[Construction of recombinant strain expressing enterotoxigenic Escherichia coli K88ac-ST1-LTB fusion protein].
K88ac genes, heat-stable enterotoxin I (ST1) mutant genes and heat-labile enterotoxin B subunit (LTB) genes from plasmids of Escherichia coli C83902 were amplified by PCR. The recombinant expression plasmid pXKST3LT5 containing K88ac-ST1-LTB fusion gene was constructed by recombinant DNA technique and then transformed into Escherichia coli BL21(DE3). The K88ac-ST1-LTB fusion protein was highly expressed in recombinant strain BL21 (DE3)(pXKST3LT5) and the expression level of the K88ac-ST1-LTB fusion protein was about 75.53% of total cellular protein by SDS-PAGE and thin-layer gel scanning analysis. More importantly, mice immunized with crude preparation containing the fusion protein inclusion bodies or inactivated recombinant strain produced antibodies that were able to recognize ST1 in vitro. These sera antibodies were able to neutralize the biological activity of native ST1 in the suckling mouse assay. Hence the fusion protein was nontoxic and immunogenic, the constructed recombinant strain could be used as a candidate of vaccine strain.
Xu
Chong-Bo
CB
Department of Biotechnology, Ningxia University, Yinchuan 750021, China. xcb921@sohu.com
Wei
Guang-Sen
GS
D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004768:Enterotoxins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005818:Genetic Engineering; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D011995:Recombination, Genetic; D012967:Sodium Dodecyl Sulfate
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C034151:heat stable toxin (E coli); D012967:Sodium Dodecyl Sulfate; C027538:heat-labile enterotoxin, E coli