Identification and validation of novel biomarkers for cold-dampness syndrome of rheumatoid arthritis based on integration of multiple bioinformatics methods / 中国中药杂志

This study aims to identify the novel biomarkers of cold-dampness syndrome(RA-Cold) of rheumatoid arthritis(RA) by gene set enrichment analysis(GSEA), weighted gene correlation network analysis(WGCNA), and clinical validation. Firstly, transcriptome sequencing was carried out for the whole blood samples from RA-Cold patients, RA patients with other traditional Chinese medicine(TCM) syndromes, and healthy volunteers. The differentially expressed gene(DEG) sets of RA-Cold were screened by comparison with the RA patients with other TCM syndromes and healthy volunteers. Then, GSEA and WGCNA were carried out to screen the key DEGs as candidate biomarkers for RA-Cold. Experimentally, the expression levels of the candidate biomarkers were determined by RT-qPCR for an independent clinical cohort(not less than 10 cases/group), and the clinical efficacy of the candidates was assessed using the receiver operating characteristic(ROC) curve. The results showed that 3 601 DEGs associated with RA-Cold were obtained, including 106 up-regulated genes and 3 495 down-regulated genes. The DEGs of RA-Cold were mainly enriched in the pathways associated with inflammation-immunity regulation, hormone regulation, substance and energy metabolism, cell function regulation, and synovial pannus formation. GSEA and WGCNA showed that recombinant proteasome 26S subunit, ATPase 2(PSMC2), which ranked in the top 50% in terms of coefficient of variation, representativeness of pathway, and biological modules, was a candidate biomarker of RA-Cold. Furthermore, the validation results based on the clinical independent sample set showed that the F1 value, specificity, accuracy, and precision of PSMC2 for RA-Cold were 70.3%, 61.9%, 64.5%, and 81.3%, respectively, and the area under the curve(AUC) value was 0.96. In summary, this study employed the "GSEA-WGCNA-validation" integrated strategy to identify novel biomarkers of RA-Cold, which helped to improve the TCM clinical diagnosis and treatment of core syndromes in RA and provided an experimental basis for TCM syndrome differentiation.

Prokaryotic expression,purification and identification of low-toxic staphylococcal enterotoxin A with D227A mutation / 中国免疫学杂志

Objective:To obtain recombinant D227A mutation Staphylococcal enterotoxin A protein(rSEAD227A) with low toxicity but still retain its immunological activity and high purity. Methods: The SEA gene containing D227A mutation was cloned by PCR. By constructing pET44a-SEAD227Avector and transfecting the expression strain Rosetta, inclusion bodies were solubilized with guanidium hydrochloride and refolded by gradient dialysis;proteins were purified using StrepⅡ affinity chromatography,and identified by Western blot and high performance liquid chromatography-mass spectrometry(LC-MS/MS). Results: The D227A mutation of SEA was cloned and the expression system of Rosetta-rSEAD227Awas constructed. The purified rSEAD227Aprotein was obtained by refolding with gradient dialysis and affinity purification. LC-MS/MS analysis confirmed that the tryptic digested rSEAD227A peptide sequences matched the sequences of SEA in the database. Conclusion: The rSEAD227A protein in high purity was obtained,which provided the ex-perimental basis for further basic research and clinical application of SEA.

Strong Expression of Recombinant Human Morphogenetic Protein-4 in Escherichia coli and its Bioassay in vivo / 中国生物工程杂志

Objective:To produce rhBMP-4 with bioactivity in E.coli. Methods: The full-length human BMP-4 gene was mutated by PCR without changes in amino acid sequence, then the synthesized gene was cloned into plasmid pET-3c, transducted into BL21(DE)plysS, and induced by adding IPTG to a final concentration of 1.0 mmol/L. The protein product was purified using ion-exchange chromatography method and then renaturated, bioactivity was checked by C2C12 differentiation in vitro and mouse ectopic bone formation in vivo. Results: A 438 bp gene fragment encoding mature peptide of hBMP-4 was cloned , the protein product was mostly in the form of inclusion body, after renaturation, the engineering protein shows better bioactivity. Conclusion:The mutant strategy can enhance the expression of bioactive rhBMP-4 in E.coli expression system.