| Enterotoxigenic Escherichia coli(ETEC) can cause diarrhea, and even lead to death, bringing serious economic losses to the swine industry. Thermal stability enterotoxin STa(heat-stable enterotoxin) is the key virulence factors. Subunit vaccines made of the STa toxoid are effective. However, subunit vaccines require immunization by injection, and need for cryopreservation and transportation, which not only makes the high cost of the vaccine but also is inconvenient for use. Therefore, developing oral vaccines which can be stored and transported at room temperature is a good strategy to solve this problem. Bacillus cereus MIL158 probiotic strain of probiotic characteristic can be used as probiotics, and oral safety. The heat-resistant spores, decide it can be stored at room temperature. S- layer protein is a good carrier which could demonstrate the exogenous proteins to the cell surface of microorganism. In this study, surface display function of S-layer protein and thermal stability of Bacillus cereus spores were utilized to construct Bacillus cereus recombinant strains expressing enterotoxigenic Escherichia coli STa toxoids and the immunogenicity in mice immunized by recombinant strains was examined, providing preliminary foundation for the development of enterotoxigenic E. coli. STa vaccine, which is safe and can be transported and preserved at room temperature.1. The construction of recombinant strains in Insertion method and determination of their immunogenicity in mice.The STa toxoid gene(msta) was amplified by Overlap PCR, the recombination plasmid p CSA-S(csa-ctc-msta) was obtained by inserting the sta gene into the vector p BMB982-304 below the Xba I site of the S-layer protein gene ctc anchoring sequence slh(S-layer homology), insert the csa AB operon which is necessary for S-layer protein anchoring on the cell wall upstream of the ctc gene. The recombinant strain MIL158/p CSA-S was acquired by electro-transferring the recombinant plasmid into the MIL158.The STa fusion protein was expressed in recombinant strain which was examined by the Western blotting method. After immunizing mice with the recombinant strain, the result showed that when compared with the control group(PBS group, MIL158 no plasmid group and MIL158/p BMB982-304 empty plasmid group), the detection value(O D450) of recombinant strain in insertion method significantly improved(P<0.05).This indicated that STa antibody was produced after immuned by recombinantstrain.2. The construction of recombinant strains in Substitution method and determination of their immunogenicity in miceIn order to investigate the immunogenicity impact of different fusion gene construction methods in mice, this study we costructed recombinant strains in Substitution method. mgfp-linker-msta, ctc-his-msta and ctc-msta-his genes amplified by overlap PC R were replaced into the the S-layer protein ctc gene of the carrier p BMB982-304 between the Xba I and Hinc II, then we inserted the operon csa AB which was necessary for S- layer protein anchoring on the cell wall upstream of the ctc gene and obtained recombinant plasmid p CSA-GS(csa-ctc-mgfp–linker-msta), p CSA-HS(csa-ctc-his-msta) and p CSA-SH(csa-ctc-msta-his) respectively. The recombinant plasmids were electroporated into MIL158 and the corresponding recombinant strains was acquired, named MIL158/p CSA-GS, MIL158/p CSA-HS and MIL158/p CSA-SH respectively. After immunizing in mice with recombinant strains, the value(OD450) of the MIL158/p CSA-GS, MIL158/p CSA-HS and MIL158/p CSA-SH groups detected by ELISA significantly increased when compared with the control group(P <0.05) indicating that STa antibody was produced. In addition, the value(OD450) significantly increased when compared with insertion method. |
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