Expression,purification and bioinformatics analysis of PE＿PGRS35 protein of Mycobacterium tuberculosis H37Rv strain / 中国生物制品学杂志

@#Abstract： Objective To clone PE_PGRS35 gene of Mycobacterium tuberculosis（MTB），construct recombinant vector pET28a⁃PE_PGRS35，express and purify the PE_PGRS35 protein of MTB H37Rv heterologously，and explore a new target against MTB after bioinformatics analysis. Methods The PE_PGRS35 coding gene was amplified by PCR and used to construct the expression vector pET28a⁃PE_PGRS35 by recombinant cloning technology，which was transformed to E. coli BL21（DE3）after successful sequencing and induced by using IPTG. The obtained PE_PGRS35 protein was purified by Ni column affinity chromatography and analyzed by bioinformatics. Results The pET28a⁃PE_PGRS35 prokaryotic expression vector was constructed correctly as identified by sequencing. The PE_PGRS35 protein was mainly expressed in the form of inclusion bodies，with a relative molecular mass of about 53 000 and a purity of 90%. Bioinformatics analysis showed that PE_PGRS35 protein was an acid⁃labile protein，with main secondary structure of β⁃sheet and random coil，and no transme⁃ mbrane region，which was presumed to be an extramembrane protein with 39 phosphorylation sites and two conserved domains. Total 10 proteins，including Rv1769，PPE8，PPE64，PPE54，PPE24，PPE16，PPE35，PPE6，PPE28 and PE2， interacted with PE_PGRS35 protein. Conclusion PE_PGRS35 protein with high purity was successfully obtained，which provided a reference for the further development of new targets for drugs against MTB.

The effect of PD-1/PD-L1 signaling axis on the interaction between CD19+B cells and CD4+T cells in peripheral blood of patients with systemic lupus erythematosus

Abstract Background The defect of B cell self-tolerance and the continuous antigen presentation by T cells (TCs) mediated by autoreactive B cells (BCs) play a key role in the occurrence and development of systemic lupus erythematosus (SLE). PD-1/PD-L1 signaling axis negatively regulates the immune response of TCs after activation and maintains immune tolerance. However, the effect of PD-1/PD-L1 signaling axis on the interaction between CD19+B/CD4+TCs in the peripheral blood of patients with SLE has not been studied in detail. Methods PD-1/PD-L1 and Ki-67 levels in peripheral blood (PB) of 50 SLE patients and 41 healthy controls (HCs) were detected through flow cytometry, and then the expression of PD-1+/−cells and PD-L1+/−cells Ki-67 was further analyzed. CD19+B/CD4+TCs were separated for cell culture and the supernatant was collected to determine proliferation and differentiation of TCs. IL-10 and IFN-γ secretion in the supernatant was also determined using ELISA. Results The PD-1, PD-L1, and Ki-67 levels on CD19+B/CD4+TCs in patients with SLE were higher than HCs. In CD19+B/CD4+TCs of SLE patients, the proliferative activity of PD-L1+ cells was higher than that of PD-L1− cells, and the proliferative activity of PD-1+ cells was higher than that of PD-1− cells. In the system co-culturing CD19+B/CD4+TCs from HCs/SLE patients, activated BCs promoted TCs proliferation and PD-L1 expression among TCs. Addition of anti-PD-L1 to co-culture system restored the proliferation of TCs, and inhibited IL-10/IFN-γ level. The addition of anti-PD-L1 to co-culture system also restored Tfh and downregulated Treg in HCs. Conclusions Axis of PD-1/PD-L1 on CD19+B/CD4+TCs in PB of SLE patients is abnormal, and cell proliferation is abnormal. In CD19+B/CD4+TCs of SLE patients, the proliferative activity of PD-L1+ and PD-1+ cells compared with PD-L1− and PD-1− cells in SLE patients, respectively. CD19+B/CD4+TCs in SLE patients can interact through PD-1/PD-L1.