CAJ | 학술논문

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(Ts LDH-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Ts LDH-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(+)-Ts LDH-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Ts LDH-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Ts LDH-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.
목적 이용생물신식학방법종저대조충성충cDNA문고중식별유산탈경매A(lactate dehydrogenase A,LDH-A),분석화예측기편마단백적결구화공능,병대기진행극륭표체화면역학특성연구.방법 이용NCBI화ExPASy중유관기인、단백적서렬화결구신식분석공구,결합기타생물신식학분석연건포,종저대조충성충전장cDNA질립문고중식별유산탈경매A(Ts LDH-A)적기인급기편마구,분석、예측해기인편마적단백적결구여공능;병장Ts LDH-A극륭도원핵표체질립pET-28a(+)중,재대장간균BL-21/DE3중유도표체,표체산물경순화후용단백인적(Western Blotting)진행면역학분석.결과 해기인전장1 332bp,편마331개안기산.기안기산서렬여기타물충LDH-A안기산서렬일치성가체54%,구유유산탈경매보수결구역.기편마적단백이론분자량위3 5461.1Da ,유3개과막구화다개린산화위점,단백적이화성질교은정.예측유4개주요적B세포항원표위,L-유산탈경매활화위점지일적His192 포함우표위190-199aa중.매최화위점적3개관건안기산재공간결구상상호고근.PCR、쌍매절급DNA측서결과균표명중조질립pET-28a(+)-Ts LDH-A구건성공.SDS-PAGE결과표명목적 기인재대장간균BL-21/DE3중획득표체,경친화층석획득료고순도단백.중조단백능피Ts LDH-A중조단백면역적SD대서혈청、감염료저대조충적병인혈청급저혈청식별,표명기구유교호적면역원성화면역반응성.결론 응용생물신식방법종저대조충성충cDNA문고중사선출료Ts LDH-A적전장서렬병예측득도기편마단백적결구여공능방면적신식,해기인가재원핵표체계통중획득구유면역학활성적표체.
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.