Optimized expression of Helicobacter pylori ureB gene in the Lactococcus lactis nisin-controlled gene expression (NICE) system and experimental study of its immunoreactivity.
In the development of an oral vaccine against Helicobacter pylori, H. pylori urease subunit B (UreB) was expressed in a food-grade delivery vehicle, Lactococcus lactis NZ3900. The ureB gene (Genbank accession no. FJ436980) was amplified by polymerase chain reaction (PCR) from MEL-Hp27. The PCR-amplified ureB gene was cloned in the E. coli-L. lactis shuttle vector pNZ8110 and transformed into E. coli MC1061. After the transformant had been identified, the recombinant plasmid was purified and electrotransformed into L. lactis NZ3900. The conditions of UreB expression in the L. lactis transformant were optimized by orthogonal experiment. The maltose binding protein (MBP)-UreB fusion protein expressed by TB(1)/pMAL-c2X-ureB was used to cultivate mice polyclonal anti-UreB serum after purification by the amylose prepacked column. The Western blot method was adopted to confirm whether the UreB expressed by L. lactis transformant had immunoreactivity. The optimized conditions for UreB expression were as follows. Nisin 40 ng/ml was added to the medium when the recombinant grew to OD(600) approximately 0.30-0.40 and the induction time lasted 5 h. As a result, the maximum yield of UreB was 27.26 microg/mL of medium, and the maximum percentage of UreB in cell extracts of the L. lactis transformant reached its peak at 20.19%. Western blot analysis showed that the UreB protein expressed by L. lactis transformant had favorable immunoreactivity. All these results make an appealing case for construction of the food-grade vaccine for H. pylori.
Zhang
Xiao Juan
XJ
Henan University of Science and Technology, Luoyang, China. gjfzxj0921@gmail.com
Duan
Guangcai
G
Zhang
Rongguang
R
Fan
Qingtang
Q
D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D001428:Bacterial Vaccines / Q000276:immunology*; D015153:Blotting, Western; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D016481:Helicobacter Infections / Q000276:immunology / Q000517:prevention & control*; D016480:Helicobacter pylori / Q000201:enzymology* / Q000276:immunology; D007114:Immunization; D013294:Lactococcus lactis / Q000235:genetics; D008320:Maltose / Q000378:metabolism; D051379:Mice; D009561:Nisin / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000276:immunology*; D014169:Transformation, Bacterial; D014510:Urease / Q000096:biosynthesis* / Q000276:immunology*
D000907:Antibodies, Bacterial; D001428:Bacterial Vaccines; D011994:Recombinant Proteins; D009561:Nisin; D008320:Maltose; D014510:Urease