CAJ | 학술논문

参考GenBank收录的单增李斯特菌Hly基因序列，设计1对引物，采用PCR技术扩增出单增李斯特氏菌的溶血素基因Hly（不含有信号肽部分），得到一条1590bp的条带。将其连入pMD18-T载体，经酶切、PCR鉴定和序列测定法进行鉴定。测序正确后，将该基因插入到pET-28a中构建原核表达载体pET-28a—sH1y，将重组质粒转化到大肠杆菌BL21（DE3），经IPTG诱导，将诱导产物用SDS-PAGE和Western-blot鉴定。结果显示，Hly基因可以在大肠杆菌中获得表达，表达产物分子质量约为65kU，与预期蛋白质分子质量大小一致。经Western、blotting鉴定可知，诱导表达产物以可溶形式存在，可被兔抗LM阳性血清特异识别，具有较好的抗原活性，为进一步研制基于溶解素蛋白的诊断抗原和特异性单克隆抗体，开展LM的致病与免疫机理研究奠定基础。
삼고GenBank수록적단증리사특균Hly기인서렬，설계1대인물，채용PCR기술확증출단증리사특씨균적용혈소기인Hly（불함유신호태부분），득도일조1590bp적조대。장기련입pMD18-T재체，경매절、PCR감정화서렬측정법진행감정。측서정학후，장해기인삽입도pET-28a중구건원핵표체재체pET-28a—sH1y，장중조질립전화도대장간균BL21（DE3），경IPTG유도，장유도산물용SDS-PAGE화Western-blot감정。결과현시，Hly기인가이재대장간균중획득표체，표체산물분자질량약위65kU，여예기단백질분자질량대소일치。경Western、blotting감정가지，유도표체산물이가용형식존재，가피토항LM양성혈청특이식별，구유교호적항원활성，위진일보연제기우용해소단백적진단항원화특이성단극륭항체，개전LM적치병여면역궤리연구전정기출。
In this study, the Hly gene ofListeria monocytogenes train C53005 was amplified by PCR using designed primers. Then, the gene was cloned into pMD18-T vector, and indentified by digestion with restriction endonuclease, PCR and sequencing. After the sequences were confirmed correct, a prokaryotic expression vector was constructed by inserting the gene into pET-28a vector. The recombinant plasmid pET-28a- silly was transformed into E.coli BL21 （DE3） competent cells, and was induced with 1PTG. The expressed product was analysed by SDS-PAGE and Western-blot. In result, the Hly gene was highly expressed in E.coli and the expressed protein was 65 Ku in size as expected. Western-blot test demonstrated that the expressed protein could specifically react with rabbit anti-LM serum. The results showed the expressed protein was in soluble form with satisfactory antigenicity.