CAJ | 학술논문

目的探讨血清Ⅱ型高尔基体膜蛋白(Golph2)在肝癌诊断中的价值.方法应用逆转录聚合酶链反应技术,从人肝癌细胞株WBF44钓取Golph2基因序列,聚合酶链反应回复突变制备全长完整基因(1206 bp),将其插入原核表达载体pET-21、转化大肠杆菌DH5α,应用异丙基硫代β-D-半乳糖苷诱导其表达,镍柱亲和层析纯化,并经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳鉴定.用纯化的蛋白质免疫家兔,制备多克隆抗血清.建立Westem blot鉴定重组蛋白和抗血清特异性,建立双抗体夹心酶联免疫吸附法测定150例肝癌、120例其他肝病、200名健康体检者血清Golph2水平.均值比较采用ONO-WAY ANOVA法. 结果从肝癌细胞中调取的基因,出现2个基因置换突变和1个碱基缺失突变,经回复突变,获得与NM 177937完全一致的序列.经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和Westem blot证实,重组Golph2与预期结果一致.抗血清能够特异地识别相对分子质量为5.2×104的重组蛋白质和7.3×104的血清蛋白质,与预期结果一致.建立的酶联免疫吸附试验方法检测肝癌、其他肝病患者、对照组血清Golph2,其吸光度值均数相差显著.以吸光度值≥0.40为阈值,其敏感度和特异性分别为44.5%和82.0%.结论 Golph2在各组人群中均有表达,在肝癌组最高,在肝癌诊断中有一定价值,但特异性和敏感度不高.
목적 탐토혈청Ⅱ형고이기체막단백(Golph2)재간암진단중적개치.방법 응용역전록취합매련반응기술,종인간암세포주WBF44조취Golph2기인서렬,취합매련반응회복돌변제비전장완정기인(1206 bp),장기삽입원핵표체재체pET-21、전화대장간균DH5α,응용이병기류대β-D-반유당감유도기표체,얼주친화층석순화,병경십이완기류산납-취병희선알응효전영감정.용순화적단백질면역가토,제비다극륭항혈청.건립Westem blot감정중조단백화항혈청특이성,건립쌍항체협심매련면역흡부법측정150례간암、120례기타간병、200명건강체검자혈청Golph2수평.균치비교채용ONO-WAY ANOVA법. 결과종간암세포중조취적기인,출현2개기인치환돌변화1개감기결실돌변,경회복돌변,획득여NM 177937완전일치적서렬.경십이완기류산납-취병희선알응효전영화Westem blot증실,중조Golph2여예기결과일치.항혈청능구특이지식별상대분자질량위5.2×104적중조단백질화7.3×104적혈청단백질,여예기결과일치.건립적매련면역흡부시험방법검측간암、기타간병환자、대조조혈청Golph2,기흡광도치균수상차현저.이흡광도치≥0.40위역치,기민감도화특이성분별위44.5%화82.0%.결론 Golph2재각조인군중균유표체,재간암조최고,재간암진단중유일정개치,단특이성화민감도불고.
Objective To study the correlation between hepatocellular carcinoma (HCC) and serum Golph2 protein. Methods Golph2 gene was cloned by RT-PCR using RNA from WBF44 cell line as template, the point mutations of the cloned sequence were corrected by PCR, then the gene (1206 bp) was cloned into pET-21 plasmid, and the resulted plasmid was transformed into E.coli DH5 α. The expression of6xHis and Golph2 fusion protein was induced by isopropylthio-β-D-galactoside (IPTG). The expression of fusion protein was detected by SDS-PAGE and Western blot, and was purified by Ni NTA chelating agarose.The rabbit antibody against Golph2 protein was obtained by immunizing 2 rabbits with the purified Golph2 protein. The specificity and titer of the antibody was determined by Western-blot and ELISA respectively.Sandwich ELISA was used to detect the level of serum Golph2 protein. Results There were two replacement mutation and 1 deletion mutation in the cloned sequence contrasted to NM 177937 in Genbank, including 644 (T → C, L → P), 970 (G → A, V → I) and 802 G deletion. The sequence was completely reversed by PCR.The sequence of Golph2 gene cloned into expression vector was confirmed by DNA sequencing. SDS-PAGE and Western blot analysis showed that Golph2 protein was expressed in E.coli DH5a. The antiserum could bind to the 52 kD recombinant protein and serum 73 kD protein specifically. The mean A450 value of ELISA for serum Golph2 protein were significantly higher in HCC and other liver diseases than that in control groups. The sensitivity and specificity for HCC were 44.5% and 82.0%, respectively, at the cut off value ≥0.40. Conclusion The polyclonal antibody against Golph2 protein is specific. The level of serum Golph2 is significantly higher in patients with HCC and other liver diseases than that in healthy controls.