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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2023, Vol. 43 Issue (1): 27-34    DOI: 10.13523/j.cb.2207053
    
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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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     
Received: 25 July 2022      Published: 14 February 2023
ZTFLH:  R446  
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Yue-yang LIN
Wen-feng KE
He REN
Zhong-hu BAI
Cite this article:

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.

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

Fig.1 Double-enzyme digestion verification of recombinant plasmid and SDS-PAGE verification of recombinant protein (a) M: DNA marker; 1: Enzyme cleavage product (b)M: Protein marker; 1: Purified recombinant protein
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
Table 1 Detection of biological activity of sFlt-1
Fig.2 Screening results of sFlt-1 antibody combinations (a) Eight antibody combinations were screened with high and low value serum (b) Serum correlation of antibody combination H
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
Table 2 Screening of used concentrations of antibody combination
Fig.3 The effect of the change in the incubation time of the reaction system on the signal value
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
Table 3 The effect of different incubation times on the pull-off ratio of the luminescence value of the samples
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
Table 4 Accuracy evaluation
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
Table 5 Precision evaluation
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
Table 6 Validation of linear range of sFlt-1 detection method
Fig.4 Linear range fitting results for the detection method
Fig.5 Correlation of this method with PerkinElmer detection results
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