Construction, Expression And Characteristic Research For Reconstituted Human SIgA Specific To H.pylori Urease Subunit B

Helicobacter pylori (H. pylori), a Gram-negative bacterial pathogen, has been associated with the development of chronic gastritis and peptic ulcers and is considered a risk factor for the development of gastric adenocarcinoma and malignant mucosa-associated lymphoid tissue lymphoma. The overall prevalence of H. pylori infection is strongly correlated with socioeconomic conditions. The prevalence among middle-age adults is over 50% in many developing countries, as compared with 40% in industrialized countries. H. pylori infection in adults is usually chronic and does not heal without specific therapy. Because gastric luminal acidity influences the effectiveness of some antimicrobial agents that are reactive against H. pylori, antibiotics are combined with proton-pump inhibitors and ranitidine bismuth citrate. Although combined antibiotic therapy is available to treat this infection, the development of an effective prophylactic or therapeutic vaccine would be of enormous benefit, especially in countries where there is a high incidence of gastric cancer, high reinfection rates, and increasing antibiotic resistance. However, although vaccination has been successful in animal models, translation to a human vaccine remains difficult. Human clinical trials with different vaccine candidates showed poor efficacy, and illustrate that a more in-depth understanding of the immune responses to H.pylori infection as well as additional information on suitable epitopes and adjuvants are required before a successful vaccine can be developed. Experiments demonstrated that a oral administrated urease specific monoclonal antibody immunoglobulin A had a ability to prevent Helicobacter felis infection to mice. Another study demonstrated immunoglobulin A in maternal milk may protect Gambian infants from early H.pylori colonization. These data suggest that sIgA antibodies in gastrointestinal tract were very important in preventing H. pylori infection. Most tests demonstrated that H. pylori urease subunit B (UreB) could be used as an oral vaccine against H. pylori. The possible role of IgA mAb and sIgA in preventing H. pylori adhesion and infection combined with the capacity to engineer complex Ab prompted us to develop entirely human recombinant sIgA Ab from a single chain Fv (scFv) polypeptide specific for H. pylori UreB. Main results are listed below:1. scFv--displaying phage library construction, selection and sequencingAfter mixed peripheral blood mRNA was isolated from sera anti- H. pylori and anti- H. pylori UreB positive patients, we obtained the code fragments of antibody heavy and light chain variable region (VH and VL) which were about 400bp and 350bp respectively by RT-PCR. Then, the VH and VL PCR products were purified and connected using overlapping method. Subsequently, the library was constructed by ligating overlapping products into the vector pComb3X, and was calculated to contain 4.6 x 108 independent clones and high diversity was generated. Later, purified H. pylori UreB was used to coat a microtiter plate, The binding sites were blocked 3% BSA (w/v), and incubated phages in each well, then washed the plates five times for the first round of panning, decreasing the coating concentration of H. pylori UreB and increasing the number of washes for each subsequent round. After 5 rounds of panning, 41 clones from the library were verified to show strong binding affinities for H. pylori UreB by enzyme-linked immunosorbent assay (ELISA), the strongest monoclonal phage, termed scFv1, was isolated. The specificity of scFv1 was further demonstrated by immunoblot. scFv1 react to H. pylori stains and UreB only, and not to other non- H. pylori bacteria .SO the scFv1 gene fragment was sequenced and analysed.2. Construction of Expression Vector pcDNA3.1(+):HSVHCα1 and pcDNA3.1(+): LSVLCλ₂The Cα1 region was amplified from neutrophilic leukocyte by RT-PCR carrying the new sites EcoRI and Xba I. Heavy chain signal peptide coding sequence was obtained by primer overlapping method, and ligating with VH fragment by overlapping approach introducing the HindIII and EcoRI sites. Expression vector pcDNA3.1(+):HSVHCα1 was assembled from a three-piece ligation including (i) plasmid pcDNA3.1(+)cleaved with HindIII / Xba I, (ii) the fragment include heavy chain signal peptide coding sequence and VH fragment treated with HindIII/ EcoRI, and (iii) the Cα1 region cut with EcoRI/XbaI. The regions coding for Cλ2 was amplified from genomic DNA of human spleen by PCR introducing the EcoRI and NotI sites. Light chain signal peptide coding sequence was obtained by same method as heavy chain, and ligating with VL fragment by overlapping approach carrying the new sites HindIII and EcoRI. Expression vector pcDNA3.1(+):LSVLCλ₂ was assembled from a three-piece ligation including: (i) Plasmid pcDNA3.1(+) cleaved with HindIII /NotI, (ii) the fragment include light chain signal peptide coding sequence and VL fragment treated with HindIII/ EcoRI, and (iii) the PCR fragment with the Cλ₂ region cut with EcoRI /NotI.3. Construction of Expression Vector pcDNA3.1(+)Hyg:J Chain and pcDNA3.1 (-)Bsd:SCThe region comprising the neoR gene in pcDNA3.1 (+) was excised by digestion with SmaI and Bpu14I. A 1.1-kb fragment containing the hygR gene was amplified from plasmid pMEP4 (Invitrogen) by using primers containing a SmaI site and a Bpu14I site. After digestion with SmaI and Bpu14I, the PCR fragment was introduced into the corresponding sites of pcDNA3.1(+), resulting in the production of pcDNA3.1(+)Hyg. A about 430bp fragment coding for human J chain was amplified from RNA of human spleen by RT-PCR using primers introducing a HindIII site and a NotI site. The RT-PCR product was digested with HindIII and NotI before ligation into pcDNA3.1(+)Hyg treated with same enzymes, creating expression vector pcDNA3.1(+)Hyg:Jchain. The secretory component (SC) coding sequence was obtained from human colon adenocarcinoma HT29 cells by RT-PCR with primers carrying the new sites HindIII and XbaI. Before ligation into pcDNA3.1(-)Bsd which was cut with HindIII and XbaI , the SC fragment cleaved with same enzymes, and then expression vector pcDNA3.1(-)Bsd:SC was constructed.4. Establishment of Stable CHO Cells Expressing sIgA specific for H. pylori UreBThe CHO cells were cultured in IMDM medium supplemented with dialyzed fetal calf serum (FCS), Hepes (pH 7.0), penicilin and phytomycin. Cells (1×10⁷) were electroporated with ScaI-linearized pcDNA3.1(+):VHCα1 and BglII-linearized pcDNA3:VLCλ₂. Selection was started 24 h later in the presence of G418 (500μg/ml), and continued for 3 more weeks. Cell clones were isolated at one cell per well in 96-well culture dishes, and emerging clones were then tested by dot-ELISA. Clone1 which produced the highest amount of IgA1, was amplified to serve as the recipient cell for subsequent transfection with BglII -linearized pcDNA3.1(+)Hygro:Jchain. Screening was carried out in the presence of G418 (500μg/ml)and hygromycin (400μg/ml). Clone 2, which secreted the largest amount dIgA, was chosen. The CHO cells were electroporated with BglII-linearized pcDNA3.1(-)Bsd:SC. Selection was started 24 h later in the presence of blasticidin (10μg/ml),and kept on for 3 more weeks, Clone 3, which produced the highest amount dIgA, was obtained. Before mixed and incubated in 37℃2h , cell culture supernatants of Clone 2 and Clone 3 were concentrated respectively. Then, sIgA was obtained by combining IgAp/d with SC. The specificity of sIgA was further demonstrated by immunoblot. sIgA only react to H. pylori stains and UreB, and not to other non- H. pylori bacteria .After reacted to sIgA, the amount of H. pylori which adhere to gastric epithelial cells GES-1 was decreased significantly, so sIgA we reconstituted prevent the H. pylori adhesion.In conclusion, we demonstrate that an active human sIgA antibody molecule can be assembled in heterologous mammalian cells, and a recombinant sIgA Ab specific for H. pylori UreB can inhibit the adhesion of the bacterium to gastric epithelial cells GES-1.Our data indicate that sIgA Ab may be valuable alternatives or complements to combined antibiotic therapy for all patients where H. pylori eradication was unsuccessful.