CAJ | 학술논문

本实验采用RT-PCR和cDNA末端快速扩增((RACE)技术克隆得到了虾夷马粪海胆(Strongylocentrotusinter-medius)溶菌酶(LYZ)基因的全长cDNA序列。结果表明，虾夷马粪海胆LYZ基因全长为912by，含有1个480by的开放阅读框(ORF)，编码159个氨基酸，其中第1-20个氨基酸为信号肤，蛋白计算分子量为17.69kD，等电点为7.750氨基酸比对分析表明，虾夷马粪海胆LYZ基因与紫球海胆(Strongylocentrotuspurpuratus)和刺参(Apostichopusjaponicus)的i型LYZ基因相似百分比分别为91.4%和59.3%，并且含有i型LYZ基因的保守序列DVGSLSCGP(Y)Y(F)QIK，所以推断本实验克隆的溶菌酶为i型。采用实时定量PCR方法，以户actin为内标，对其在虾夷马粪海胆各组织中的表达进行研究，发现LYZ基因在围口膜中表达量最高，其次是齿间肌、管足、肠、体腔液、雄性性腺和雌性性腺。利用脂多糖(LPS)刺激虾夷马粪海胆，取刺激后不同时间的海胆体腔液，对该基因的表达差异进行分析。结果表明，虾夷马粪海胆的LYZ基因在LPS刺激后8h时表达量最高，12h时开始逐步回落，至36h时回落至对照组相近水平。本结果可为虾夷马粪海胆免疫学研究及抗病相关分子标记的开发提供参考依据。
본실험채용RT-PCR화cDNA말단쾌속확증((RACE)기술극륭득도료하이마분해담(Strongylocentrotusinter-medius)용균매(LYZ)기인적전장cDNA서렬。결과표명，하이마분해담LYZ기인전장위912by，함유1개480by적개방열독광(ORF)，편마159개안기산，기중제1-20개안기산위신호부，단백계산분자량위17.69kD，등전점위7.750안기산비대분석표명，하이마분해담LYZ기인여자구해담(Strongylocentrotuspurpuratus)화자삼(Apostichopusjaponicus)적i형LYZ기인상사백분비분별위91.4%화59.3%，병차함유i형LYZ기인적보수서렬DVGSLSCGP(Y)Y(F)QIK，소이추단본실험극륭적용균매위i형。채용실시정량PCR방법，이호actin위내표，대기재하이마분해담각조직중적표체진행연구，발현LYZ기인재위구막중표체량최고，기차시치간기、관족、장、체강액、웅성성선화자성성선。이용지다당(LPS)자격하이마분해담，취자격후불동시간적해담체강액，대해기인적표체차이진행분석。결과표명，하이마분해담적LYZ기인재LPS자격후8h시표체량최고，12h시개시축보회락，지36h시회락지대조조상근수평。본결과가위하이마분해담면역학연구급항병상관분자표기적개발제공삼고의거。
A full-length cDNA coding lysozyme (LYZ) was cloned from sea urchin Strongylocentrotus intermedius by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends(RACE) methods. The full-length cDNA of LYZ was 912 by with a 480 by open reading frame(ORF) encoding 159 amino acids, which included a signal peptide of 20 amino acids at the N-terminus and a mature peptide of 139 amino acids. The deduced amino acid sequence had a putative size of 17.69 kD and the theoretical isoelectric point was 7.75. The multiple alignments revealed identity of 91.4% between S. intermedius and S. pu}pu}atus and 59.3% identity between S. intermedius and Apostichopus japonicus in the i-type LYZ amino acid sequence. The i-type lysozyme conserved sequence DVGSLSCGP(Y)Y(F)QIK was detected in the S. intermedius amino acid sequence. These results indicate that the cDNA sequence cloned from S. intermedius is a member of the i-type lysozyme family. Real-time quantitative PCR was carried out to measure LYZ mRNA expression in different tissues and monitor LYZ mRNA expression patterns in the coelomic fluid after a lipopolysaccharide (LPS) challenge. LYZ mRNA expression levels in peristome membrane were significantly higher than those of muscle in the Aristotle's lantern, tube-foot, intestines, coelomic fluid, male gonad, and female gonad (P<0.05). LYZ expression in the coelomic fluid was up-regulated after the LPS challenge and reached its maximum level at 8 h post-stimulation, and decreased gradually thereafter. Thirty-six hours after the LPS challenge, LYZ levels in the coelomic fluid were similar to those of the control.