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目的通过去除降钙素原(PCT)的降解位点,获得重组高稳定性PCT抗原,提高PCT免疫诊断试剂的准确性。方法设计降解位点缺失的PCT抗原序列,采用基因工程技术纯化该重组PCT抗原。采用加速热稳定性试验和荧光免疫层析检测技术评价该抗原在诊断试剂中的应用。结果加速热稳定性试验显示:37℃破坏3 d后,重组PCT抗原和完整抗原的稳定率分别为89.99%±0.25%、19.46%±0.73%;破坏7 d后,两者的稳定率分别为62.53%±0.41%、5.67%±0.07%;重组PCT抗原的稳定性显著高于完整PCT抗原,差异有统计学意义(P<0.01)。采用重组PCT抗原制备标准品可通过二次多项式方程与荧光强度进行有效拟合,两者呈正相关(r=0.991 3,P<0.01)。结论研制的重组降钙素原具有较高稳定性,在PCT定量检测中具有很好的应用前景。
OBJECTIVE To improve the accuracy of PCT immunodiagnostic reagents by removing the degradation site of procalcitonin (PCT) and obtaining recombinant high-stability PCT antigens. METHODS: The PCT antigen sequence deleted from the site of degradation was designed and the recombinant PCT antigen was purified by genetic engineering. Accelerated thermal stability test and fluorescence immunochromatographic test were used to evaluate the application of this antigen in diagnostic reagents. Results The accelerated thermal stability test showed that the stability rates of recombinant PCT antigen and intact antigen were 89.99% ± 0.25% and 19.46% ± 0.73%, respectively, after 3 days of destruction at 37 ℃. After 7 days of destroying, the stability rates of the two were 62.53% ± 0.41% and 5.67% ± 0.07%, respectively. The stability of recombinant PCT antigen was significantly higher than that of intact PCT antigen (P <0.01). The preparation of standard with recombinant PCT antigen can be fitted by the quadratic polynomial equation with the fluorescence intensity, and there is a positive correlation between them (r = 0.991 3, P <0.01). Conclusion The developed recombinant procalcitonin has high stability and has a good application prospect in the quantitative detection of PCT.