Expression of Helicobacter pylori hspA Gene in Lactococcus lactis NICE System and Experimental Study on Its Immunoreactivity.

Xiao-Juan Zhang,Zhi-Tao Li,Shu-Ying Feng,Yan-Ming Feng

doi:10.1155/2015/750932

Abstract

Aim. The aim of this study was to develop an oral Lactococcus lactis (L. lactis) vaccine against Helicobacter pylori (H. pylori). Methods. After L. lactis NZ3900/pNZ8110-hspA was constructed, growth curves were plotted to study whether the growth of recombinant L. lactis was affected after hspA was cloned into L. lactis and whether the growth of empty bacteria, empty plasmid bacteria, and recombinant L. lactis was affected by different concentrations of Nisin; SDS-PAGE and Western blot were adopted, respectively, to detect the HspA expressed by recombinant L. lactis and its immunoreactivity. Results. There was no effect observed from the growth curve after exogenous gene hspA was cloned into L. lactis NZ3900; different concentrations of Nisin did not affect the growth of NZ3900 and NZ3900/pNZ8110, while different concentrations of Nisin inhibited the growth of NZ3900/pNZ8110-hspA except 10 ng/mL Nisin. No HspA strip was observed from SDS-PAGE. Western blot analysis showed that HspA expressed by recombinant bacteria had favorable immunoreactivity. Conclusion. The growth of recombinant L. lactis was suppressed even though a small amount of HspA had been induced to express. Therefore recombinant L. lactis only express HspA which was not suitable to be oral vaccine against Helicobacter pylori.

Highlights

Helicobacter pylori (H. pylori) is a spiral and motile Gramnegative bacterium, which colonizes the human stomach mucosa and is the main cause of a range of gastric and duodenal diseases including chronic gastritis, stomach and duodenal ulcers, MALT lymphoma, and gastric adenocarcinoma, and it is considered as the second most frequent cause of cancer death worldwide [1, 2]
The sequencing result showed that the sequences of hspA gene in this experiment were correct compared with the sequences of MEL-Hp27 hspA
To express the HspA protein in L. lactis transformant, the hspA gene was inserted into the E. coli-L. lactis shuttle vector pNZ8110 and the recombinant plasmid was transformed into the E. coli MC1061

Summary

Introduction

Helicobacter pylori (H. pylori) is a spiral and motile Gramnegative bacterium, which colonizes the human stomach mucosa and is the main cause of a range of gastric and duodenal diseases including chronic gastritis, stomach and duodenal ulcers, MALT lymphoma, and gastric adenocarcinoma, and it is considered as the second most frequent cause of cancer death worldwide [1, 2]. Heat shock proteins (Hsp) of H. pylori play important roles in H. pylori adhesion to gastric epithelial, assembling of urease enzyme subunit and stimulating the body to produce an immune response; at the same time they are the main immunogens of H. pylori. HspA is the main functional unit of Hsp and it locates bacterial surface, is relatively conservative, and has immunogenicity, so it is an important candidate vaccine antigen of H. pylori. Lactococcus lactis (L. lactis) is a Gram-positive, noninvasive, nonpathogenic, and food-grade bacterium and is generally recognized as safe (GRAS), which has been used in the food industry for production of fermented milk products for many years. Nisin has been successfully applied to a variety of strains, including Lactococcus, Lactobacillus, Streptococcus, and Enterococcus [9, 10]

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Conclusion