Identification And Immunoprotective Effect Of Prokaryotic Expression Product Of LTB-hpaA Fusion Gene

In China, Chronic gastritis and peptic ulcer are the most prevalent gastric diseases and gastric cancer is one of the malignant tumors with high morbidities. Helicobacter pylori, a microaerophilic, spiral and gram-negative bacillus, is recognized as a human-specific gastric pathogen that colonizes the stomaches of at least half of the world's populations. Most infected individuals are asymptomatic. However, in some subjects, the infection causes acute, chronic gastritis or peptic ulceration, and plays an important role in the development of peptic ulcer and gastric adenocarcinoma, mucosa-associated lymphoid tissue (MALT) lymphoma and primary gastric non-Hodgkin's lymphoma. This organism is categorized as a class I carcinogen pronounced by the World Health Organization, and direct evidence of carcinogenesis was recently demonstrated in an animal model. Immunization against the bacterium represents a cost-effective strategy to prevent H.pylori-associated peptic ulcer diseases and to reduce the incidence of global gastric cancers. The selection of antigenic targets is critical in the design of H.pylori vaccine. To date, this area is scarcely touched upon.H. pylori adhesin (HpaA) is one of the major structural outer membrane proteins of Kpylori and plays an important role in adhesion of the microbe. hpaA gene islocated in genome DNA of and considerably conservative for its nucleotide and amino acid sequences. Furthermore, antibody against HpaA could be found in approximate 86% of H.pylori infected patients' sera and this proportion was obviously higher than those of the heat shock protein (68%) and vacuolating cytotoxin (68%). Therefore, it is possibility that HpaA plays an ideal antigen candidate in H.pylori vaccine.For lower immunization effects of genetic engineering vaccines usually composed of a single protein antigen, it may be necessary to increase the immunogenicity of the vaccine antigens by co-administration of an appropriate adjuvant, such as E.coli heat-labile toxin B subunit (LTB) and cholera toxin B subunit (CTB). Several previous studies demonstrated that CTB activates Th2 pathway, in which causing IL-4 production at a high level. It is well known that IL-4 can selectly promote IgE synthesis and IgE imediates allergic reaction type I. Therefore, CTB was considered to be probably associated with IgE-imediated allergic reaction and LTB mainly stimulates Thl pathway, so it could hardly induce IgE-imediated allergic reaction. Some studies showed that the mucosal adjuvanticity of LTB is stronger than that of CTB, therefore LTB is more suitable for the adjuvant in genetic engineering vaccine than CTB.MATERIALS AND METHODS1. Source of bacteria! strain and cultureClinical isolate Y06 and SSI (sydney strain 1) of H.pylori were smeared on H.pylori selective Columbia agar plate containing 10% of sheep blood and antibiotic additive. The plate was cultivated under microaerobic conditions for 5d. The bacterium with gram-negative, curve seagull-like shape, positive for rapid urease and oxidase assay and immunoactive to the antiserum of H.pylori as well as its colony on the plate with tiny and semi-pellucid was identified as H.pylori.Kcoli strain 44815 was inoculated on LB plate and incubated for 24 h at 37*C. A single colony on the plate was identified by Gram-staining method and microscopy. The bacterial strain was inoculated on LB plate again if the microbe was a Gram-negative, moderate-size bacillus.2. Preparation of Bacterial Genomic DNAsThe routing Phenol-chloroform method was applied to prepare genomic DNAs of Kpylori strain Y06 and E.coli strain 44815. The DNA preparations was digested with DNase-free RNase and then extracted by Phenol-chloroform method. The DNAs used as templates in PCR were dissolved in TE buffer and the concentration and purification of the two DNA solutions were determined by ultraviolet spectrophotometry.3. Porymerase chain reactionOligonucleotide primers were designed to amplify the whole length of hpaA and LTB genes according to the published corresponding nucleotide sequences. The hpaA and LTB genes from Kpylori strain Y06 and Kcoli strain 44815 were amplified by PCR, respectively. The results of PCR were observed after electrophoresis in 15 g?L*' agarose pre-stained with ethidium bromide.The sequences of primers for hpaA gene amplification: 5 '-CCGGGATCCATGA GAGCAAATAATC-3'(BamH I, sense);5'-CCGGAAJTCTTCTTATGCGTrATTTG -3'(EcoR I, antisense). The total volume per PCR was 100 ^1 containing 2.5 mol'L*1 each dNTP, 250 nmol'L'1 each the primers, 20 mol'L"1 MgCl2, 3.0 U 3.0 U EX Taq polymerase,200 ng DNA template and IX PCR buffer (pH8.3). The parameters of PCR was described as the following: 94"C5min,X 1 cycle;94"C30sec, 50*C30sec, 72 "C60sec, X10 cycles;94*C30sec, 50*C30sec,, 72*C70sec, X 20 cycles (each of the following cycles added 10 seconds each);72"C10min, XI cycle. The size of expected target amplification fragment was 802bp.The sequences of primers for LTB gene amplification: 5'-CCGGATATCATGAA TAAAGTAAAATGTTA-3'(EcoR V, sense);5'-CCGCTCGAGGAGCTAGTTTTCC ATACTGA-3' (Xhol, antisense). The total volume per PCR was 100 ul containing 2.5 mol'L"1 each dNTP, 250 nmol'L'1 each the primers, 20 mol'L"1 MgCl2, 3.0 U 3.0 U EX Taq polymerase, 200 ng DNA template and 1 X PCR buffer (pH8.3). The parameters of PCR was described as the following: 94 °C 5min, X 1 cycle;94°C30sec, 48*C30sec, 72*C45sec, X 10 cycles;94 V 30sec, 48 ^ 30sec, 72*C50sec, X20cycles (the following cycles add 5 seconds each);72*C7min, X1 cycle. The size of expected target amplification fragment was 375 bp.4. T-A cloning and sequencing of hpaA and ltB genesThe target DNA amplification fragments were recovered with 3S PCR Product Purification Kit V2.0 (SNBC) and ligated to pUCm-T vectors, respectively. The two recombinant pUCm-T vectors for sequencing were respectively named as pUCm-T-hpaA and pUCm-T-ltB, and transfer into E.coli DH 5a.The obtained positive colonies by blue-white screening were amplified and then the plasmides in the bacteria were respectively extracted by alkaline-denature method. The plasmides were identified with restriction endonucleases and then the inserted target fragments were sequenced by dideoxynucleotide chain termination method. Homologies of nucleotide sequences of the inserted fragments were compared with the corresponding sequences from GeneBank.5. Construction of HB-hpaA fusion geneTwo plasmids pUCm-T-hpaA and pUCm-T-ltB in two different strains of E.coli DH5a after amplified in LB medium were extracted by the conventional phenol-chloroform method and DNase-free RNase treatment. The whole sequence of ltB gene was amplified by PCR using pUCm-T-ltB DNA template. Its sense primer sequence was 5'-CCGGGAlCCTGAATAAATAAATGTGTA-3'(BamH I). Its antisense primer sequence was 5'-CTTTAAAATGATTATTTGCTCTGTTTTCCATA CTGATTGCCGC-3'. The other reagents and reaction volumes used and the reaction parameters in PCR were the same as ltB gene amplification mentioned previously. The results of PCR were observed under UV light after electrophoresis in 15 g^L"1 agarose pre-stained with ethidium bromide. Then the target fragment was recovered by DNA Agarose Gel Purification Kit (BBST). The pUCm-T-hpaA plasmid were extracted and digested with BamH I and Xho I, then the hpaA target fragments was recovered. Concentration and purity of the DNA preparations were determined by ultraviolet spectrophotometry. Total volume per PCR was 90ul containing all reagents but primers, thereinto, the templates were lOOng including two recovered target fragments with equal volume, ltB and hpaA. Parameters for PCR to be convenient forcomposing compound template: 94*C for 5min, X1;94'C for 30 sec, 45'C for 30 sec, 72"C for 150 sec, X 10;72"C for 10 min, XI. Then ltB sense primer mentioned previously and hpaA antisense primer: 5'-CCGAAGCTTTCGGTTTCTC TTGTTTTTC-3'(Hind El) were added in, 250 nmol'L"1 each. Parameters for PCR of UB-ureB fusion gene: 94T3 min,X 1;94'C30 sec, 50'C30 sec, 72^90 sec, X10;94 °C30 sec, 50T?30 sec, 72X?180sec (15 sec addition for the each of the following cycles), X15;72*C lOmin, X1. The results of PCR were observed under UV light after electrophoresis in 15 g'L'1 agarose pre-stained with ethidium bromide. The expected size of ltB-hpaA gene amplification fragment was 1177 bp.6. T-A Cloning , sequencing of ltB-hpaA gene and Construction of prokaryotic expression vectorThe ltB-hpaA amplification DNA fragments were cloned, transcripted, amplified and extracted the plasmids (pUCm-T-ltB-hpaA) by using the methods mentioned above. After nucleotide sequence analysis of the inserted fragments, the two plasmids pUCm-T-ltB-hpaA and expression vector pQE32 were extracted digested with BamH I and Hind IE, respectively. Then the ltB-hpaA target fragments and pQE32 were recovered for ligation. The recombinant expression vector pQE32-ltB-hpaA was transformed into Rcoli Ml5, which was named as pQE32-ltB-hpaA-M15. The target ltB-hpaA gene fragment inserted in pQE32 plasmid was sequenced again.7. Expression and purification of die target recombinant proteins pQE32-ltB-hpaA-M15 was rotatively cultured in LB medium at 37"C induced byisopropylthio- P -D-galactoside (IPTG) at different concentrations of 1.0, 0.5 and 0.1 mmol'L"1. The supernatant and precipitate were separated through centrifugation after the bacterial pallet was ultrasonically broken. The molecular weight and output of rLTB-HpaA were measured by SDS-PAGE. Ni-NTA affinity chroma-tography was applied to extract and purify the recombinant proteins.8 . Identification of antigenicity, hnmunoreactivity and ajuvanticity of rLTB-HpaA The commercial rabbit antiserum against whole cell of//, pylori and HRP-labelingsheep anti-rabbit IgG were used as the first and second antibodies, respectively, toUdetermine the immunoreactivity of rLTB-HpaA by Western blot. Rabbits were immunized with rLTB-HpaA to prepare the antiserum and Western blot was applied again to determine the antigenicity of rLTB-HpaA.Plates were coated by bovine GMi(Sigma) and then added with 4 ug rLTB-HpaA each. The rabbit anti-rLTB-HpaA sera was used as the first antibodies and the commercial HRP-labeling sheep anti-rabbit IgG was applied as the second antibody. The values at OD490 nm were detected in the plates. Negative controls without addition of rLTB-HpaA were set up and their OD490 means plus three-fold SD values were used as the positive standard for the corresponding tested groups.9. Immunoprotective effects of rLTB-HpaA on miceBALB/c mice were divided into 4 groups which were tested (rHpaA), tested (rLTB-HpaA), infection, normal, each group has 12. rHpaA and rLTB-HpaA were dissolved into PBS (pH 7.4, 0.01 mol/L), respectively. The mouse of tested group were oral immunized with rHpaA and rLTB-HpaA, respectively. 200 ul 5 X1010 CFU/ml Hp SSI liquid was taken orally after the mouse were immunized with rHpaA and rLTB-HpaA in 28,30,32 d. The mouse were executed 4 weeks after the last infection, then the stomach tissue were triturated and inoculated into selective Columbia agar plate. The plate was cultivated under microaerobic conditions for 5-7d. The identification methods of Hp were the same as what mentioned previously.10. ELISABy using rLTB-hpaA as antigen at the coated concentration of 20 ng/ml, a patient serum sample (1:200 dilution) as the first antibody and HRP-labeling sheep anti-human IgG as the second antibody, the specific antibodies against HpaA in sera from the 126 patients infected with H. pylori were detected by ELISA. The result of ELISA for a patient's serum sample was considered as positive if the optical density value at 490 nm (OD490) was over the mean plus 3 SD of six negative serum samples.HpaA expression in the H. pylori isolates was examine by ELISA using the ultrasonic supernatant of each the H. pylori isolate (50 ug/ml) as a coated protein antigen, the self-prepared rabbit anti-rLTB-HpaA serum as the first antibody and HRP-labeling sheep anti-rabbit IgG as the second antibody. The result of ELISA for aH. pylori ultrasonic supernatant sample was considered as positive if its OD490 value was over the mean plus 3SD of six ultrasonic supernatant samples at the same protein concentration of Kcoli ATCC 25922.RESULTSl.PCRThe sizes of target fragments amplified from E.coli strain 44815 ltB gene and Hp hpaA gene and ltB-hpaA gene were approximate 375bp, 802bp and 1177bp, respectively.2. Rresults of the nucleotide and putative amino acid sequence analysisIn comparison with the corresponding sequences from GeneBank, homologies of the nucleotide sequences of the cloned hpaA and ltB genes were 94.25%~99.19%, 99.12%~99.71%, while their putative amino acid sequence homologies were 95.38%~98.46%, 97.58%~99.19%, respectively. The nucleotide sequences of ltB-hpaA were completely the same as that of ltB and hpaA genes.3. Identification of tile expression vectorsThe target genes were inserted into pQE32 with correct sequencing and direction confirmed by the results of the digestion with two restriction endonucleases and sequence analysis.4. Expression of target fusion proteinThe results of SDS-PAGE demonstrated that EPTG can efficiently induce the expression of rLTB-HpaA in pQE32-ltB-hpaA-M15 system. The product of rLTB-HpaA was mainly presented in ultrasonic precipitates and the output was approximate 30% of the total bacterial proteins.5. Antigenicity, immunoreactivity and ajuvanticity of rLTB-HpaAThe commercial rabbit antibody against the whole cell of H. pylori could combine with rLTB-HpaA and induce rabbit to produce specific antibody as confirmed by Western blot, respectively.The results of GMrELISA showed that when the dilutions of the rabbit antisera were 1:10, 1:20, 1:40 and 1:80, the positive standard values were 0.68, 0.52, 0.55,0.19 for rLTB-HpaA. The OD490values of rLTB-HpaA , using the same different dilutions of the antisera, were 1.64, 1.55, 1.52, 1.10, strongly indicating that the two recombinant proteins had the ability of binding to bovine GM,.6. Immunoprotectrve effects of rLTB-HpaA on miceThe protective rate in the mice immunized with rHpaA alone was as low as 66.7%. When immunized with rLTB-HpaA in the mice, the protective rate was increased to 83.3%.7. ELISAThe mean±SD at OD490 of the negative serum samples was 0.11 ±0.03 and the positive reference value was 0.20. The mean±SD at OD490 of the negative bacterial control was 0.04+0.04 and the positive reference value was 0.16. According to the reference values, 81.6% (102/125, another one serum sample was contaminated ) of the tested patients' serum samples were positive for antibodies against HpaA fusion protein with a range of 0.54-1.84 and 100%, of the tested Kpylori isolates were detectable for HpaA with a range of 0.52-1.47.CONCLUSION1. A prokaryotic expression systems with high efficiency of ltB-hpaA fusion gene was successfully established in this study.2. The expressed rLTB-HpaA showed well immunogenicity and adjuvanticity, and stronger immunoprotective effect in H. pylori infected mice.