Construction Of A Helicobacter Pylori 6S RNA Gene-deficient Strain And Preliminary Study Of Its Biological Function

Objective:To investigate the effects of different culture conditions on the expression of 6S RNA gene in Helicobacter pylori?Hp?.The 6S RNA mutant strain was constructed by genetic engineering to explore the biological function of 6S RNA in the pathogenesis of Hp.Methods:1.Effects of different culture conditions on Hp ATCC 26695 6S RNA gene expression:The transcriptional expression of Hp ATCC 26695 6S RNA were analyzed by RT-qPCR at different growth times?24h,26h,48h,60h?,different pH?5.0,5.5,6.0,7.5?,different temperatures?25?,30?,37?,42??to study the induction of Hp-6S RNA to external signals.2.Construction and phenotypic analysis of Hp-6S RNA gene-deficient strains:?1?Plasmid pSD-6S was constructed by homologous recombination technique,and Hp ATCC 26695 was electrotransformed.Hp-6S RNA-deficient strain??6S RNA?was screened and identified by colony PCR and RT-qPCR.?2?Comparison of phenotypic differences between wild strain Hp ATCC 26695and mutant strain?6S RNA:?1?The OD₆₀₀00 value of Hp ATCC 26695 and?6S RNA in BHI liquid medium was measured every 12h to prepare a growth curve at microaerobic atmosphere.?2?The scanning electron microscope was used to observe the morphological changes of the two strains at 24h,36h,48h,and 60h.?3?The total protein of Hp ATCC 26695 and?6S RNA were extracted,and the expression of CagA protein was detected by Western-Blot at 24h,36h,48h,and 60h.The urease enzyme immunoassay kit was used to detect the difference of urease content and activity.?4?The culture supernatant and bacterial cell disruption of Hp ATCC 26695and?6S RNA at 24h,36h,48h,and 60h were collected into GES-1 cells,the Hp medium and RPMI1640 medium were used as negative control and blank control respectively.The changes of cell morphology were observed at 12h,24h,36h and 48h,and the same time the viability of the cells was detected by CCK-8 method.Results:1.?1?Determination of the growth curve of Hp ATCC 26695:Hp ATCC 26695showed a lag phase,log phase,and stationary phase in the microaerobic environment at 37?with the prolongation of the culture time,and reached the logarithmic peak at48h.After 60h,it entered a stable period.?2?RT-qPCR was used to detect the effect of different culture conditions on Hp ATCC 26695 6S RNA gene expression:?1?One-way analysis of variance showed that the expression of Hp ATCC 26695 6S RNA gene was statistically different at 24h,36h,48h and 60h?P<0.05?,and the expression level was the highest at 24h,and the expression decreased gradually with the prolongation of culture time.?2?Under different pH?5.0,5.5,6.0,7.5?conditions,pH7.5 was used as a control,the expression of 6S RNA gene was the highest?45.026±22.014,t=-12.454,P=0.000<0.05?.)when pH=6.0 and cultured for 36h.?3?One-way analysis of variance,Hp ATCC 26695 6S RNA gene expression was not statistically significant?F=0.475,P=0.708>0.05?at 25?,30?,37?,42?for 24h.2.?1?Construction of Hp-6S RNA-deficient strains:The Hp-6S RNA-deficient strain was successfully constructed by colony PCR,RT-qPCR and sequencing analysis,and the Hp-6S RNA-deficient strain was named?6S RNA.?2?Determination of?6S RNA and Hp ATCC 26695 growth curve:Hp ATCC 26695and?6S RNA showed a lag phase,log phase,and stationary phase in the microaerobic environment at 37?with the prolongation of culture time.One-way analysis of variance showed that the growth of Hp ATCC 26695 and?6S RNA was statistically different after 12h of culture?P<0.05?,?6S RNA was delayed by 12h compared with Hp ATCC 26695;Hp ATCC 26695 reached a logarithmic peak at 48h while?6S RNA at 60h.?3?Hp ATCC 26695 and?6S RNA at different time points?24h,36h,48h,60h?were similar in morphology under the scanning electron microscope,and no significant difference was found.?4?There was no significant difference in the expression of?6S RNA and Hp ATCC 26695 CagA protein at different time points?24h,36h,48h,60h??P>0.05?.There was a statistical difference of?6S RNA CagA protein expression was at 24h and 60h by One-way analysis?F=2.201,P=0.043<0.05?.?5?There was statistical difference between?6S RNA and Hp ATCC 26695 urease content OD₄₅₀50 in culture for 48h?t=5.164;P=0.026<0.05?;urease activity OD₄₅₀50 was at 24h?t=22.486;P<0.05?,36h?t=6.995;P<0.05?and 60h?t=3.497;P<0.05?.?6?The toxic effects of supernatant and disrupted solution of Hp ATCC 26695 and?6S RNA on GES-1 cells at different time points were different.The toxic effect of?6S RNA supernatant and disrupted solution on GES-1 cells is weak compared with Hp ATCC 26695.Conclusions:1.Hp-6S RNA is an essential transcriptional regulator that responds to different culture conditions.2.Hp-6S RNA gene has a specific regulatory effect on the growth,and expression of virulence factors of Hp.