Objective To prepare humanized monoclonal antibodies(Mabs)targeting Acinetobacter baumannii(Ab)based on single memory B cell sequencing technology,construct the immune repertoire of the core protein of Ab,hemolysin-coregulated protein(Hcp),and express its Mabs with binding activity.Methods E.coli BL21 harboring the recombinant plasmid pGEX-6p-1-Hcp was constructed.Hcp protein was obtained using protein expression and affinity chromatography.Female SPF BALB/c mice(6～8 weeks old,weighing 18～20 g)were immunized intramuscularly with antigen Hcp to generate specific memory B cells.Single antigen-specific memory B cells were sorted using flow cytometry.The immune repertoire of Hcp was constructed using single-cell sequencing technology,and bioinformatics analysis was performed on the sequencing results.Mabs were obtained using antibody humanization techniques.The in vitro binding activity of the antibodies was detected by ELISA.Results The target protein Hcp with a purity>95%was obtained after expression and purification.The immune repertoire of Hcp was successfully constructed,and the results of BCR clonotype identification and analysis,CDR3 region characteristic analysis,and V-J gene pairing characteristic analysis were achieved.Antibody humanization got 7 Mabs,that is,IgG1-1,IgG1-2,IgG2-1,IgG2-2,IgG3-1,IgG4-1 and IgG4-2.ELISA results showed IgG1-1,IgG3-1,IgG4-1,and IgG4-2 had an antibody binding titer of 1∶1 280,IgG2-2 of 1∶10 240,IgG2-1 of 1∶5 120,and IgG1-2 of 1∶160.Conclusion Single-cell sequencing technology enables rapid,accurate,and efficient construction of an Hcp protein immune repertoire containing extensive antibody information.Utilizing this immune repertoire allows for the expression of Mabs with binding activity.

Phage antibody display technology is currently the most widely used in vitro antibody screening technology,which uses bacteriophages as a vector,and inserts exogenous antibody library genes into phage capsid protein genes,and expresses the capsid protein on the phage surface while also displays the antibody protein.Antibody drugs play an important role in tumor immunity and microbial immunity due to their targeting advantages,which is also an important driving force for them to become a hot spot in the field of pharmaceutical research and development.Therefore,this article reviews the background,basic principles,antibody library types and antibody fragment types of phage display technology,and looks forward to the latest progress and application prospects of fully human antibodies.

Chronic inflammation induced by Helicobacter pylori is considered to be one of the main causes of gastric cancer,and CagA is a main virulence factor of H.pylori.The study aimed to investigate the role and mechanism of CagA in host inflammatory response.Mass spectrometry was used to identify the interacting proteins of CagA in AGS cells.By immunoprecipitation and immunofluorescence,the interaction was validated.Pathway expression was detected by immunoblotting after knockdown by using siRNA,and mRNA levels of inflammatory cytokines were detected by quantitative PCR.CagA-induced inflammatory responses were detected in clinical samples using hemoglobin-eosin staining(H&E).Data showed that CagA interacted with SHARPIN.And CagA activated the NF-κB signaling pathway and upregulated the mRNA and protein levels of the inflammatory cytokines IL-6,IL-8,and TNF-α,as compared with the CagA knockout strain(all P<0.05).Knockdown of SHARPIN by siRNA reduced inflammation levels and partially inhibit NF-κB signaling.In clinical samples,CagA-positive samples exhibited stronger inflammatory responses.To sum up,CagA promoted the host inflammatory response,and CagA-induced inflammatory response was reduced when SHARPIN was partially inhibited,suggesting that CagA activates the NF-κB signaling pathway through binding to SHARPIN.

Objective To investigate the correlation between Helicobacter pylori (HP) infection-associated gastritis and the ap-optotic genes in gastric mucosa .Methods Forty-five patients with chronic gastritis were registrated in our study from November 2013 to December 2014 .HP infection status in the patients was detected by using urease test and 13C-urea breath test .The degree of gastritis in the gastric mucosa with HP infection was confirmed via histopathology .qRT-PCR was used to measure the mRNA ex-pressions of Bax ,Bak and Bcl-2 in the gastric mucosa with HP infection and matched normal gastric mucosa .Person analysis was used to assess the correlation between the HP infection-associated gastritis and the mRNA expressions of Bax ,Bak and Bcl-2 in the gastric mucosa .Results Forty-five patients with HP infection in antrum and 45 patients (100% ) with chronic antrum gastritis were identified ,including 28 patients (62 .2% ) with light gastritis ,16 patients (35 .6% ) with moderate gastritis ,1 patient (2 .0% ) with severe gastritis .9 patients (20 .0% )with metaplasia ,5 patients(11 .1% ) with low grade intraepithelial neoplasms .The urease tests were negative in the gastric body of 45 patients ,6 patients (13 .3% )were mild chronic gastritis in the body ;Patient with meta-plasia and intrapithelial gastritis was not found .The Bax expression in the HP-infected gastric mucosa was markedly increased when compared with the normal gastric mucosa (P< 0 .01) ,and positively correlated with the degree of gastritis (P< 0 .01) , whereas the expressions of Bak and Bcl-2 have no significantly deferences bttween two groups(P>0 .05) .Conclusion HP infec-tion-associated gastritis positively correlated with the expressions of apoptotic genes in gastric mucosa ,suggesting that HP infection might result in increasing the Bax expression and further enhancing the cell apoptosis .

Objective To isolate and purify VacA protein secreted by Helicobacter pylori or recombinant VacA , and to investigate the effect of VacA-induced cell vacuolar change and apoptosis .Methods VacA proteins were separated and pu-rified from the culture supernatant of H.pylori ( ATCC26695 ) or from the split products of genetically engineered bacteria (pQE30-VacA-E.coli M15) expressing recombinant VacA.The VacA protein obtained was acidified and then incubated with AGS cells for 24 h at different final concentrations of 5 and 10 ng/ml before the vacuolar change and apoptosis of AGS cells were detected via microscopy and flow cytometry assay , respectively .Results H.pylori-secreted VacA and recombi-nant VacA were successfully separated and purified .The H.pylori-secreted VacA significantly induced the vacuolar change and apoptosis of AGS cells (P<0.01) while the recombinant VacA did not.Conclusion H.pylori-secreted VacA protein can effectively induce cell vacuolar change and apoptosis, but recombinant VacA can not, suggesting that the purified VacA protein secreted by H.pylori can be used to explore VacA-induced pathogenesis.