| Objective:To analysis the sequence characteristics of Hsp60 gene in Helicobacter pylori (Hp) isolated from different patients and to express recombinant Hsp60 by prokaryotic expression system in E. coli BL21 (DE3) pLysS, in order to investigate the different effects between the two kinds of recombinant protein in the production of IL-8 in gastric epithelial cells.Methods:1. Gastric mucosal specimens obtained from different patients with gastroduodenal diseases, were grinded and inoculated onto Columbia-selective agar plates at 37℃ for 3 days, under microaerophilic condition. Cultures were identified as Hp by colonial morphology, Gram staining, rapid urease test (RUT) and oxidase test;2. Genomic DNA of these strains was extracted and a pair of primers was designed by Primer Premier 5.0 according to the sequence of Hsp60 gene of Hp ATCC 26695. Then the Hsp60 genes were amplified by PCR method and the products were sequenced after gel extraction. All sequenced fragments were subjected to a multiple alignment with the program MegAlign of DNAstar 5.0 software;3. Two sets of primers were designed by Primer Premier5.0 and mismatches were introduced into primers, mutagenesis was performed in a three-step PCR. In addition, we also cloned the wild-type Hsp60 gene;4. The amplified fragments were sequenced and analyzed, sub-cloned into the pEASY-E1 vector and transformed into E. coli BL21 (DE3) pLysS. The recombinant transformants were induced by IPTG and detected by SDS-PAGE. Subsequently, the recombinant proteins were purified with desalting and endotoxin-removal column;5. The purified Hsp60 proteins (W-Hsp60 and T-Hsp60) were co-cultured with GES-1 cells in different concentrations. The contributions of IL-8 secreted by GES-1 cells were detected by ELISA.Results:1. Single colonies were grown at the Columbia-selective agar plates after 3 days, these colonies were identified as Hp. By Gram staining, curved and S-shaped Gram-negative bacterium can be observed under microscope, in addition, the urease and oxidase tests were positive.2. The Hsp60 genes were successfully cloned and purified. According to the DNA sequencing and alignment, we found there exist differences in the Hsp60 gene isolated from non-gastric cancer and gastric cancer patients, at the site 877,1399 and 1579, respectively. In the Hp isolated from non-gastric cancer patients, at the site 877, 1399 and 1579, there were 8 strains (8/45,17.78%) showed G to A change,9 strains (9/45,20.00%) showed C to G change and 7 strains (7/45,15.56%) showed A to G change, respectively; while in the gastric cancer patients, in these 3 sites, there were 3 strains (3/18,16.67%) showed G to A change,l strains (1/18,5.55%) showed C to G change and 2 strains (2/18,11.11%) showed A to G change, respectively. In the deduced amino acid sequences of these 3 sites, there exist Val to Ile change, Gln to Glu change and Thr to Ala change, correspondingly.3. According to the DNA sequencing, the nucleotide sequence of Hsp60 gene of Hp ATCC26695 was successfully changed from AG to GC at the sites 1 398th and 1 399th.4. The prokaryotic expression vector pEASY-E1-Hsp60 was efficiently transformed into E. coli BL21 (DE3) pLysS. The protein was expressed under the optimum condition:OD600, the final concentration of IPTG and induction time is 0.4, 1.0mM and 4h, respectively.5. In the Hsp60 group, the concentration of IL-8 is higher than PBS group (P<0. 05), which was not inhibited by PMB; at the concentration of 50μg/ml of recombinant protein, the concentration of IL-8 was significantly higher in T-Hsp60 group than that in W-Hsp60 group (P<005).Conclusions:1. A total of 63 Hp strains were isolated from different patients with gastroduodenal diseases in different geographic regions of Shandong Province, which lays the foundations for studies in the pathogenic difference of Hp.2. In the Hp isolated from different patients, there exist single nucleotide polymorphisms (SNPs) at the site 877,1399 and 1579, respectively.3. The recombinant Hsp60 can induces GES-1 cell to produce inflammatory cytokine IL-8, which is unrelated to LPS; the function of wild and mutant rHsp60 in induction of inflammation has a significant differences. |
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