| Infection of Helicobacter pylori(Hp)can lead to serious health problems.By the year of 2022,Helicobacter pylori has been colonized in the stomach of 4.4 billion people around the world.At present,the treatment of Hp is mainly antibiotics,but with the abuse of antibiotics,the drug resistance of Hp increases,and the failure of treatment may gradually lead to the development of primary infection into more serious complications.Urease is the key factor of Hp colonization and pathogenesis in the stomach.Inhibiting urease activity is very important to reduce the risk of Hp infection.In this study,the gene engineering expression system of recombinant nano antibody Ab specific for Hp urease subunit(UreB)was constructed,and its expression in food grade probiotics was explored.The main research contents are as follows:(1)Preparation and activity determination of Hp urease: Hp were broken by ultrasound,and the supernatant was dialyzed,desalted and concentrated to prepare urease.The content of urease was 0.73 mg/ml determined by quick start Bradford method.The activity of the extracted urease was determined qualitatively and quantitatively by phenol red indicator method and indophenol method.The results showed that the prepared urease had high activity,and its activity was 27.77 U/mg.(2)Structural analysis of anti UreB nano antibody(UreB-Ab): I-TASSER software analysis shows that the matching score between UreB-Ab and 6EHW structural model in PDB database is the highest,so the structural model with the highest score is obtained.Then,Cluss Pro2 software is used for UreB-Ab docking prediction,and pmyol software is used for intermolecular force analysis.The results showed that there was a strong interaction force(hydrogen bond)between UreB and Ab.(3)Heterologous expression and activity detection of UreB-Ab in E.coli: recombinant vectors such as p ET-28a(+)-Ab,Pe-SUMO-Ab and p Mal-c2x-Ab were constructed respectively,and Ab,SUMO-Ab and MBP-Ab were obtained after induced expression in E.coli Rosetta(DE3).The results of one-way immunodiffusion experiment showed that urease and recombinant antibody could form a white precipitation ring around the hole.The activity measurement results showed that the inhibition rates of Ab,SUMO-Ab and MBP-Ab on urease were 48.0%,74.0% and 12.5%,indicating that the recombinant antibody had good affinity and inhibitory activity to HP UreB antigen.(4)Heterologous expression and activity detection of UreB-Ab in probiotics:recombinant vectors such as PTM151-Ab,PNZ8148-Ab and PNZ8148-Usp45-Ab were constructed and expressed in probiotic strains E.coli Nissle 1917(Ec N)and Lactococcus NZ9000.Unidirectional immunodiffusion experiment confirmed that the obtained antibodies could effectively bind to UreB antigen.The activity determination results showed that the inhibition rate of Ab expressed in ECN to urease was 67.4%,while the inhibition rate of Ab expressed in Lactococcus lactis to urease was 85.7%.It can be used as a potential oral probiotic delivery system for Helicobacter pylori antibody drugs.Conclusion: In this study,the recombinant nano antibody(UreB-Ab)probiotic expression system specific for HP urease subunit B(UreB)was obtained by using genetic engineering method,which is expected to be delivered into the body in the form of live bacteria to achieve direct and accurate prevention and control of Hp.At the same time,probiotics will also play a beneficial role in the gastrointestinal tract,so as to "double Swords" quickly kill Hp.In addition,antibodies that specifically recognize Hp antigen can not only deal with infection,but also overcome the development of bacterial drug resistance.Therefore,this study has laid a good foundation for the development of immunodrug therapy for HP,and has good innovation and application potential. |
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