ABSTRACT
Objective To observe humoral and cellular immune responses induced in mice by a TSOL18 recombinant Mycobacterium smegmatis (MS) vaccine of Taenia solium.Methods Sixty specific pathogen-free (SPF) Kunming mice (18-22 g,half male and half female) were divided into 3 groups by random number table method,20 mice in each group.One group was administered with recombinant MS-TSOL18 vaccine,one group was administered with MS as control,and one group was administered phosphate buffer saline (PBS) as control.Kunming mice were vaccinated once every two weeks for 2 times through intragastric administration.At 0,2,4,6,and 8 weeks after immunization,blood sample was collected and serum was separated.The levels of IgG and IgG2a in serum were detected using enzyme linked immunosorbent assay (ELISA).The spleen was separated to prepare spleen suspension.The proliferation of spleen lymphocyte with the stimulation of a specific antigen was detected by CCK-8.The levels of interleukin (IL)-2 and IL-4 in spleen cell culture supernatant with the stimulation of a specific antigen were detected by ELISA.Results Compared with MS control group (IgG:0.160 ± 0.019,0.187 ± 0.038,0.193 ± 0.050,0.170 ± 0.005;IgG2a:0.213 ± 0.010,0.198 ± 0.012) and PBS control group (IgG:0.159 ± 0.015,0.184 ± 0.029,0.191 ± 0.025,0.165 ± 0.018;IgG2a:0.198 ± 0.032,0.178 ± 0.025),the levels of specific IgG (0.310 ± 0.034,0.391 ± 0.029,0.443 ± 0.030,0.373 ± 0.021) in recombinant MS-TSOL18 vaccine group increased at 2,4,6,and 8 weeks after immunization (P ＜ 0.05),the levels of specific IgG2a (0.446 ± 0.056,0.339 ± 0.026) in recombinant MS-TSOL18 vaccine group increased at 6 and 8 weeks after immunization (P ＜ 0.05),and reached the highest level by the 6th week.After antigen stimulation,compared with MS and PBS control groups,the levels of spleen lymphocyte proliferation increased at 2,4,6,and 8 weeks after immunization (P ＜ 0.05),and reached the highest level by the 6th week.After antigen stimulation,compared with MS and PBS control groups,the levels of IL-2 and IL-4 in spleen lymphocyte culture supernatant increased at 2,4,6,and 8 weeks after immunization (P ＜ 0.05),and reached the highest level by the 6th and 4th weeks,respectively.Conclusion The recombinant MS-TSOL18 vaccine of Taenia solium might induce mice to produce humoral and cellular immune responses.
ABSTRACT
Objective To investigate the effect of myeloid differentiation factor 88 inhibitor ST2825 on the autophagy of THP?1 cells infected by re?combinant mycobacterium smegmatis. Methods The myeloid differentiation factor 88 inhibitor ST2825 was applied on the THP?1 cells infected by recombinant mycobacterium smegmatis,and three groups were defined:the test group with ST2825 treatment,the control group without ST2825 treatment,and the blank group. Autophagosomes were observed under the fluorescence microscope,and the mRNA expression of Beclin?1 gene and Bcl?2gene was analyzed by RT?PCR. Results Compared with the control group,the number of autophagy fluorescent dots in the test group was ob?viously reduced(P<0. 05),and the expression levels of Beclin 1 gene and Bcl?2 gene were declined as indicated by the RT?PCR detection. Con?clusion The myeloid differentiation factor 88 inhibitor ST2825 might inhibit the autophagy of THP?1 cells through interfering the separation of Be?clin?1 and Bcl?2.
ABSTRACT
Objective To construct a recombinant bacterial vaccine which can express specific 10-23 deoxyribozyme(DZ) in macrophage, identify the intracellular production of specific 10-23DZ and detect the activity of this recombinant bacterial vaccine on inhibiting the expression of TACO gene in macrophage.Methods The pSDE02 was obtained by inserting the replicon of Mycobacterium into pSDE01, a plasmid which can express 10-23DZ in eukaryotic cells. The expression sequence of DZ1, a 10-23DZ targeting the TACO mRNA of macrophage designed in our previous study was synthesized and inserted into pSDE02. The resulted plasmid was named pDZM01. pDZM01 was then transferred into Mycobacterium smegmatis by electroperation. The recombinant M. smegmatis, named rMs-DZ1 was screened on low-salt LB medium containing Zeocin and identified by Colony PCR. The targeted delivery property of recombinant M. smegmatis was observed by Ziehl-Heelson stain and GFP expression observation via fluorescence microscope. rMs-DZ1 was used to infect RAW264.7 cells and the expression of DZ1 in macrophage was identified by dot-blot assay. At 24 h and 48 h after infection, total RNA and proteins were extracted and the TACO mRNA and protein expression level was assayed by RT-PCR and western-blot respectively. Results Restrictive analysis and sequencing data showed that the Mycobacterium-eukaryotic cell shuttle plasmid pSDE02 and pDZM01 was successfully constructed. rMs-DZ1 was confirmed by colony PCR. When engulfed by macrophage, rMs-DZ1 would express DZ1 in RAW264.7 cells and inhibit the expression of taco gene. When compared to uninfected macrophage, rMs-DZ1 significantly reduced the taco mRNA by 67.90% and 57.14% and down-regulated the expression of TACO protein by 53.85% and 68.92% at 24 h and 48 h respectively. Conclusion A recombinant M. smegmatis vaccine was successfully constructed which could generate specific 10-23DZ in macrophage and inhibit the expression of target gene of interest. To our knowledge, this is the first bacterial vector which can express intracellularly 10-23DRz in targeted manner. This study may further prompt the feasibility of using 10-23 DNAzyme to achieve effective and targeted gene silence.