Preparation And Identification Of Polyclonal Antibody Against Methyl-accepting Chemotaxis Signal Transduction Protein Of Helicobacter Hepaticus

Objective and significanceSince the discovery of Helicobacter pylori, various enterohepatic Helicobacter spices have been detected in the guts of humans and animals. Some enterohepatic Helicobacters have been associated with inflammatory bowel disease or liver disease in mice. However, these studies are limited to animal models. To date, the pathogenicity and epidemiology of Helicobacter hepaticus (H.hepaticus) on human are not known yet. In this study we aim at prepare a polyclonal antibody against methyl-accepting chemotaxis signal transduction protein(MCP) of Helicobacter hepaticus, extract cell surface proteins(CSPs) of H.hepaticus, H.bilis and H.pylori and finally identify the specificity of the antibody by western blotting analysis from CSPs probed with anti-MCP. This is the foundation of detecting people's infection rate of Helicobacter hepaticus in our country and approaching the etiopathogenisis of liver diseases and intestinal non-specificity inflammation of human.Materials and methods1. Main materials:Helicobacter hepaticus strain and Helicobacter pylori strain, Campylobacter jejuni selective blood agar medium, Freund's complete adjuvant, Freund's incomplete adjuvant, goat anti-rabbit IgG labelled by horse radish peroxidase(HRP), Balb/C mice, New Zealand white rabbits.2. Methods2.1 Isolation and identification of Helicobacter bilis.The method of isolating Helicobacter bilis (H.bilis) is according to reference. C.jejuni selective agar supplemented with defibered 7-8% sheet blood was used to isolate H. bilis from the intestine of specific-pathogen free laboratory Balb/c mice under microaerobic conditions at 37℃. After subculture, suspected bacterial colony was identified by the following experiments. Gram's staining was performed to identify the Gram stain properties of the suspected strain. Biochemical characterization including repid urease test, oxidase test, peroxidase experiment and nitrate reductase test were performed. The colonies which were transparent and needle tip-like was scraped into the EP tube with cotton bud, then washed three times with PBS. The genome DNA of Helicobacter hepaticus was extracted using bacteria genome DNA extracting kit. With the genome DNA of Helicobacter hepaticus as template, the 16srDNA gene was amplified by PCR. The PCR conditions were initial denaturation at 94℃for 10min, followed by denaturation at 94℃for 30sec, annealing at 54℃for 30sec, and extension at 72℃for 55s for 35 cycles, with the final extension at 72℃for 10min after the last cycle of amplification. Gene sequencing was proceeded to determine the species of the suspected bacterial.2.2 Bacterial culture.100μl bacterial liquid was spreaded to Campylobacter jejuni(C. jejuni) selectivity blood agar medium which contained 7% freeze thawing dis-fibrilla sheep blood,1% selective antibiotic including vancomycin 10mg/L, Polymyxin B 2500U/L, Amphotercin B 10mg/L and TMP 5mg/L. The bacteria were cultivated under microaerobic condition(5%O2,10%CO2, and 85%N2) at 37℃and observed everyday.2.3 Extraction of cell surface proteins(CSPs) of bacteria.Cell surface proteins were extracted following the method mentioned before. After 48 to 72 hours' culture on Campylobacter jejuni(C. jejuni) selectivity blood agar medium, the bacterial colonies were scraped and washed three times with Sterile double-distilled water. The bacteria were collected by centrifuging 4000r/min for 20 minutes at room temperature. Added 100ml Glycine-HC1 buffer to every 4 gram bacteria and Magneticly stirred at 4℃for 30 minutes. And then centrifuged 12000r/min at 4℃for 15 minutes to remove debris and dialysed against PBS. Finally concentrated and measured protein concentration.2.4 Expression of recombinant MCP protein in E.coliThe pET22b+/MCP plasmid was transformed into E.coli BL21(DE3) and plated on LB agar medium containing ampicillin(100μg/ml). Mono-colony was cultivated in 10ml LB medium at 37℃,200rpm for half and three hours until that OD600 of culture solution was between 0.6 and 1.0, then 100ul culture solution was continued to cultivate in 10ml LB medium at 37℃,200rpm for half and one hours until OD600 was 0.6 approximately.500mmol/L IPTG was added into the culture solution which was continued to culture at 37℃,200rpm for four hours. The induced solution was centrifuged at 4℃,12000rpm for five minutes, then identified by SDS-PAGE and Western blot.2.5 Purification of recombinant MCP protein1000ml induced solution was centrifuged at 4℃,12000rpm for five minutes, in which bacteria lysate and PMSF was added subsequently. After crushed by sonicator on the ice and centrifuged at 4℃for fifteen minutes, the supernatant of solution was abandoned. The sedimentum was put at room temperature for thirty minutes after added bacteria lysate and PMSF again, then centrifuged at 4℃for fifteen minutes. The supernatant was put in the chromatography, and eluted by UrNTA-20, UrNTA-40, UrNTA-60, UrNTA-100, UrNTA-200, UrNTA-500 respectively. The target protein of eluted solution was definited by SDS-PAGE. The eluted solution was dialysed by gradient Urea-PBS. After measured protein concentration, the purified protein was stored at-70℃.2.6 Preparation of polycolonal antibody against recombinant MCP protein New Zealand White rabbits were injected subcutaneously with 800μg of purified MCP mixed with an equal volume of complete Freund's adjuvant after preimmune serum had been collected. The immunization was repeated at 4 and 6 weeks after the first injection. The animals were bled 1 week after the third injection and then once monthly. Antibody specificity and titers were determined by Western blotting and ELISA, and the animals were sacrificed when the specific antibody had produced and there was no further incease in antibody titers. The serum was preserved at -80℃.2.7 Purification of anti-serum2.7.1 Purification of anti-serum with ammonium sulfate precipitationThe anti-serum was Preliminary purified by adding equal volume of ammonium sulfate solution to anti-serum, reacting at 4℃for 30 minutes and collecting the IgG by centrifuging at 4℃,2000rpm for 15 minutes. The precipitation was re-desolved with cold PBS.2.7.2 Purification of polyclonal antibody by antigen-specific method.Firstly adding Carbonate buffer (PH 8.3) to CNBr-activated Sepharose 4B to pre-equalize chromatography. The purified MCP recombinated protein was add in to react overnight. The next day removed the unbound protein by centrifuging. The Preliminary purified antibody were mixed in after blocking and shaking at 4℃overnight. The sepharose was centrifuged after added in eluate. The supernatant was the antibody which would specifically combined with MCP recombinant protein. The antibody were preserved at -70℃after mixed in equal volum of glycerol and sodium azide(final concentration 0.02%).2.8 Western blotting Analysis of the specificity of rabbit anti-MCP antibody.A total of 20μg of the bacterial cell surface protein from H.hepaticus, H.bilis and H.pylori were mixed with 2×sample buffer, denatured at 95℃for 5 min, and separated on 12.5% SDS-PAGE. Proteins were transferred to PVDF membranes (Milipore). Membranes were blocked with 1% BSA in PBS for 2hr and then incubated for 1hr with anti-MCP antiserum diluted 1:500 at room temperature. The membranes were washed three times with PBS/T (PBS with 0.1% Tween-20) and then incubated for 1hr with 1:5000 dilution of horseradish peroxidase-conjugated anti-rabbit antibody (Cell Signal). The bound antibody complexes were detected by DAB (Boster) or using the enhanced chemiluminescence (ECL) system from Amersham.Results1. Isolation and identification of H.bilisAfter 3 days culture of helicobacters in C.jejuni selective agar, H.bils has been isolated .The colonies were transparent and needle tip-like. It was Gram's stain negative and was "S" or "C"-shaped spiral which was similar with H.Pylori except for longer bacterial and with more sprials. Biochemical identification showed that the bacteria were positive for repid urease test, oxidase test, peroxidase experiment and nitrate reductase test. The bacteria genomic DNA was amplified using helicobacter genus primer and H.bilis specific primer .The PCR-generated DNA was electrophoretic separated in 1.5% Agarose gel. The result showed prositive bands at 780bp, respectively. Sequence analysis was compared with genome published on GenBank for homology and showed 99% similarity with H.bilis standard strain, ATCC43879, which comfirmed the bacterial isolated was H.bilis.2. Extraction of cell surface proteins(CSPs) of bacteria.Cell surface proteins were concentrated and seperated on 17.5% SDS-PAGE. Coomassie brilliant blue staining showed that the proteins were Mainly distributed in 75 KD to14 KD.3. Expression of recombinant MCP protein in E.coliRecombinant protein rhMCP of which the relative molecular mass was 14118D was expressed from E.coli BL21(DE3) containing recombinant plasmid after IPTG inducing, it was mainly in the sediment through SDS-PAGE identification, so the existence pattern of rhMCP was non-solubility cytorrhyctes. There was one specific strap of which the position was about 14KD by Western blot identification.4. Purification of recombinant MCP proteinrhMCP induced by IPTG was dissolved by 8M urea because its existing form was insoluble inclusion bodies and eluted by gradient urea-PBS according to purification method of degenerated protein. The best elution liquid was UrNTA200. The soluble rhMCP was acquired by dialysis with gradient urea-PBS after purification. The protein concentration of rhMCP was 2.08mg/ml by BCA.5. Immunizations and collection of antiserumNew Zealand White rabbits were injected subcutaneously with purified MCP for 4 times and Serum was collected and purified of IgG with ammonium sulfate precipitation and CNBr-activated Sepharose 4B. The purity and specificity of antibody werevanalysis by SDS-PAGE and ELISA. The results showed a high-specificity and high-titer rabbit anti-MCP polyclonal antibody was prepared.6. Further analysis of specificity of antibody using western blottingCell surface proteins were prapared and seperated by western blotting. As shown in Fig.2, specific reactivity with a protein of the anticipated relative molecular weight of approximately 14kD was detected in the extracts of H.hepaticus.ConclusionOur study successfully isolated H.bilis from the intestine of Balb/c mice. For production of the polyclonal antibody, a highly purified recombinant MCP protein was used as the immunogen. Injection of this MCP protein into rabbits produced anti-serum. The polyclonal antibody was purified and analyzed by Western blotting analyses using recombinant MCP as antigen revealed a single protein with a relative molecular weight of 14 kD. And a dilution of 1:32,000 was detected on ELISA. Western blotting analysis of cell surface protein of H.hepaticus, H.bilis and H.pylori probed with purified anti-MCP revealed only H.hepaticus showed positive protein. These results indicate that purified anti-MCP can detect MCP protein expressed in H.hepaticus and is highly specific.