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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2023, Vol. 43 Issue (1): 27-34    DOI: 10.13523/j.cb.2207053
技术与方法     
子痫前期循环系统标志物sFlt-1的重组表达及化学发光检测方法的建立
林月阳,柯文锋,任和,白仲虎*()
江南大学粮食发酵与食品生物制造国家工程研究中心 无锡 214122
Recombinant Expression of sFlt-1, a Marker of Circulatory System in Preeclampsia, and Establishment of a Chemiluminescence Detection Method
LIN Yue-yang,KE Wen-feng,REN He,BAI Zhong-hu*()
National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing, Jiangnan University, Wuxi 214122, China
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摘要：

目的: 表达可溶性fms样酪氨酸激酶-1(soluble fms-like tyrosine kinase-1,sFlt-1)重组蛋白,建立针对sFlt-1的化学发光检测方法。方法: 构建pcDNA3.4-sFlt-1-His*6表达载体,转染至HEK293细胞进行重组表达。利用链霉亲和素-生物素亲和体系及双抗体夹心法,实现对sFlt-1的定量检测。结果: 该方法的最低检测限为2 pg/mL,线性范围为20~40 000 pg/mL,平均回收率为92%,批内及批间精密度变异系数(variable coefficient,CV)均小于10%,与珀金埃尔默sFlt-1检测试剂盒进行比对的相关系数R2为0.925 2。结论: 获得了具有天然生物活性的sFlt-1蛋白,建立了具有良好性能的sFlt-1检测方法,后续进一步开发可应用于子痫前期的诊断筛查。
关键词： 子痫前期 sFlt-1;  重组表达;  化学发光免疫分析    
Abstract:

Objective: To express soluble fms-like tyrosine kinase-1 (sFlt-1) recombinant protein and establish a chemiluminescence immunoassay detection method for sFlt-1. Methods: The pcDNA3.4-sFlt-1-His*6 expression vector was constructed and transfected into HEK293 cells for recombinant expression of sFlt-1. Using streptavidin-biotin affinity system and double antibody sandwich method, the quantitative detection of sFlt-1 was realized. Results: The minimum detection limit of the method is 2 pg/mL, the linear range is 20 ~ 40 000 pg/mL, the average recovery is 92%, the intra-assay and inter-assay precision coefficients of variation (CV) are less than 10%, and the correlation coefficient R2 compared with the PerkinElmer diagnostic kit is 0.925 2. Conclusion: The sFlt-1 protein with natural biological activity was successfully recombinantly expressed, and a sFlt-1 CLIA detection method with good performance was established, which can be further developed and applied to the diagnosis and screening of preeclampsia.
Key words: Preeclampsia sFlt-1    Recombinant expression    Chemiluminescence immunoassay
收稿日期: 2022-07-25 出版日期: 2023-02-14
ZTFLH:  R446  
通讯作者: *白仲虎 电子信箱:baizhonghu@jiangnan.edu.cn   
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引用本文:

林月阳, 柯文锋, 任和, 白仲虎. 子痫前期循环系统标志物sFlt-1的重组表达及化学发光检测方法的建立[J]. 中国生物工程杂志, 2023, 43(1): 27-34.

LIN Yue-yang, KE Wen-feng, REN He, BAI Zhong-hu. Recombinant Expression of sFlt-1, a Marker of Circulatory System in Preeclampsia, and Establishment of a Chemiluminescence Detection Method. China Biotechnology, 2023, 43(1): 27-34.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2207053    或    https://manu60.magtech.com.cn/biotech/CN/Y2023/V43/I1/27

图1  重组质粒双酶切验证及重组蛋白SDS-PAGE验证
Dilution factor of sFlt-1 Relative light unit
2 117 397 206
10 32 820 336
100 3 380 248
1 000 308 014
Negative control 168 219
表1  sFlt-1生物活性检测
图2  sFlt-1抗体组合的筛选结果
Sample
/(pg·
mL-1)		 Ab-HRP/(μg·mL-1) Ab-HRP/(μg·mL-1) Ab-HRP/(μg·mL-1)
Ab-biotin
/(μg·mL-1)		 Ab-biotin
/(μg·mL-1)		 Ab-biotin
/(μg·mL-1)		 Ab-biotin
/(μg·mL-1)		 Ab-biotin
/(μg·mL-1)		 Ab-biotin
/(μg·mL-1)		 Ab-biotin
/(μg·mL-1)		 Ab-biotin
/(μg·mL-1)		 Ab-biotin
/(μg·mL-1)
587 864 805 833 905 781 940 1 565 258 1 563 540 1 470 320 2 308 847 2 367 402 2 245 319
1 396 2 293 985 2 445 753 2 220 136 3 857 769 3 866 457 4 216 600 5 510 167 6 652 155 5 580 414
2 285 3 662 354 3 218 309 3 312 971 6 159 777 6 338 059 5 943 730 7 620 010 8 486 838 7 765 782
表2  抗体组合使用质量浓度的筛选
图3  反应体系孵育时间的变化对信号值的影响
Object Ratio of sample
concentrations		 Ratio of sample RLU for different incubation times
10 min 20 min 30 min
Sample 2/Sample 1 2.4 2.9 2.7 2.6
Sample 3/Sample 2 1.6 1.6 1.4 1.5
Sample 3/Sample 1 3.9 4.5 3.8 3.9
S3/S2 2.5 2.8 2.6 2.5
S4/S3 2.5 2.8 2.7 2.7
S5/S4 4.0 3.8 3.4 3.1
S6/S5 4.0 3.1 3.2 3.2
S6/S1 100.0 91.5 77.3 69.1
表3  不同孵育时间对样本发光值拉开比例的影响
Sample The oretical
concentration
/(pg·mL-1)		 Detection
concentration
/(pg·mL-1)		 Recovery
rate/%
Sample 1 5 144.56 4 689.21 90
Sample 2 9 456.17 8 903.73 94
表4  准确度评价
Proposed
concentration
/(pg·mL-1)		 Batch 1 Batch 2 Batch 3 Inter-assay precision
sFlt-1
/(pg·mL-1)		 CV/% sFlt-1
/(pg·mL-1)		 CV/% sFlt-1
/(pg·mL-1)		 CV/% sFlt-1
/(pg·mL-1)		 CV/%
1 215 1 251.32 5.71 1 170.46 6.37 1 260.00 4.69 1 227.26 6.42
21 824 21 783.53 3.04 20 899.19 3.26 22 083.88 2.96 21 588.87 3.83
表5  精密度评价
Theoretical concentration/(ng·mL-1) Measured concentration/(ng·mL-1) Deviation/%
0.020 0.019 -4.26
0.578 0.600 3.82
1.136 1.239 9.07
2.251 2.099 -6.76
4.483 4.319 -3.64
11.177 10.121 -9.44
22.333 24.245 8.56
33.490 31.543 -5.81
44.646 40.846 -8.50
表6  sFlt-1检测方法线性范围评价
图4  检测方法的线性范围拟合结果
图5  本研究方法与珀金埃尔默检测结果的相关性
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