RESUMEN
Aim To express and purify recombinant hCGH-CTP fusion protein in high-density suspension culture of Chinese hamster ovary cells (CHO-S), and to verify the lipid accumulation effect of rhCGH-CTP on 3T3-L1 mature adipocytes. Methods The recombinant protein expression vector (pcDNA3. 1-rhCGH-CTP) was constructed, achieved by fusing the human glycoprotein hormone beta 5/alpha 2 cDNA with CTP Linker. The expression plasmid was transiently transfected into the suspended CHO-S to express rhCGH-CTP protein and then purified, and the protein biological activity was verified. Intervention with 3T3-L1 mature adipocyte cells for 24 h was performed to detect the changes of intracellular triglyceride (TG) level. Results Western blot results showed that rhCGH-CTP protein was successfully expressed in CHO-S cells, and the yield was up to 715. 4 mg • L~ . The secreted protein was purified by AKTA pure system with higher purity that was up to 90% as identified by SDS-PAGE. In addition, the intracellular cAMP content of mature adipocytes with high expression of TSHR gene significantly increased after intervention with different concentrations of rhCGH-CTP protein by ELISA kit, indicating that rhCGH-CTP protein had biological activity. Oil red 0 staining showed that compared with the control group, the lipid content of mature adipocytes in the intervention groups with different concentrations of rhCGH-CTP protein significantly decreased (P < 0. 05) . Conclusions The rhCGH-CTP protein has been successfully expressed and purified with biological activity, and effectively reduce TG. This research provides an important theoretical basis for further revealing the physiological role of CGH protein and its potential application in clinical practice.
RESUMEN
Aim To express and purify rhα-Gal A with a 6 X His tag via using a serum-free expression system in high-density suspension culture of Chinese hamster ovary cells ( CHO-S) , and to verify the scavenging effect of rhα-Gal A on globular trisaccharide ceramide (Gb3 or GL3) . Methods The construction of recombinant protein expression vector, pcDNA4-GLA, was achieved by fusing the human α-galactosidase cDNA, gla, with 6 X His tag and artificial DNA synthesis. The expression plasmid was transfected into the suspended CHO-S to express rhα-Gal A and then purified. Following this procedure, we determined rhα-Gal A's expression, the enzymatic activity, and the glycosylation of the recombinant enzyme. Co-incubation with cultured cells was performed to examine whether rhα-Gal A could be taken up into the cells and effectively remove Gb3 substrates. Results rhα-Gal A was successfully expressed and purified after transiently transfecting pcDNA4-GLA into the suspended CHO-S, and the yield was up to (100 ±20. 6) mg • L
RESUMEN
<p><b>OBJECTIVE</b>To investigate the significance of early screening of pediatric developmental dysplasia of the hip (DDH) and congenital muscular torticollis (CMT) using ultrasonography and establish a simultaneous screening model for pediatric DDH and CMT.</p><p><b>METHODS</b>From January, 2013 to January, 2016, a total of 5060 pediatric patients with suspected DDH and CMT underwent ultrasonic examinations. The diagnostic results of the two diseases were classified into different clinical types, and Chi-square test was used to analyze the one-way relationship between different types of DDH and CMT; correspondence analysis was used for multivariate analysis of the variables. Chi-square test was used to analyze the difference between the detection rates in suspected CMT patients and the normal population.</p><p><b>RESULTS</b>GrafIIa type DDH was associated with mass-type CMT in the children (χ=331.800, P<0.001). DDH of GrafIIb, GrafIIc, Graf III, and Graf IV types were related with non-tumor type of CMT. The children with a suspected diagnosis of CMT showed a significantly higher detection rate of DDH than the normal subjects (χ=321.889, P<0.001).</p><p><b>CONCLUSION</b>DDH is closely related with CMT. Early simultaneous screening of DDH and CMT can help to improve the early diagnosis rate of CMT in children.</p>