用NCBI和ExPASy中有关基因、蛋白的序列和结构信息分析工具,结合其它生物信息学分析软件包,从猪带绦虫成虫全长cDNA质粒文库中识别乳酸脱氢酶A(TsLDH-A)的基因及其编码区,分析、预测该基因编码的蛋白的结构与功能;并将TsLDH-A克隆到原核表达质粒pET-28a(+)中,在大肠杆菌BL-21/DE3中诱导表达,表达产物经纯化后用蛋白印迹(Western Blotting)进行免疫学分析。结果该基因全长1 332bp,编码331个氨基酸。其氨基酸序列与其它物种LDH-A氨基酸序列一致性可达54%,具有乳酸脱氢酶保守结构域。其编码的蛋白理论分子量为3 5461.1Da,有3个跨膜区和多个磷酸化位点,蛋白的理化性质较稳定。预测有4个主要的B细胞抗原表位,L-乳酸脱氢酶活化位点之一的His192包含于表位190-199aa中。酶催化位点的3个关键氨基酸在空间结构上相互靠近。PCR、双酶切及DNA测序结果均表明重组质粒pET-28a(+)-TsLDH-A构建成功。SDS-PAGE结果表明目的基因在大肠杆菌BL-21/DE3中获得表达,经亲和层析获得了高纯度蛋白。重组蛋白能被TsLDH-A重组蛋白免疫的SD大鼠血清、感染了猪带绦虫的病人血清及猪血清识别,表明其具有较好的免疫原性和免疫反应性。结论应用生物信息方法从猪带绦虫成虫cDNA文库中筛选出了TsLDH-A的全长序列并预测得到其编码蛋白的结构与功能方面的信息,该基因可在原核表达系统中获得具有免疫学活性的表达。

The structure and properties about encoding protein of lactate dehydrogenase A from Taenia solium（Ts LDH-A）were analyzed and predicted by bioinformatics in this study.The immunological characteristics of this novel gene were also analyzed by cloning and expressing.The full-length cDNA encoding Ts LDH-A was identified from the cDNA plasmid library by blastx and rpsblast programs provided by NCBI.The physico-chemical properties and structures of Ts LDH-A were analyzed by tools provided by ExPASy.And the B cell epitopes of Ts LDH-A were predicted by the B Cell Epitope Prediction Tools provided by IEDB Analysis Resource.The PCR amplified coding region of Ts LDH-A was cloned into the prokaryotic expression vector pET-28a（＋） and expressed in E.coli BL21 with IPTG induction.The immunogenicity of the purified recombinant protein was analyzed by Western Blotting.It was demonstrated that the amino acid sequence of Ts LDH-A had identity with that of LDH-A from other specie and there was a conserved LDH domain in the deduced amino acid sequence.The full-length cDNA sequence encoding Ts LDH-A included a complete open reading frame（ORF）of 1332 bp and coded to a putative protein with 331 amino acids.The molecular weight of Ts LDH-A was predicted to be 35461.1 Da and the coding protein was demonstrated to contain 3 trans-membrane regions and 4 main B cell epitopes.The active site of L-lactate dehydrogenase located at the epitope aa190-199.The 3 key residues in the catalytic site of enzyme were conserved in different species and located near to each other in spatial position.PCR,double enzyme restriction and DNA sequencing were used to identify pET28a（＋）-Ts LDH-A.The recombinant protein could react with the rat＇s sera as well as the sera from the patients and the swine infected Taenia solium.It is clear that the full-length cDNA sequence encoding Ts LDH-A can be screened from the cDNA library of adult Taenia solium by bioinformatics analysis and can be used to investigate the structure and properties about gene and encoding protein of Ts LDH-A as well as the immunological activities of gene expression in the prokaryotic system.