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Preparation and Identification of Polyclonal Antibody Against Methyl-accepting Chemotaxis Signal Transduction Protein of Helicobacter Hepaticus

Author: ChenYaHua
Tutor: WangJiDe；YaoYongLi
School: Southern Medical University,
Course: Internal Medicine
Keywords: Gall Helicobacter Separate Helicobacter hepaticus MCP Polyclonal antibody
CLC: R378
Type: Master's thesis
Year: 2010
Downloads: 39
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Abstract

Purpose and significance of liver Helicobacter pylori (Helicobacter hepaticus, Hh) pathogenesis and epidemiological characteristics is not yet entirely clear. This study preparation Helicobacter hepaticus methyl groups to accept chemotaxis signal transduction proteins (methyl-accepting chemotaxis signal transduction protein, MCP) rabbit antiserum in laboratory animals and humans to further explore the Helicobacter hepaticus infection and clinical testing The development kit provides an experimental basis. Materials and Methods, the main material: strains of bacteria (Helicobacter hepaticus, Helicobacter pylori), Campylobacter bacteria agar base, Freund's complete / incomplete adjuvant, horseradish peroxidase-labeled goat anti-rabbit IgG. Balb / C mice, New Zealand white rabbits. Isolation and identification methods 2.1 Bacterial isolates and culture 2.1.1 gall Helicobacter reference to the literature method separating the gall Helicobacter. Balb / C mice were fasted 48h after SPF (special pathogene free) level, take the intestinal tissue homogenates, coated on Campylobacter jejuni selective blood agar medium (containing 7% of freezing and thawing of defibrinated sheep blood, selective antibiotics vancomycin 10 mg / L, polymyxin B 2500 units / L, amphotericin B 10mg / L, TMP 5mg / L), 37 ° C microaerophilic conditions (5% O2.10% CO2 and 85% N2 ) for 3-5 days under daily observation of the presence or absence of colony growth. Picked suspicious colonies subculture. Bacterial identification: 1, morphological identification of suspected bacterial smear Gram stain, oil microscope bacterial form. 2 biochemical identification of suspicious bacteria by rapid urea enzyme oxidase, peroxidase and nitrate reductase test (test kit provided by the ring Kay biochemical company) 3 the PCR amplification the bacteria 16srRNA gene transparently, the size of the tip of the colonies with swab scraping to the EP tube, the PBS washed three times, with bacterial genomic DNA extracted bacterial DNA extraction kit. Suspicious bacterial genomic DNA as a template using Helicobacter specific primers for PCR amplification. The reaction conditions for 94 ℃ 10min denaturation at 94 ° C for 30, at 54 ℃ for 30 and 72 ° C at 55s was a total of 35 cycles, 72 ℃ 10min total elongation. The PCR product was purified on a 1.5% agarose gel electrophoresis, and the electrophoretic conditions 80V 30min. The positive results of sequencing analysis (sequencing work completed by the handsome biotech companies). 2.1.2 bacteria pass cultured on liver Helicobacter cholestatic Helicobacter, Helicobacter species preservation solution 100μl coated on Campylobacter jejuni selective blood agar medium, and cultured for 3 days at 37 ° C under microaerophilic conditions, bacterial coating piece Gram staining of bacterial growth. 2.2 extracted bacterial outer membrane proteins (cell surface proteins, CSPs) bacteria in the jejunum Campylobacter selective blood agar culture (culture conditions with the former) 48-72h, scraped with sterile double distilled washed bacterial lawn, room temperature and 4000 r / min, 20 min the centrifugal washing broth. Washing was repeated once. The supernatant was discarded, collecting bacteria mud, weighed by adding per 4g bacteria of mud 0.2mol / L, pH 2.2 glycine - HCl buffer, 100 ml, at 4 ° C magnetic stirring for 30 min, after 12,000 r / min at 4 ℃ centrifugation 15 min. Dialysis, freeze-drying method is not concentrated. BCA method to measure protein concentration. 2.3 MCP gene expression in E. coli of my room preserving recombinant plasmid pET22b / MCP transformed into E. coli competent E. coli BL21 (DE3) pick monoclonal inoculated in 10 ml LB medium containing ampicillin 100μg/ml, 37 ℃ 200rpm cultured for 3 and a half hours to OD600 of approximately 0.6-1.0. Transfer 100μl 200rpm cultured in 37 ° C for 1.5 hours to OD600 of approximately 0.6, by adding 500mmol / L IPTG to a final concentration of 1 mmol / L, 37 ° C 180rpm shock cultured for 4 hours in 10ml of LB medium containing ampicillin 100μg/ml. Induced culture 12000rpm 4 ℃ centrifugation for 5 min, 1ml of broth precipitated by SDS-PAGE. The recombinant protein rhMCP contains the expression vector pET22b () in the 6 × His tag, therefore the expression of rhMCP by Western blot identification again. 2.4 rhMCP protein purification 1000ml induction of expression of the bacterium, and centrifuged at 12000rpm 4 ° C for 5 min, the bacterial precipitate was added to the volume of 1/20 of bacterial growth bacterial lysates and PMSF ice sonicated bacteria, 10000rpm 4 ℃ centrifugal 15min. Supernatant, 1/20 of the bacterial growth volume UrNTA-0 Buffer and PMSF was added to precipitate, the cells were suspended, at room temperature for 30min, 10000rpm 4 ℃ centrifugal 15min, supernatant. 30ml of UrNTA-0 Buffer balance 3ml NTA chromatography, the supernatant on the column, to collect the penetrating portion for the case of a combination of SDS-PAGE analysis of protein. 15ml UrNTA-0 Buffer eluting unbound portion with 15mlUrNTA-20, and then were-40 UrNTA, the UrNTA-60 UrNTA-100 the UrNTA-200-500 UrNTA elution, the eluate collected is determined by SDS-PAGE target protein distribution eluate. Gradient PBS-urea of ??4 ℃ dialysis (6M-4M-2M-1M), every 4 hours, the medium was changed once, and finally dialyzed with PBS 4 ° C for 24 hours. BCA method to measure protein concentration, -70 ℃ save. Preparation of the 2.5 rabbit anti-recombinant fusion protein MCP antiserum MCP recombinant protein 800μl (800μg) and the 800μl Freund's complete adjuvant thoroughly emulsified, multi-point injected subcutaneously into New Zealand white rabbits. 2nd immunization, 4 weeks after the first immunization, take the purified fusion protein MCP 400μl (400μg) thoroughly emulsified with 400μl incomplete Freund's adjuvant, and after 6,8 weeks respectively MCP plus an equal volume of Freund. complete adjuvant booster immunization, the last immunization without adjuvant. 5-7 days after the last time strengthen the immune ear vein blood collection 2mL, the line Western blot analysis of serum specific antibodies, using indirect ELISA antibody titers in the serum. Specific antibodies detected carotid intubation bloodletting, the serum was separated and stored at -80 ℃ home. 2.6 ELISA Determination of the titer of the purified fusion protein MCP 5μg/ml, 50μl / hole, coated microtiter plates, each with different multiples of the diluted serum, PBST, after flushing the HRP-labeled goat anti-rabbit IgG secondary antibody incubation, Finally add the TMB Substrate Yexian color, was measured using a microplate reader after termination OD450nm, is equal to the negative control wells 2.1 times judged as positive. 2.7 Rabbit anti-fusion protein MCP antibody purification 2.7.1 saturated pure antibody to 10ml immune rabbit serum, plus an equal amount of PBS (pH 7.4); equal amount of saturated ammonium sulfate solution was added dropwise, in the beginning of the ammonium sulfate at 4 ℃ Static set 30min; 4 ° C 2000rpm/min centrifuged 15 min; supernatant was precipitated with 20 ml of cold PBS was dissolved, was added dropwise 10 mL of saturated ammonium sulfate solution, set a 4 ° C refrigerator 30min; 2000rpm centrifugal 15min, the precipitate was collected, the precipitate was washed with 20ml cold PBS dissolve, to obtain the preliminary purified IgG antibody solution. 2.7.2 antigen-specific purification method of the purified antibody to CNBr-activated Sepharose 4B chromatography column pre-equilibrated, with the PH8.3 the carbonate buffer and adding purified MCP protein 5mg, 4 ° C overnight. The next day by centrifugation to remove unbound protein, the closure of the non-specific binding sites was added the crude purified antibody 5mL shaker overnight at 4 ℃. Add to the column eluate was centrifuged, draw the supernatant was combined with the recombinant protein MCP specific rabbit anti-MCP antibody. Add an equal volume of glycerol and sodium azide to a final concentration of 0.02%, and aliquots stored at -70 ℃. 2.8 rabbit anti-MCP antibody characterization by Western blot antibody specificity take MCP recombinant protein, Helicobacter hepaticus, Helicobacter pylori, and bile screw bacteria bacterial outer membrane protein CSPs degeneration underwent SDS-PAG gel electrophoresis, electrophoretically transferred to a PVDF membrane, a blocking solution (50 mmol / L Tris-Buffered-of Saline, pH 7.5, containing 1% BSA and 0.1% Tween 20) at 37 ℃ oscillation closed 2h, then added to the blocking solution was diluted (1:500) purified rabbit anti-MCP antibodies were incubated overnight at 4 ℃, washing the membrane plus 1:5000 dilution of HRP-goat anti-rabbit IgG (Wuhan Boster) at 37 ° C and incubated 1h, washed thoroughly ECL luminous, developing. Results 1 gall Helicobacter isolation and identification of suspicious bacteria in the jejunum Campylobacter microaerophilic selective blood agar after 3 days of culture, the visible tip size of the mist transparent colonies of bacteria smear Gram stain (Figure 3-1 ), see bacterial Gram stain was negative, was the \The biochemical identification showing bacterial urease test positive, oxidase the peroxidase test positive, nitrate reductase negative. Helicobacter-specific primers and bile Helicobacter-specific primers amplified 16s rRNA, 1.5% agarose gel electrophoresis, positive bands (3-2, 3-3) at 780b products were sequenced and results GeneBank library genomic homology comparison, results with Helicobacter bilis ATCC43879 homology reaches 99%, the isolated bacteria of the present experiment is confirmed bile Helicobacter (Figure 3-4). Helicobacter hepaticus, bile Helicobacter and Helicobacter pylori Campylobacter bacteria blood agar cultured for 3 days, and the visible needle the size of the mist transparent colonies. Preparation of bacterial outer membrane proteins of the bacteria bacterial outer membrane protein CSPs concentrated underwent SDS-PAGE electrophoresis, Coomassie blue staining results shown in Figure 3-5. Induced expression of the recombinant fusion protein induced by IPTG, E.coli BL21 (DE3), E. coli containing the recombinant plasmid capable of expressing the relative molecular mass M: 14118D's the recombinant protein rhMCP, pre the tests identified rhMCP in insoluble inclusion bodies form. Western blot analysis, a specific band is seen in about 14 KD. 4, the separation and purification of the recombinant protein due to the induced expression of the rhMCP in the form of insoluble inclusion bodies exist, therefore dissolved inclusion bodies with 8M urea, and then denatured protein purification methods elution gradient of urea, wherein the concentration of UrNTA200 (imidazol 200mM ) eluting effect gradient of urea-PBS dialysis refolding the purified rhMCP dialysis bag, refolded soluble rhMCP, BCA method for measuring protein concentration of 2.08mg/ml. 5, preparation of purified rabbit anti-MCP polyclonal antibody with purified fusion protein His-MCP immunize rabbits, resulting polyclonal antiserum, the line Western blotting and EL1SA detection (Figure 3-6, Figure 3-7) visible specific bands at 14KD the antisera titer of 1:32000, prove the experimental preparation of successful high specificity, high titers of rabbit anti-MCP polyclonal antibody. 6, Western blotting to further analysis of the antibody specificity by preparative bacterial outer membrane protein of CSPs and MCP line SDS-PAGE electrophoresis, transferred to a PVDF membrane, followed by adding to the preparation of an anti-goat and sheep anti-rabbit secondary antibody, ECL light emitting, film photosensitive, developing, see specific bands of the Helicobacter hepaticus and MCP proteins in the 14KD at visible, cholestatic Helicobacter Helicobacter pylori bands, indicating that the preparation of the antibody has a good activity, with the immunogen specificity combined (Figure 3-8). Conclusions This study successfully isolated from Balb / c mouse colon cholestatic Helicobacter. Separation and purification of recombinant protein MCP immunize New Zealand white rabbits were successful anti-MCP polyclonal antibody serum, the antibody specificity by Western blotting detect only a specific band of purified, and the titer of 1:32000 ( ELISA). Later experiments, we found that the antibody used for Western blotting analysis of bacterial outer membrane protein CSPs, the only liver HH group positive bands, indicating no cross-reactivity of the antibody with other common Helicobacter. The experiments important experimental basis for further in-depth study of the epidemiology of Helicobacter hepaticus and preparation of simple detection kit.

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