Tumor Necrosis Factor-α Inducing Protein Of Helicobacter Pylori Induces Cytokines Production In Macrophages Through Activation Of NF-κb

Objective This study aimed to clone and express Tipα protein of Helicobacter pylori(Hp) in Escherichia coli(E.coli), and to observe the effects of recombinant Tipα protein on cytokine expression and mechanisms involving in macrophages.Methods The Tipα gene sequence was amplified from the Hp chromosomal DNA using the specific primers for Tipα, followed by double enzyme digestion and cloned into p ET28 a resulting the plasmid p ET28a-Tipα. The recombinant vector p ET28a-Tipα was identified by PCR, double enzyme digestion and DNA sequencing then was transformed into E.coli BL21 for Tipα expression by IPTG induction. The recombinant Tipα protein was purified by Ni-NTA affinity column and identified by Western-blot. The culture of human THP-1 cell was differentiated into macrophage with PMA. The ELISA and Real time PCR were used for investigating the expressions of TNF-α,IL-1β and IL-6 when the macrophages were pretreated by 0~200 μg/ml Tipα protein for 24 h or 100 μg/ml Tipα protein for 0~36 hours. The nuclear translocations of the p65 subunit in Tipα-treated macrophage were detected by Western blot. The effects of Tipα protein on the expressions of TNF-α,IL-1β and IL-6 were measured in macrophages preincubated with a NF-κB inhibitor PDTC.Results(1)The Tipα gene was amplified from Hp 26695 chromosomal DNA by PCR andcloned into p ET28 a resulting in the recombinant p ET28a-Tipα. The PCR, double enzyme digestion and DNA sequencing results proved that the p ET28a-Tipα was constructed successfully. The coding sequences of p ET28a-Tipα were identical to the sequences of Tipα in NCBI. SDS-PAGE showed a 25 k Da fusion protein was expressed in recombinant E.coli BL21 which harboring the recombinant plasmid. The fusion protein was purified successfully by Ni-NTA metal-affinity chromatography. The Western-blot result proved that the expressed protein was the His-tagged target protein.(2)The Tipα protein stimulates the macrophages secrete pro-inflammatory cytokines in a dose and time dependent manner. The expressions of TNF-α、IL-1β and IL-6 were positively related to the Tipα protein concentration within a certain range. The pro-inflammatory cytokines levels were increased with the extension of stimulated time within 0-24 h. The m RNA levels of TNF-α、IL-1β and IL-6 are consistent with the results of ELISA.(3) The nuclear translocation of the p65 subunit of NF-κB in the macrophages began to increase at 2h and peaked at 4h after stimulation by Tipα protein. At the same time, the level of p65 in the cytosolic reduced gradually.(4)The pretreatment of PDTC could inhibit the Tipα protein induced expression of TNF-α、IL-1β and IL-6 in macrophage.Conclusion: Tipα protein of Hp induces the secretion of cytokines in macrophages through activation of NF-κB.