Construction of Bifidobacterium Infantis/CD Targeting Gene Therapy System

Objective： To construct Bifidobacterium Infantis/CD targeting gene therapy system. Methods： CD gene was amplified from E. Coli K12λ using PCR method, pGEX-1LamdaT plasmid and CD gene were digested with dual restriction endonucleas of EcoR Ⅰ and BamH Ⅰ and two segments of 4.9 kb and 1.3 kb were obtained. T4 DNA ligase was added to these two segments to make a recombinant CD/pGEX-1LamdaT plasmid. Then the recombinant plasmid was transfected into Bifidobacterium Infantis by electroporation. The recombinant plasmid was extracted from the positively transfected Bifidobacterium Infantis and digested with dual restriction endonucleases. Then the size of digested fragments was detected and sequencing of the gene segment inserted in extracted recombinant plasmid was performed according to the method of Sanger dideoxynucleotide triphosphate chain termination. Results： 6.2 kb recombinant plasmid was obtained from the positively transfected bacterial colony of Bifidobacterium Infantis. After being digested with dual restriction endonucleases, two segments of approximate 4.9 kb and 1.3 kb were gained from the extracted recombinant plasmid, which were equal to the size of pGEX-1LamdaT plasmid and CD gene, respectively. The full length and sequence of nucleotide acid of the inserted gene in extracted recombinant plasmid was completely identical to the CD gene. Conclusion： The foreign gene, CD gene was correctly inserted into pGEX-1LambdaT plasmid and transferred into Bifidobacterium Infantis. Bifidobacterium Infantis/CD targeting gene therapy system was successfully constructed.

Protective effect of Bifidobacterium infantis CGMCC313-2 on ovalbumin-induced airway asthma and b-lactoglobulininduced intestinal food allergy mouse models

AIM To determine whether oral administration of Bifidobacterium infantis CGMCC313-2(B. infantis CGMCC313-2) inhibits allergen-induced airway inflammation and food allergies in a mouse model.METHODS Ovalbumin(OVA)-induced allergic asthma and b-lactoglobulin-induced food allergy mouse models were used in this study. Following oral administration of B. infantis CGMCC313-2 during or after allergen sensitization, histopathologic changes in the lung and intestine were evaluated by hematoxylin and eosin(HE) staining. In the allergic asthma mouse model, we evaluated the proportion of lung-infiltrating inflammatory cells. OVAspecific IgE and IgG1 levels in serum and cytokine levels in bronchoalveolar lavage fluid(BALF) were also assessed. In the food allergy mouse model, the levels of total Ig E and cytokines in serum were measured.RESULTS Oral administration of B. infantis CGMCC313-2 during or after allergen sensitization suppressed allergic inflammation in lung and intestinal tissues, while the proportion of infiltrating inflammatory cells was significantly decreased in the BALF of allergic asthma mice. Moreover, B. infantis CGMCC313-2 decreased the serum levels of total Ig E in food allergy mice, and reductions in IgE and IgG1 were also observed in OVA-induced allergic asthma mice. The expression of interleukin-4(IL-4) and IL-13 in both serum and BALF was suppressed following the administration of B. infantis CGMCC313-2, while an effect on serum IL-10 levels was not observed.CONCLUSION B. infantis CGMCC313-2 inhibits the secretion of allergen-induced IgE, IL-4 and IL-13, and attenuates allergic inflammation.

Bifidobacterium infantis regulates the programmed cell death 1 pathway and immune response in mice with inflammatory bowel disease

BACKGROUND Inflammatory bowel disease(IBD)is caused by an abnormal immune response.Programmed cell death 1(PD-1)is an immunostimulatory molecule,which interacts with PD ligand(PD-L1)playing a prime important role among autoimmune diseases.Bifidobacterium infantis(B.infantis)can promote the differentiation of CD(cluster of differentiation)4^(+)T cells into regulatory T cells(Tregs).Tregs participate in the development of IBD and may be related to disease activity.B.infantis amplify the expression level of PD-1,PD-L1 and Tregs’nuclear transcription factor forkhead box protein 3(Foxp3).But the mechanism of B.infantis on PD-1/PD-L1 signaling remains unclear.AIM To explore the mechanism of B.infantis regulating the immune response in IBD.METHODS Forty-eight-week-old BALB/c mice were randomly divided into five groups:The control group,dextran sulphate sodium(DSS)model group,DSS+B.infantis group,DSS+B.infantis+anti-PD-L1 group,and DSS+anti-PD-L1 group.The control group mice were given drinking water freely,the other four groups were given drinking water containing 5%DSS freely.The control group,DSS model group,and DSS+anti-PD-L1 group were given normal saline(NS)400μL daily by gastric lavage,and the DSS+B.infantis group and DSS+B.infantis+anti-PDL1 group were given NS and 1×109 colony-forming unit of B.infantis daily by gastric lavage.The DSS+B.infantis+anti-PD-L1 group and DSS+anti-PD-L1 group were given 200μg of PD-L1 blocker intraperitoneally at days 0,3,5,and 7;the control group,DSS+anti-PD-L1 group,and DSS+B.infantis group were given an intraperitoneal injection of an equal volume of phosphate buffered saline(PBS).Changes in PD-L1,PD-1,Foxp3,interleukin(IL)-10,and transforming growth factorβ(TGF-β)1 protein and gene expression were observed.Flow cytometry was used to observe changes in CD4^(+),CD25^(+),Foxp3^(+)cell numbers in the blood and spleen.RESULTS Compared to the control group,the expression of PD-1,Foxp3,IL-10,and TGF-β1 was significantly decreased in the intestinal tract of the DSS mice(P<0.05).Compared to the control group,the proportion of CD4^(+),CD25^(+),Foxp3^(+)cells in spleen and blood of DSS group was visibly katabatic(P<0.05).B.infantis upgraded the express of PD-L1,PD-1,Foxp3,IL-10,and TGF-β1(P<0.05)and increased the proportion of CD4^(+),CD25^(+),Foxp3^(+)cells both in spleen and blood(P<0.05).After blocking PD-L1,the increase in Foxp3,IL-10,and TGF-β1 protein and gene by B.infantis was inhibited(P<0.05),and the proliferation of CD4^(+),CD25^(+),Foxp3^(+)cells in the spleen and blood was also inhibited(P<0.05).After blocking PD-L1,the messenger ribonucleic acid and protein expression of PD-1 were invariant.CONCLUSION It is potential that B.infantis boost the proliferation of CD4^(+),CD25^(+),Foxp3^(+)T cells in both spleen and blood,as well as the expression of Foxp3 in the intestinal tract by activating the PD-1/PD-L1 pathway.

Construction of dual suicide gene therapy system pTRKH2/CD and pTRKH2/UPRT in Bifidobacterium infantis and its characterization

Objective:We recombine the suicide gene CD,UPRT into plasmid pTRKH2 and clone the recombinant dual suicide gene therapy system into tumor-hypoxia-targeting vector Bifidobacterium infantis and characterize its function.Methods:CD gene,UPRT gene and lactic acid bacteria expression plasmid pTRKH2 were digested by restriction endonuclease BamH I and Sal I,and constructed recombinant plasmids pTRKH2/CD and pTRKH2/UPRT in E.coli.The recombinant plasmids were then transfected into Bifidobacterium Infantis by electroporation.Identification of pTRKH2/CD and pTRKH2/UPRT was processed by dual restriction endonuclease digesting and sequencing.RT-PCR and SDS-PAGE were used to examine the expression of CD and UPRT genes at RNA and protein levels.The killing effects on Melanoma B16-F10 cells by pTRKH2/CD and pTRKH2/UPRT suicide gene therapy system with 5-FC were examined by MTT assay.Results:The CD gene and UPRT gene was successfully recombined into lactic acid bacteria expression plasmid pTRKH2.After dual endonuclease digestion of plasmid purified from the positively transfected E.coli,two fragments of 6.9 Kb and 1.3 Kb were found for CD gene and two fragments of 6.9 Kb and 620 bp were found for UPRT gene.The sequencing of CD gene and UPRT gene proved consistent sequences with Genebank published data.A fragment of 1.3 Kb for CD gene and fragment of 620 bp for UPRT gene was found in recombinant Bifidobacterium by RT-PCR.A 52 KDa protein for CD gene was identified in whole-cell protein of recombinant Bifidobacterium and a 26 KDa protein for UPRT gene was identified in supernatant fluid of recombinant Bifidobacterium.The survival rate of tumor cells treated by extracts from culture of recombinant Bifidobacterium with 5-FC showed a strong killing effects of pTRKH2/CD and pTRKH2/UPRT dual suicide gene therapy system on Melanoma B16-F10 cells.Conclusion:CD gene and UPRT gene are successfully inserted into pTRKH2 and transfected into tumor-hypoxia-targeting vector Bifidobacterium Infantis.This dual suicide gene therapy system shows a high efficiency for tumor cells killing.

A self-guidance biological hybrid drug delivery system driven by anaerobes to inhibit the proliferation and metastasis of colon cancer

Colorectal cancer is often accompanied by multiple organ metastasis.Anaerobic Bifidobacterium Infantis(BI)bacterial can selectively grow in hypoxic colorectal tumor microenvironment(TME),to own the natural advantage of preferentially colorectal tumor targeting.Herein,a self-guidance biological hybrid drug delivery system(BI-ES-Fe Alg/DOX)based on BI was constructed to inhibit the proliferation and metastasis of colon cancer.Results demonstrated that BI-ES-Fe Alg/DOX could overcome physical barriers to target and accumulate in colon tumor tissues.Then DOX was released to kill tumor cells along with the phase transition(solid to liquid)of Fe Alg hydrogel,due to Fe3+was reduced to Fe^(2+)by intracellular GSH.Meanwhile,BI-ES selectively colonized into tumors and expressed endostatin(ES)protein to down-regulate VEGF and b FGF expression,exerting anti-angiogenic effect.Moreover,Fe Alg catalyzed H_(2)O_(2)in the local tumor to generate cytotoxic·OH,further enhancing the antitumor effect.The pharmacodynamic result in AOM/DSS model proved that BI-ES-Fe Alg/DOX had the best therapeutic effect,with the final V/V0of 2.19±0.57,which was significantly lower than the other groups.Meanwhile,on CT-26tumor-bearing model,it also showed an outstanding anti-tumor effect with inhibition rate of 82.12%±3.08%.In addition,lung metastases decreased significantly in tumor metastasis model after BI-ES-Fe Alg/DOX treatment.

Oral Administration Recombinant Bifidobacterium-LTB (B Subunit of Heat-Labile Enterotoxin) Enhances the OVA (Ovalbumin)-Specific sIgA in Jejunal Mucosa of Sprague-Dawley (SD) Rat

The LTB of enterotoxigenic Escherichia coli (ETEC) expressed in Bifidobacterium infantis (BI) has been testified as mucosal adjuvant with co-vaccination BI-CfaB (the major fimbrial subunit) together in vivo in our previous study. In order to investigate the mucosal adjuvant effect of BI-LTB to purified antigens, we oral vaccinated SD rats with recombinant BI-LTB plus OVA (rBI-LTB + OVA), and wild type BI plus OVA (wBI + OVA), OVA and PBS (Phosphate buffered saline) were vaccinated as controls, respectively. The OVA-specific sIgA in jejunal mucosa and specific IgG in serium were measured with ELISA (Enzyme-linked immunosorbent assay) and the sIgA producing cells were detected with immunohistochemistry technology (IHC) and Qwin image manipulation tools subsequently. The results shown rBI-LTB could stimulate SD rats produce high titer OVA-specific sIgA in rBI-LTB + OVA group and the OVA-specific sIgA titer in rBI-LTB + OVA group was found significant greater than that of the wBI + OVA group or OVA single group (p < 0.05). However no such significant difference was detected between the group wBI + OVA and OVA. IHC results suggested that intestinal mucosa and submucosa was the main field of sIgA secretion. These results suggested that recombinant LTB expression in BI could be used as a wide range mucosal adjuvant with different form antigens.