| Background and Subject:Based on intestinal mucosal immune system characteristics(including: ①primary type of mucosal immune respone being secretory immune, ②oral immune tolerance to normal flora microbial, ③normal flora microbial being able to translocate to the intestinal submucosa, ④the weak intestinal mucosal memory response to normal flora microbial etc.), the auxotrophic E.coli DH10 B Δasd(p DV_nirb_hly_cmv_gene) strain was designed,which strain was able to translocate to the intestinal submucosa, to break the phagosome through and to release the carried plasmid into the cell matrix. The infected cells could express the interesting protein following the gene carried by the plasmid. The expressed protein could take biological effect. A serial of tests were carried out to confirm the design.Method and results:This paper was divided into three parts. Firstly, the Non-invasive E.coli DH10 B strain was changed by the recombinant engineering(the p KD46 system and kan / kil two-screening system). The key enzyme(ASD) gene was knocked out. Because of no key enzyme gene,the DAP(the important ingredient of bacterial peptidoglycan for the bacterial wall forming) synthesis reaction halted in the changed bacterial strain. The changed bacterial strain could not synthesize the good bacterial wall in the no DAP condition. We obtained the changed bacterial strain, the auxotrophic E.coli DH10 B Δasd strain, through a lot of screening work. The characteristics of the strain were detected. The strain is auxotrophic gram-negative bacillus against streptomycin. The proliferation rate of the strain was 21.1minutes per generation. The half cracking time is about 24 hours at 37℃ in the normal LB culture media. The biochemical reaction characteristics are consistent with normal E.coli metabolism characteristics except for the auxotroph of the cell wall.Secondly, the eukaryotic expression plasmid(p DV_nirb_hly_cmv_gene) was constructed,containing the hly gene prokaryotic expression cassette controlled by Nir B promoter(Hypoxia promoter), the epo/egfp gene eukaryotic expression cassette controlled by CMV promoter, prokaryotic replicon and kanamycin screening marker. Furthermore, the plasmids, p DV_nirb_hly_cmv_epo and p DV_nirb_hly_cmv_egfp were obtained. They were detected by some identity tests. The results of DNA sequencing and restriction endonuclease mapping confirmed that constructed plasmids were correct. The LLO protein test results of the reduction SDS-PAGE, the Western blot and the N-terminal amino acid sequencing confirmed the expressed LLO was correct. The EPO protein test results confirmed that the expressed EPO was correct, including the Dot Immunobinding Assay(DIBA) results and the N-terminal amino acid sequencing results. The observation,through fluorescence microscopy, showed the enhanced green fluorescent protein(EGFP)was expressed in the cells transfected by pDV_nirb_hly_cmv_egfp.Thirdly, the plasmids(pDV_nirb_hly_cmv_epo and pDV_nirb_hly_cmv_egfp) were respectively electroplused into the E.coli DH10 B Δasd strain. After being screened, the E.coli DH10 B Δasd(p DV_nirb_hly_cmv_epo) strain and the E.coli DH10 B Δasd(p DV_nirb_hly_cmv_egfp) strain were gotten. The following tests indentify the strain characteristics.The E.coli DH10 B Δasd(p DV_nirb_hly_cmv_epo) strain is auxotrophic gram-negative bacillus against streptomycin and kanamycin. The proliferation rate of the strain is 29.6minutes per generation. The half cracking time is about 24 hours at 37℃ in the normal LB culture media. The biochemical reaction characteristics are consistent with normal E.coli metabolism characteristics except for the auxotroph of the cell wall.The safe oral gavage dose was confirmed using the observation of femal BALB/c mice responses after different oral gavage doses. The test results demonstrated that the safe oral gavage dose was less than 2 × 109 CFU per mouse.The E.coli DH10 B Δasd(p DV_nirb_hly_cmv_ egfp) strain infecting cells(including Caco-2 cells, Hela cells, NIH/3T3 cells, Ana-1 cells, mouse peritoneal macrophages)experiments were carried out. It was observated, through microscope, that mouse peritoneal macrophages engulfed the strain. The EGFP in the infected cells was observed by the fluorescence microscope, which demonstrated the released plasmid had been transcripted and the interesting protein had been expressed by the infected cells. Then, the subsequent results of the flow cytometry showed that infectional ratioes were respectively0.5%, 0.2%, 0.2%, 0.1% and 0.2%. The results of laser scanning confocal microscopy showed that the E.coli DH10 B Δasd(p DV_nirb_egfp_cmv_ epo) could be engulfed by the cells.For the intestinal tissue slice and immunohistochemistry, we took the intestinal tissue of the female BALB/c mice which had been respectively infected with the E.coli DH10BΔasd(p DV_nirb_hly_cmv_epo) strain and the E.coli DH10 B Δasd(p DV_nirb_hly_cmv_egfp) strain. The HE staining tissue section results showed that intestinal mucosal structure was intact, the intestinal cell membrane was intact and the intestinal cell structure was clear. The results indicated that the strain didn’t damage the intestinal mucosal immune barrier. The immunohistochemistry results showed that some intestinal cells contained protein LLO coden by the plasmid. The results indicated that the engulfed strain was able to break the phagosome through and to release the bacterial protein into the cellular matrix. The HE staining intestinal tissue sections of the mice infected by the E.coli DH10 B Δasd(p DV_nirb_hly_cmv_egfp) strain showed some cells had expressed green fluorescent protein coden by the plasmid. The results indicated that the engulfed strain break the phagosome through and release the plasmid into the cellular matrix. The interesting protein was expressed by the engulfing-the-bacterial-strain cell.The above results confirmed that the E.coli DH10 B Δasd(p DV_nirb_hly_cmv_egfp) strain or the E.coli DH10 B Δasd(p DV_nirb_hly_cmv_epo) strain had the ability to translocate,to break the phagosome through and to release the contents(bacterial protein, plasmid,etc.).Based on optimization experimental conditions, we tested the female BALB/c mice reticulocyte percentage change after oral gavage of the E.coli DH10 B Δasd(p DV_nirb_hly_cmv_epo) strain. The results showed the mean female BALB/c mice reticulocyte percentage raised 113% after the first oral gavage. At interval of 21 days, the mean female BALB/c mice reticulocyte percentage raised 53% after the second oral gavage in the same condition.Using viable bacterial culture, we tested the distribution of the E.coli DH10 B Δasd(p DV_nirb_hly_cmv_epo) strain in the infected female BALB/c mice body. The results showed that the viable bactceria could be detected in the liver and mesenteric lymph nodes(including some mesentery) of the infected mice, but could not be detected in more distant locations. Using RT q-PCR, we tested the distribution of the plasmid,p DV_nirb_hly_cmv_epo, in the infected mice body. The results showed that the plasmid could be detected in the more distant locations than the liver. These two experiments confirmed that the distribution of the plasmid were from the gut tract to the whole body through the portal vein way and intestinal lymphatic ways. The plasmid-expressed-locations(p DV_nirb_hly_cmv_egfp) were detected though immunohistochemistry test. The results showed that the liver and the spleen were the primary expressing organs.Conclusion:In this study, the E.coli DH10 B Δasd strain was obtained using homologous recombinant technology. p DV_nirb_hly_cmv_epo/egfp were constructed respectively. The E.coli DH10 B Δasd(p DV_nirb_hly_cmv_ epo/egfp)strain were obtained as a oral delivery system for gene therapy. The current study demonstrated that the oral delivery system could delivery the plasmid into the eukaryotic cells and the infected cells could express the interesting protein following the gene of the plasmid. Based on the experiement results, it is feasibility that the modified bacterial strain is used as the oral delivery system. If the strain can be modified furtherly, there will be a better prospect.Comparing with the supplying-interesting-gene way of the traditional gene therapy, this system(oral delivery system) doesn’t supply the interesting gene to the body directly. The interesting gene was supplied to the intestinal bacterial flora microbial and was deliveried to the intestinal cell through the natural translocation way.It is the advantage of this system that the interesting protein, the protein expressing way and the protein modifying way all belong to the the interesting body. Other advantages are to reduce the production cost, transportation cost, storage cost and so on. It is the limitation of this current system that the amount of expressed-interesting-protein need to be further improved.In addition, it’s hypothesis on intestinal mucosal immune barrier strength automatic control that was put forward. In the condition that the intestinal mucosal immune barrier is intact, peyer’s patches can be regarded as “intestinal cavity bio-threat intensity receptors”. They can evaluate the risk from intestinal cavity bio-threat though contacting micro-organisms in the intestinal tract. They can excite the effect chain for maintaining intestinal mucosal immune barrier strength against the intestinal cavity bio-threat intensity in order to limit the intestinal cavity bio-threat intensity under the control and in order to adapt to environmental changes,especially, changes of the intestinal micro-environment. |
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