摘要目的 构建人铁蛋白轻链基因的原核表达载体,并评价与探讨能在大肠埃希菌(E.coli)中过表达的人铁蛋白轻链所制备的基因rFL质控品的均匀性、稳定性和精密度以及应用价值.方法 采用RT-PCR从人新鲜全血总RNA克隆铁蛋白轻链(ferritin L)基因,构建亚克隆质粒pGM-T/ferritin L;用测序鉴定后,将重组质粒pET-30a/ferritin L转化E.coli BL21(DE3),经异丙基硫代-β-D半乳糖苷(IPTG)诱导rFL表达;采用SDS-PAGE对rFL进行鉴定,用WB分析其抗原性,ACCESS免疫分析仪测定其浓度.同时,采用随机数表从制备的rFL质控品中抽取15支,用单因素方差分析评价质控品的均匀性;用一元线性回归分析评价质控品的稳定性,再根据评价结果计算均匀性和稳定性的不确定度,然后进行精密度评价.结果 构建的铁蛋白轻链原核表达体系经测序显示,扩增的ferritin L基因片段与GenBank中的ferritin L编码序列(NM_000146.3)一致;PCR扩增出与ferritin L DNA的527 bp相符的片段;用IPTG诱导表达后,可得到表达量高的相对分子质量为19 000的rFL,WB证明其具有良好的抗原性;用ACCESS测定rFL稀释1 000倍后的浓度为(1 115.84±38.38) ng/ml.均匀性评价结果显示,高低2个浓度基因rFL质控品样本内和样本间的差异均无统计学意义(高浓度和低浓度基因rFL质控品的F值分别为2.336、0.730,P均>0.05).稳定性结果表明,质控品样本随贮存时间(5个半月)变化的线性趋势差异无统计学意义(F=1.755,P>0.05).精密度评价为高浓度基因rFL质控品批内CV为2.06%,批间CV为2.12%,日间CV为0%,总CV为2.96%;低浓度基因rFL质控品批内CV为2.03%,批间CV为2.08%,日间CV为0%,总CV为2.90%.结论 基因rFL质控品的均匀性、稳定性及精密度符合临床检测要求,可成为具有我国自主知识产权的铁蛋白质控品,同时也为铁蛋白混合质控品的制备提供了原料.

abstractsObjective To construct a prokaryotic expression system of human ferritin L which could be overexpressed in E.coli, and then prepare quality control materials of rFL and evaluate the homogeneity, stability, precision and application value of the rFL.Methods RT-PCR was used to clone ferritin L gene with total RNA from peripheral blood.Ferritin L gene was inserted into plasmid pGM-T to construct subclone plasmid pGM-T/ferritin L, which was identified by sequencing.The recombinant plasmid pET-30a/ferritin L was transformed into E.coli BL21 (DE3) and efficiently expressed under IPTG induction.rFL was identified by SDS-PAGE, and its antigenicity was examined by WB.The concentration of rFL was measured by ACCESS immunoassay system.Fifteen control samples were randomly selected using random number table.The homogeneity and stability of rFL were evaluated by one-way analysis of variance (ANOVA) and a linear regression model, respectively.The uncertainties of homogeneity, stability and the precision were evaluated.Results A prokaryotic expression system of ferritin L was successfully constructed.Sequence analysis showed that the ferritin L gene inserted was identical with the one from GenBank(NM_000146.3).The PCR products were 527 bp long, in complete agreement with length of ferritin L gene.The molecular weight of rFL highly expressed in E.coli induced by IPTG was 19 000 Da.The WB analysis indicated that this rFL protein had good antigenicity.The concentration of rFL(1 000 times dilution) measured by ACCESS immunoassay system was (1115.84±38.38) ng/ml.Homogeneity evaluation of low-concentration QC sample and high-concentration QC sample of rFL showed that there were no statistical significances in the within-groups and between-groups (for high-concentration F=2.336, P>0.05 and for low-concentration F=0.730, P>0.05).A linear regression based on the stability test indicated that there was no statistically significant trend of instability in five and a half months (F=1.755, P>0.05). Precision analysis showed the within-run CV, the between-run CV, the between-day CV and the total CV of high-concentration QC sample were 2.06%, 2.12%, 0% and 2.96% respectively; the within-run CV, the between-run CV, the between-day CV and the total CV of low-concentration QC sample 2.03%, 2.08%, 0% and 2.90%, respectively.Conclusion This rFL for quality control materials with independent intellectual property rights could meet the clinical requirement of homogeneity, stability and precision, and could provide raw materials for preparation of mixed ferritin for quality control.