RESUMO
Objective:To construct a prokaryotic expression vector for human retinol binding protein 4 (hRBP4) that allows technicians to obtain hRBP4 purified protein with low cost, high efficiency, high concentration and high purity.Methods:The hRBP4 coding sequence provided by National Center for Biotechnology Information was optimized by E. coli codons, and a synthetic DNA fragment was cloned into the PET-28A (+) prokaryotic expression vector to construct a recombinant hRBP4 expression plasmid. The recombinant protein was transformed into E. coli BL21, and the induced expression conditions (temperature, rotate speed and isopropyl β-d-thiogalactoside concentration) were optimized. The recombinant protein was purified by His fusion tag. Results:The recombinant hRBP4 prokaryotic expression plasmid was successfully constructed, and the expression concentration and induction temperature of the recombinant protein were optimized. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that a band with a relative molecular weight of 26 000 daltons was clearly visible in the purified product. The purified hRBP4 protein could be detected clinically, and there was a good linear relationship between the dilution ratio and the detection concentration.Conclusions:The recombinant hRBP4 protein has high purity, high concentration, and short production cycle. It has the potential to become a candidate for reference materials for laboratory quality evaluations.
RESUMO
Objective:To develop a self-made plasma quality control material for non-invasive prenatal testing (NIPT) and evaluate its performance.Methods:139 NIPT-negative maternal plasmas stored in the genetic department of Shaoxing maternal and child health hospital from January 1, 2019 to June 30, 2021 were divided into male groups (19 cases) and female groups (120 cases) according to the neonatal gender. 9360 cases from September 2020 to September 2021 were enrolled as clinical validation cases.First step, 200 μl plasma from a 47 years-old non-pregnant healthy women was used as a matrix. Different amounts (0.1, 0.2, 0.5, 2.5, and 5 μl) of positive DNA from fetal chromosome aneuploidy (T21, T18, T13) detection kit were added. The appropriate volume of positive DNA was 0.5 μl according to the test results. Second step,Plasma in male and female group was treated as matrix. 0.5 μl positive DNA was added per 205 μl. Plasma matrix from female group showed good repeatability and the sensitivity was 100%.Third step, evaluate the self-made plasma quality control material, including storage stability, matrix uniformity and repeatability, and the effect of different batch numbers of positive DNA, by calculating Z score and the CV of fetal DNA concentration (FF).Results:Plasma matrix from female group showed good repeatability and the sensitivity was 100%, while the sensitivity of male group was only 84%. The CV of FF in female matrix was 3.9% in the repetitive experiments. After adding 0.5 μl positive DNA, the mean FF of self-made positive plasma quality control was 5.63%±0.42%, Z values>6, and the CV was 7% after storage of three months. Considering the concentration variation of positive DNA in different lots, 1 μl of positive DNA should be added when the FF of positive DNA is lower than 10%.Used in 9360 clinical cases from September 2020 to September 2021, all positive plasma quality control materials showed positive results, and the positive predictive value of trisomy 21 was 100%.Conclusions:The NIPT self-made positive plasma quality control material has been successfully developed in this study. The preliminary experimental results show that it has good repeatability and stability, which is suitable for clinical application.