Primary Application And Expression Of Escherichia Coli Heat-labile Enterotoxin B Subunit Protein From A Recombinant Strain

Heat-labile enterotoxin(LT) is a enterotoxin that can secrete to the periplasm in Escherichia coli. LT consists of A and B subunits and it can cause severe diarrhea of the travellers and livestocks. No vaccine is created to treat the disesae. LT has strong immunogenicity and it is a good mucosal immunization adjuvant. The potent toxicity of LTA limit its application. In this report, we try to express a recombinant protein LTB used for the vaccine to prevent livestocks from diarrhea and the mucosal immunization adjuvant of attenuated salmonella vaccine.Objective:Recombination protein LTB can be used for the vaccine to prevent from diarrhea induced enterotoxigenic Escherichia coli and the the mucosal immunization adjuvant activity of LTB when it vaccinates with attenuated salmonella vaccine.Methods:Refer to the sequence of LTB gene in GeneBank, we uesed the special software to design a pair of primers to amplify the gene by polymerase chain reaction (PCR). Both the pCold-TF vector and the PCR product were digested with BamHⅠ and HindⅢ. LTB gene fragment was inserted into the digested pCold-TF vector to construct a recombinant plasmid (pCold-TF/LTB). Then identificated the recombinant plasmid and transformed it into the Escherichia coli BL21 (DE3) for expression under the induction of IPTG. The TF/LTB protein was expressed in the supernatant and it was detected by Sodium dodecyl sulfate-polyacrylamide gelelectrophoresis(SDS-PAGE) and then purified by His tag affinity using BIO-RAD protein purifier. The antigen activity of the protein was tested by indirect ELISA and the protein was uesd to vaccinate the BALB/c mice. The IgG in the serum and IgA in the feces were detected by indirect ELISA after three times of the immune. The immune mice were vaccinated with Escherichia coli K88 to study the protection of the vaccine LTB.The mice were vaccinated with the purified TF/LTB protein and attenuated salmonella vaccine together and then IgA level in feces of the vaccinated mice was detected by indirect ELISA after three times of the immune to research the mucosal immunization adjuvant activity of LTB.Results:The LTB gene was amplified by PCR; The recombinant plasmid pCold-TF/LTB was reconstructed successfully; The recombinant protein TF/LTB was harvested; The TF/LTB has a good effect when it becomes a vaccine to prevent from diarrhea induced enterotoxigenic Escherichia coli; The TF/LTB has mucosal immunization adjuvant activity when it vaccinated the mice with attenuated salmonella vaccine together.Conclusion:The recombinant protein TF/LTB provides a tool to prevent the livestock from diarrhea induced enterotoxigenic Escherichia coli. The TF/LTB can become the attenuated salmonella vaccine’s adjuvant.