CAJ | 학술논문

目的：从恶性疟原虫FCC1／HN株环子孢子蛋白（circumsporozoite protein，CSP）的全长编码基因中扩增4个CSP基因编码片段，克隆至原核表达载体pET28 a／EGFP 中进行表达纯化，观察环子孢子蛋白肽段与EGFP融合蛋白对肝细胞的靶向结合能力，探讨其作为原发性肝癌基因治疗的靶向分子载体的可行性。方法根据恶性疟原虫FCC1／HN株环子孢子蛋白的编码序列设计4对引物，利用聚合酶链反应（ PCR ）扩增出4段CSP 的编码基因，将其克隆到原核表达载体pET28 a／EGFP中，与EGFP融合表达，在大肠杆菌BL21中用IPTG诱导表达，表达产物用Ni2＋螯合柱亲和纯化，采用SDS-PAGE对纯化的融合蛋白检测；并观察重组环子孢子蛋白对不同组织细胞的结合能力。结果从恶性疟原虫FCC1／HN株CSP基因中成功扩增到4段分别为300、90、120、80 bp的基因片段，且在原核表达载体pET28 a／EGFP中经诱导表达出相对分子质量约39×103、31×103、33×103和30×103大小的融合蛋白： CSR1a-EGFP、CSR1b-EGFP、CSR2a-EGFP及CSR2b-EGFP；通过Ni2＋亲和柱纯化获得重组CSP融合蛋白，能被疟原虫阳性血清特异识别，且CSR2 a-EGFP能够与肝癌和正常肝细胞特异性地结合，与其他组织来源的细胞则未见反应。结论重组环子孢子蛋白CSR2a-EGFP能够与肝组织特异性地结合，作为原发性肝癌基因治疗的靶向分子具有一定的潜在应用价值。
목적：종악성학원충FCC1／HN주배자포자단백（circumsporozoite protein，CSP）적전장편마기인중확증4개CSP기인편마편단，극륭지원핵표체재체pET28 a／EGFP 중진행표체순화，관찰배자포자단백태단여EGFP융합단백대간세포적파향결합능력，탐토기작위원발성간암기인치료적파향분자재체적가행성。방법근거악성학원충FCC1／HN주배자포자단백적편마서렬설계4대인물，이용취합매련반응（ PCR ）확증출4단CSP 적편마기인，장기극륭도원핵표체재체pET28 a／EGFP중，여EGFP융합표체，재대장간균BL21중용IPTG유도표체，표체산물용Ni2＋오합주친화순화，채용SDS-PAGE대순화적융합단백검측；병관찰중조배자포자단백대불동조직세포적결합능력。결과종악성학원충FCC1／HN주CSP기인중성공확증도4단분별위300、90、120、80 bp적기인편단，차재원핵표체재체pET28 a／EGFP중경유도표체출상대분자질량약39×103、31×103、33×103화30×103대소적융합단백： CSR1a-EGFP、CSR1b-EGFP、CSR2a-EGFP급CSR2b-EGFP；통과Ni2＋친화주순화획득중조CSP융합단백，능피학원충양성혈청특이식별，차CSR2 a-EGFP능구여간암화정상간세포특이성지결합，여기타조직래원적세포칙미견반응。결론중조배자포자단백CSR2a-EGFP능구여간조직특이성지결합，작위원발성간암기인치료적파향분자구유일정적잠재응용개치。
Objective To amplify four fragments of circumsporozoite ( CSP) protein gene from Plasmodium falciparum FCC1/HN strain and express recombinant CSP proteins in prokaryotic vector pET28 a/EGFP for further evaluation of their binding activities to hepatic cells .Methods Four pairs of primers were designed according to the cDNA sequence of CSP protein from Plasmodium falciparum FCC1/HN strain and used to amplify the gene fragments by PCR .The cloned gene fragments were inserted into pET28a/EGFP to construct the recombinant expression plasmids .The transformed E.coli BL21 strains carry-ing expression plasmids were induced by IPTG to express CPS proteins .The recombinant CSP proteins were purified with Ni2+chelating HiTrap HP column and detected by SDS-PAGE.The binding activities of the ex-pressed proteins to different tissues were also detected .Results Four gene fragments encoding CSP protein were successfully amplified and expressed in E.coil BL21 strain as fusion protein CSR1a-EGFP, CSR1b-EGFP, CSR2a-EGFP and CSR2b-EGFP with a relative molecular mass of about 39×103, 31×103, 33×103 and 30 ×10 3 , respectively .The purified fusion proteins reacted specifically with Plasmodium falciparum-posi-tive serum samples.Moreover, the recombinant protein CSR2a-EGFP could bind to the hepatic cells specif-ically rather than other cells.Conclusion The purified recombinant CSR2a-EGFP protein has a strong binding activity to hepatocytes , indicating its potential value as a targeting molecule for hepatic gene therapy .