Interactions of highly pathogenic avian influenza H5N1 nucleoprotein with NF-κB-inducing kinase(NIK) / 军事医学

Objective To study the interactions between the highly pathogenic avian influenza H5N1 nucleoprotein (H5N1 NP) and NF-κB-inducing kinase (NIK),and to reveal the effect of H5N1 NP on NIK-induced NF-κB transcriptional activity.Methods The gene encoding NIK protein was amplified by RT-PCR from total RNA of HeLa cell line.Eukaryotic expression plasmid pCMV-Myc-NIK and prokaryotic expression plasmid pGEX-4T-1-NP (GST-NP) were constructed by cloning from HeLa cell cDNA and pcDNA3-Flag-NP vector,respectively.Co-immunoprecipitation (co-IP) and GST pull-down were used to test the interactions between H5N1 NP and NIK.Dual-luciferase reporter gene analysis system was used to test the effect of H5N1 NP on NIK-induced NF-κB transcriptional activity.Results Co-IP and GST pull-down showed that pCMV-Myc-NIK and pGEX-4T-1-NP (GST-NP) could express Myc tagged NIK protein and GST tagged NP protein in HEK293T cells and E.coli,respectively,and that H5N1 NP was associated with NIK in vivo and in vitro.Dual-luciferase reporter gene analysis suggested that H5N1 could inhibit NIK-induced NF-κB transcriptional activity.Conclusion H5N1 NP interacts with NIK and inhibits NIK-induced NF-κB transcriptional activity.This finding can facilitate further study of H5N1.

Construction and preliminary study of biological functions of O157∶H7 typeⅢsecre-tion system effector NleF gene knockout mutant / 军事医学

Objective To construct Escherichia coli O157∶H7 T3SS effector NleF gene knockout mutant and its com-plementary strain, and probe its effects on bacterial growth and cell death .Methods T3SS Effector NleF gene knockout mutant ΔnleF was constructed with λ-Red homologous recombination .Complementary strain ΔnleF/NleF was constructed by transferring pET-24a(+)-NleF into ΔnleF competent cells.Wild type,ΔnleF and ΔnleF/NleF were cultured in LB and DMEM(10%FBS) respectively,D600 was measured every hour , and the growth curve was drawn .HeLa cells were infected with three kinds of strains , the supernatant of LDH release was detected with cytotoxicity detection kit ,and the cytotoxicity was calculated .Results ΔnleF and ΔnleF/NleF were constructed .The growth rates of wild type , ΔnleF and ΔnleF/NleF was not significantly different .Wild type O157 infection induced cell death .Cytotoxicity was increased as much in ΔnleF in-fected cells as in ΔnleF/NleF infected cells.Conclusion EHEC O157∶H7 T3SS Effector NleF has no significant effect on bacterial growth ,but might inhibit host cell death caused by bacterial infection .

Prokaryotic expression, purification, identification, and polyclonal antibody prepa-ration of enterohemorrhagic Escherichia coli effector NleB1 / 军事医学

Objective To construct a prokaryotic plasmid expressing the recombinant protein of enterohemorrhagic Escherichia coli(EHEC) effector NleB1 and to prepare the polyclonal antibody of mouse anti-NleB1.Methods The nleB1 (990 bp) gene was amplified from the genome EHEC O157∶H7 and cloned into the expression plasmid pET24a to construct the recombinant plasmid pET24a-nleB1 that was transformed into E.coli BL21(DE3).After induction with isopropylthio-gelactoside( IPTG) , the His-tag fusion proteins were purified by Ni+affinity chromatography and gel slices.The polyclonal antibody was prepared by immunizing BALB/c mice with purified recombinant proteins and analyzed by Western blotting and ELISA.Results The pET24a-nleB1 recombinant plasmid was successfully constructed, the fusion protein was ex-pressed and purified,and the polyclonal antibody was obtained by immunizing mice with purified fusion protein.Western blotting and ELISA staining demonstrated that the polyclonal antibody was successfully obtained.Conclusion The prepara-tion of the polyclonal antibody against EHEC O157∶H7 NleB1 will be of help for further studies on the function of NleB1 protein.