ABSTRACT
Objective To establish a prokaryotic expression system of interstitial lung disease associated autoantigen human bactericidal/permeability-increasing fold-containing B1 (BPIFB1), providing tools for the study on its function in immune responese. Methods The coding region of BPIFB1 gene was amplified with specific primers from recombinant pGEM-C20ORF114 plasmid and cloned into the pET28a-MBP-His and pGEX-5X-1 vectors. The recombinant pET-BPIFB1-MBP-His and pGEX-BPIFB1-GST plasmids were transfected into Top10 cells. The positive clones were selected and sequenced. The correct clones of pET-BPIFB1-MBP-His and pGEX-BPIFB1-GST were transfected into prokaryotic expression strain Rosetta (DE3) and induced by Isopropyl β-D-Thiogalactoside (IPTG). The expression of recombinant BPIFB1 fusion protein was analyzed by SDS-PAGE and Western blotting, and purified by urea modified and renaturation and affinity chromatography of nickel NTA-resin. Results The polymerase chain reaction (PCR) produced specific product with the molecular weight equivalent to that of BPIFB1. The recombinant pET-BPIFB1-MBP-His and pGEX-BPIFB1-GST plasmids were cloned by double restriction enzyme digestion and ligation and confirmed by sequencing. The SDS-PAGE result showed that both BPIFB1-MBP and BPIFB1-GST fusion proteins were mainly expressed in the form of inclusion bodies. The Western blotting result revealed that the recombinant BPIFB1-MBP-His protein could be recognized by Anti-6 ×His antibody. The purified soluble BPIFB1-MBP fusion protein was obtained by urea denaturation, affinity chromatography of nickel NTA-resin and then renaturation after purification. Conclusion The BPIFB1 prokaryotic expression system is established by construct recombinant plasmid pET-BPIFB1-MBP-His, and an approach of renaturation after nickel resin affinity purification in denatured condition.
ABSTRACT
ObjectiveTo evaluate the influence of different hemoperfusion (HP) intensity on 7-day and 28-day mortality for patients with paraquat (PQ) poisoning, and examine the factors that may affect the decision of the clinicians to prescribe a high intensity HP.Methods A retrospective cohort study was conducted. The patients admitted to the department of critical care medicine of Anhui Provincial Hospital Affiliated to Anhui Medical University with the diagnosis of PQ poisoning from August 2012 to August 2014, fulfilling the following criteria were enrolled in the study: older than 18 years, interval from ingestion PQ to hospital admission shorter than 12 hours, and receiving HP treatment within 24 hours, and expecting surviving time exceeding 24 hours after admission, and data of the patients available for at least 28 days after admission. Depending on the intensity of HP, patients were assigned to either lower intensity HP group (LHP, defined as receiving HP for less than 4 hours, 2 columns) or higher intensity HP group (HHP, defined as receiving HP longer than 6 hours, 3 columns). Patients' data were retrieved from hospital's electronic database after hospital admission, and the results at 7th day and 28th day were recorded. Multiple logistic regression model was used to determine factors with which the clinician decided to choose the intensity of HP for the patients, and Cox regression model was used to evaluate 7-day and 28-day mortality.Results Data of 60 patients was finally available for this study. LHP group consisted of 28 patients, with a 7-day mortality of 53.6%(15 patients) and 28-day mortality of 64.3% (28 patients); 32 patients were assigned to HHP group with 7-day mortality of 43.8% (14 patients) and 28-day mortality of 62.5% (20 patients). Twenty-eight patients constituted as the HHP group, with higher PQ concentration in plasma, higher incidence of respiratory alkalosis and acute kidney injury (AKI), and higher level of lactate (Lac) compared with LHP group. However, a lower 7-day mortality was observed in the HHP group. Multiple logistic regression model indicated that at admission, interval from ingestion PQ to hospital admission longer than 4 hours [odds ratio (OR) = 1.461, 95% confidence interval (95%CI) = 1.132-1.435,P 10×109/L (OR = 1.222, 95%CI = 1.032-1.275, P = 0.018), Lac> 2.0 mmol/L (OR = 2.392, 95%CI = 2.090-2.734,P 50 years old (HR = 1.282, 95%CI = 1.050-1.530,P = 0.043), PQ concentration increased by 1 mg/L (HR = 2.521, 95%CI = 2.371-3.825,P = 0.012), AKI on admission (HR = 3.850, 95%CI = 2.071-5.391,P10×109/L (HR = 1.932, 95%CI = 1.782-2.171,P = 0.006), Lac> 2.0 mmol/L (HR = 2.981, 95%CI =2.210-3.792,P = 0.002), and PaCO2< 35 mmHg (HR = 1.772, 95%CI = 1.483-2.516,P = 0.008; 1 mmHg =0.133 kPa) were independent risk factors for 28-day mortality.Conclusions Though HHP was helpful in lowering mortality rate in patients with PQ poisoning within 7 days, it did not influence on 28-day mortality. Clinicians' decisions on HP intensity need further investigation, and more perfect clinical evaluation system is required for reasonable use of expensive medical resources such as HP.