The Immunological Activation And Antitumour Activity Of Staphylococcal Enterotoxin-Like Q

BackgroundStaphylococcal enterotoxins(SEs)are a group of superantigen-active exotoxins encoded by staphylococcus aureus.As typical superantigens,SEs have a wide range of anti-tumor activities in clinic.However,their unavoidable side effects,such as SEs-induced fever and emesis,seriously limit their extensive application in treating malignant tumours.Fortunately,the identification of staphylococcal enterotoxins-like toxin(SEls),which possess the similar amino acid sequences as classical SEs but retain no or low emetic activity,has provided potential immunomodulatory candidates for malignant tumours therapy.Objective1.Construction of prokaryotic expression vector of staphylococcal enterotoxin-like Q,(SElQ).2.To examine the effect of SElQ on the activation of mouse splenic lymphocyte and human PBMCs.3.To further confirm the antitumor activity of SElQ in vitro and in vivo.Methods1.The selq gene fragment amplified by PCR was cloned into the plasmid of pET28a,and transformed into E.coli DH5?.After being confirmed by colony PCR,double digestion and sequence,the successfully constructed pET28a-SElQ was further transformed into E.coli BL21.After induction by IPTG,the SElQ was purified by BeaverBeads?IDA-Nickel.2.The proliferation of spleen lymphocytes and human PBMCs were examined by MTT assay,CD4~+and CD8~+T lymphocytes were tested by flow cytometry.3.The expression of IL-2 and IFN-?in the supernatant of mice spleen lymphocytes and human PBMCs in vitro were detected by ELISA.Simultaneously,the expression of IL-2 and IFN-?in the serum of mice received SElQ via tail vein were also were detected by ELISA.4.The gene expression of IL-4,IL-6,IL-10,TNF-?,GA and GB in mouse splenic lymphocytes stimulated by SElQ for 48 hours were detected by qPCR.Furthermore,the specific gene expression profile of TCR V?in human PBMCs stimulated by SElQ for 48hours was detected by qPCR.5.MTT assay was used to detect the anti-tumor activity of SElQ-activated PBMCs on human breast cancer cell line(BT474)and human lung cancer cell line(A431)in vitro;then non-contact co-culture was used to detect the effect of cytokines secreted by SElQ on the proliferation or apoptosis of A431 after PBMCs were stimulated by SElQ;furthermore,whether SElQ stimulated human PBMCs directly killed A431 tumor cells was observed by live cell imaging system.6.After the successful establishment of the mouse tumor model,the experimental mice received SElQ via tail vein were sacrificed at the end of the experiment,then the tumor,liver and spleen were isolated,imaged and weighted.The anti-tumor activity of SElQ in vivo was evaluated by tumor inhibition rate.Additionly,the toxicity and side effects of SElQ were preliminarily evaluated by the indexs of BWVI,MFC,SI and LI.Results1.The plasmid of pET28a-SElQ was successfully constructed,and high purity his-taged SElQ protein was achieved by BeaverBeads?IDA-Nickel.2.The results of MTT assay showed that SElQ can significantly stimulate the proliferation of mouse splenic lymphocytes and human PBMCs.The results of flow cytometry showed that SElQ can significantly promote the increase of percentage and absolute number of CD4~+and CD8~+T mouse-derived splenic lymphocytes.3.The results of ELISA showed that the expression of IL-2 and IFN-?were significantly up-regulated in vitro and in vivo after SElQ treatment.The results of qPCR indicated that incubation with SElQ significantly promote the gene expression of IL-4,IL-6,IL-10,GA and GB.In addition,the expression of IL-2 and IFN-?in PBMCs supernatant stimulated by SElQ at different concentrations were also up-regulated.3.The results of qPCR also showed that the T cells bearing TCR V?5(P<0.01),V?14(P<0.05),V?17(P<0.05),V?18(P<0.05)and V?21(P<0.05)were significantly activated by SElQ stimulation,particularly the T lymphocytes bearing TCR V?5.4.SElQ exhibited marked antitumor activity both in vitro and in vivo.The treatment with low concentrations of SElQ can inhibit the growth of tumours by approximately 30%,and no significant toxicity was observed.ConclusionSElQ can significantly induce T cell activation and cytokine release,which further elicit substantial antitumour activity.