Shiga toxin B-subunit （STxB） from Shigella dysenteriae targets in vivo antigen to cancer cells, dendritic cells （DC） and B cells, which preferentially express the globotriaosylceramide （Gb3） receptor. This pivot...Shiga toxin B-subunit （STxB） from Shigella dysenteriae targets in vivo antigen to cancer cells, dendritic cells （DC） and B cells, which preferentially express the globotriaosylceramide （Gb3） receptor. This pivotal role has encouraged scientists to investigate fusing STxB with other clinical antigens. Due to the challenges of obtaining a functional soluble form of the recombinant STxB, such as formation of inclusion bodies during protein expression, scientists tend to combine STxB with vaccine candidates rather than using their genetically fused forms. In this work, we fused HPV16 E7 as a vaccine candidate to the recombinantly-produced STxB. To minimize the formation of inclusion bodies, we investigated a number of conditions during the expression procedure. Then various strategies were used in order to obtain high yield of soluble recombinant protein from E. coli which included the use of different host strains, reduction of cultivation temperature, as well as using different concentrations of IPTG and different additives （Glycin, Triton X-100, ZnC12）. Our study demonstrated the importance of optimizing incubation parameters for recombinant protein expression in E. coli; also showed that the secretion production can be achieved over the course of a few hours when using additives such as glycine and Triton X-100. Interestingly, it was shown that when the culture mediums were supplemented by additives, there was an inverse ratio between time of induction （TOI） and the level of secreted protein at lower temperatures. This study determines the optimal conditions for high yield soluble E7-STxB expression and subsequently facilitates reaching a functionally soluble form of STxB-based vaccines, which can be considered as a potent vaccine candidate for cervical cancer.展开更多
The mature Shiga toxin B （StxB） gene was optimized and generated by overlapping PCR. Recombinant expression vector pQE40-DHFR/StxB was constructed when the gene was cloned into pQE fusion expression vector. Induced ...The mature Shiga toxin B （StxB） gene was optimized and generated by overlapping PCR. Recombinant expression vector pQE40-DHFR/StxB was constructed when the gene was cloned into pQE fusion expression vector. Induced by Isopropyl β-D-thiogalactoside （IPTG）, the DHFR/StxB fusion protein was highly expressed to the level of 41.36% in E. coli MI5 cells. The 35 kDa fusion protein with a 6 His-tag was one-step purified from inclusion bodies using Ni-NTA affinity chromatography column under denaturing conditions, and was refolded by dialyzing with a decreasing urea gradient. Purified DHFR/StxB fusion protein was used to immunize Kunming mice for generating the ascitic polyclonal antibody against recombinant StxB protein by injecting sarcoma 180 cells and the titer ofascitic polyclonal antibody is up to 1： 1× 10^6 detected by the indirect enzyme linked immunosorbent assy （ELISA）. Western immunoblotting analysis revealed that the ascitic polyclonal antibody against StxB had a specific affinity for a 70 kDa shiga toxin protein of Shigella dysenteriae type 1. It is a new simple and quick method to produce a large amount of ascitic polyclonal antibody. The antibody is used to develop immunological method for detecting shiga toxin.展开更多
基金supported by Research Council of Shiraz University of Medical Sciences (91-01-36-4417), Shiraz, Iran
文摘Shiga toxin B-subunit （STxB） from Shigella dysenteriae targets in vivo antigen to cancer cells, dendritic cells （DC） and B cells, which preferentially express the globotriaosylceramide （Gb3） receptor. This pivotal role has encouraged scientists to investigate fusing STxB with other clinical antigens. Due to the challenges of obtaining a functional soluble form of the recombinant STxB, such as formation of inclusion bodies during protein expression, scientists tend to combine STxB with vaccine candidates rather than using their genetically fused forms. In this work, we fused HPV16 E7 as a vaccine candidate to the recombinantly-produced STxB. To minimize the formation of inclusion bodies, we investigated a number of conditions during the expression procedure. Then various strategies were used in order to obtain high yield of soluble recombinant protein from E. coli which included the use of different host strains, reduction of cultivation temperature, as well as using different concentrations of IPTG and different additives （Glycin, Triton X-100, ZnC12）. Our study demonstrated the importance of optimizing incubation parameters for recombinant protein expression in E. coli; also showed that the secretion production can be achieved over the course of a few hours when using additives such as glycine and Triton X-100. Interestingly, it was shown that when the culture mediums were supplemented by additives, there was an inverse ratio between time of induction （TOI） and the level of secreted protein at lower temperatures. This study determines the optimal conditions for high yield soluble E7-STxB expression and subsequently facilitates reaching a functionally soluble form of STxB-based vaccines, which can be considered as a potent vaccine candidate for cervical cancer.
文摘The mature Shiga toxin B （StxB） gene was optimized and generated by overlapping PCR. Recombinant expression vector pQE40-DHFR/StxB was constructed when the gene was cloned into pQE fusion expression vector. Induced by Isopropyl β-D-thiogalactoside （IPTG）, the DHFR/StxB fusion protein was highly expressed to the level of 41.36% in E. coli MI5 cells. The 35 kDa fusion protein with a 6 His-tag was one-step purified from inclusion bodies using Ni-NTA affinity chromatography column under denaturing conditions, and was refolded by dialyzing with a decreasing urea gradient. Purified DHFR/StxB fusion protein was used to immunize Kunming mice for generating the ascitic polyclonal antibody against recombinant StxB protein by injecting sarcoma 180 cells and the titer ofascitic polyclonal antibody is up to 1： 1× 10^6 detected by the indirect enzyme linked immunosorbent assy （ELISA）. Western immunoblotting analysis revealed that the ascitic polyclonal antibody against StxB had a specific affinity for a 70 kDa shiga toxin protein of Shigella dysenteriae type 1. It is a new simple and quick method to produce a large amount of ascitic polyclonal antibody. The antibody is used to develop immunological method for detecting shiga toxin.
