Soluble expression,purification and immunological properties of recombinant H.pylori adhesin A / 中国生物制品学杂志

@#ObjectiveTo obtain recombinant H.pylori adhesin A(rHpaA)by molecular cloning,protein expression and purification,immunize BALB/c mice to prepare anti-HpaA polyclonal antibody,and analyze its antibody specificity.MethodsThe three-dimensional structure and antigenic properties of rHpaA were analyzed by bioinformatics softwares such as Phyre2 and DNAstar;Adhesin HpaA gene was obtained by PAS(PCR-based accurate synthesis)and inserted into plasmid pCzn1.The prepared recombinant plasmid pCzn1-rHpaA was transformed to E.coli Artic Express(DE3),induced by IPTG and purified by Ni-IDA affinity chromatography to obtain rHpaA protein,which was identified for reactivity by Western blot.Six male BALB/c mice were immunized with rHpaA plus Freund's adjuvant to prepare anti-HpaA polyclonal anti-body,and the antibody specificity was identified by ELISA.ResultsrHpaA showed good three-dimensional structure and antigenic properties.Restriction analysis and gene sequencing showed that the recombinant plasmid pCzn1-rHpaA contained completely correct HpaA gene sequence.The recombinant strain pCzn1-rHpaA/Arctic Express expressed the soluble target protein rHpaA,which accounted for about 68.3% of total protein in the supernatant,with a purity of 98.1%.rHpaA bound to anti-His antibodies and anti-H.pylori antibodies;The anti-HpaA polyclonal antibody specifically recognized rHpaA and H.pylori lysates.ConclusionrHpaA protein with high purity can be obtained by induction at low temperature and purification.The prepared anti-HpaA polyclonal antibody had good specificity,which laid an experimental foundation of the development of H.pylori-related diagnostic reagents.

Phage display and immunological identification of efficient T- and B-combined antigenic epitopes in Helicobacter pylori adhesin A / 中华微生物学和免疫学杂志

Objective To analyze and determine the efficient T- and B-combined (T/B) antigenic epitopes in Helicobacter pylori adhesin A. Methods Recombinant HpaA (rHpaA) was expressed for immunizing rabbit to generate antiserum. T- and B-cell epitopes in HpaA molecule were predicted by using bioinformatic technique. The segments to encode T/B combined epitope peptides were amplified by PCR and the phage display systems of T/B combined epitopes were subsequently constructed. PEG/NaCl precipitation method was applied to extract the recombinant phage PⅢ (rPⅢ) that displayed T/B combined epitopes. By using either commercial IgG against whole-cell of Helicobacter pylori or rHpaA antiserum as the primary antibody, the T/B combined epitopes displayed in rP Ⅲ s were screened and identified by Western blot and ELISA. MTT was applied to determine the proliferation of rHpaA-immunized mouse splenocytes after stimulation of the different recombinant rPⅢ proteins. Results In the HpaA molecule there were five T/B combined epitopes: HpaA10, HpaA37, HpaA79, HpaA116 and HpaA143. All the T/B combined epitopes were successfully displayed on the surface of PⅢ protein of phage M13. The results of Western blot, ELISA and MTT confirmed that HpaA116 was the predominant antigenic epitope, both HpaA37 and HpaA79 were the efficient antigenic epitopes. However, HpaA10 and HpaA143 were identified as ineffective antigenic epitopes. Conclusion The phage display systems of T/B combined epitope peptides of H. pylori adhesin A have been successfully generated in this study. HpaA37 and HpaA79, especially HpaA116 are the efficient T/B combined antigenic epitopes in HpaA of H. pylori.

Immune response to pT-ureB and pT-hpaA DNA vaccine in mice / 第三军医大学学报

Objective To investigate and compare the humoral immune response induced by eukaryotic expression plasmid pTCAEureB (pTureB)and pTCAEhpaA (pThpaA) injected intramuscularly in mice. Methods The ureB gene fragment was inserted into a eukaryotic expression vector pTCAE, and pTCAEureB was constructed. A total of 60 BALB/c mice were randomly assigned to be immunized by intramuscular injection of blank plasmid pTCAE, plasmid pTureB and pThpaA (n=20 in each group). Titers of specific IgG antibody in serum of each group were detected through ELISA respectively on week 2, 4, 6 after immunization. Results Specific IgG were observed in pTureB and pThpaA immunized groups and titers of antibody increased as time prolonged. IgG level was significantly higher in pT-ureB immunized group than in pT-hpaA group on week 6 after immunization. Conclusion Plasmid pT-ureB possesses good immunogenicity and induces a higher immune response than pT-hpaA.

RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) OF ADHESIN GENE HPA A FROM DIFFERENT HELICOBACTER PYLORI STRAINS / 微生物学通报

To assess the variability of adhesin gene hpaA in differene H pyloristrains with PCR restriction fragment length polymorphism (RFLP) A 710 bp gene hpaA , obtained from 9 different H pylori strains, were digested by Hha Ⅰand Hae Ⅲ individually and analyzed by agarose gel electrophoresis Four different polymorphic types were found in hpaA digested with Hae III and five types with Hha I Clinical isolates of H pylori from Chongqing showed difference among them and remarkably distinguished from foreign standard strains Mongolia gerbil adapted H pylori strain,which were obtained from Mongolia gerbil infected with clinical isolate, also showed inconsistence in hpaA RFLP The hpaA gene from different H pylori strains revealed 1 variability, and this might provide an effective method for developing molecular epidemiology of H pylori

Construction and identification of eukaryotic expression plasmid of hpaA of H.pylori / 第三军医大学学报

Objective To clone hpaA gene of H.pylori and construct its eukaryotic expression plasmid. Methods Gene hpaA amplificated from genome of H.pylori 11637 strain by PCR was subcloned into pMD18-T vector. Then hpaA cut down from the vector with restriction enzyme was inserted to pTCAE, and the product confirmed by restriction enzyme digestion and sequence was transformed to E.coli DH5?. pT-hpaA was transfected into CHO cell by electroporation, and HpaA protein was detected by Western blot. Results hpaA gene was inserted into pTCAE and the immunological reaction band with anti-HpaA serum at MW 30 000 was detected by Western blot. Conclusion Eukaryotic expression plasmid of hpaA of H.pylori is successfully constructed. Western blot analysis demonstrates that the expression of HpaA protein can be detected in culture supernatants of transfeced CHO cells, which lays the foundation for the further study.

In vitro studies of IgY against fusion VacA-HpaA protein of Helicobacter pylori / 中国免疫学杂志

Objective:Researching physical-chemic and biologic speciality of VacA-HpaA IgY,which provide a solid experimental basis for the preparation of vaccine against H.pylori infection.Methods:The purified antigen of fusion protein VacA-HpaA was used to immunize hens and the VacA-HpaA IgY was extracted from the yolk by water dilution methods.(1)After the VacA-HpaA IgY was purified by deposition technique with ammonium sulfate,the purity of IgY was analyzed by SDS-PAGE,and protein content of IgY was analyzed by Bradford method.(2)The indirect ELISA was used to detect its heat-stability,acid-stability and pepsin-stability.(3)The specificity of VacA-HpaA IgY to the antigens was identified by Western blot.(4)MTT was applied to assay the neutralization of VacA-HpaA IgY to The cytotoxity of H.pylori.(5)The neutralization of IgY to AGS cells was observed with scanning electron microscopy(SEM).Results:(1)The purity of VacA-HpaA IgY was about 60%,and the protein content of IgY was 22g/L;(2)The IgY showed a good heat-stability,a favorable acid-stability,and a well ability of anti-pepsin digest;(3)Western blot exhibited the protein bands with molecular weight of 27 000 and 30 000.The titer of VacA-HpaA IgY to VacA and HpaA were 1∶3 200 and 1∶6 400;(4)A dose and time dependent correlation of the IgY to counteract the cytotoxic activity of VacA;(5)VacA-HpaA IgY could interrupt the adhesive attraction of H.pylori.Conclusion:The VacA-HpaA IgY has good stabilities and well biologic speciality,which imply that the IgY could be used to prepare treatmental antibody against H.pylori infection.