Production and cell surface display of recombinant anthrax protective antigen on the surface layer of attenuated Bacillus anthracis.

To investigate the surface display of the anthrax protective antigen (PA) on attenuated Bacillus anthracis, a recombinant B. anthracis strain, named AP429 was constructed by integrating into the chromosome a translational fusion harboring the DNA fragments encoding the cell wall-targeting domain of the S-layer protein EA1 and the anthrax PA. Crerecombinase action at the loxP sites excised the antibiotic marker. Western blot analysis, fluorescence-activated cell sorting and immunofluorescence analysis confirmed that PA was successfully expressed on the S-layer of the recombinant antibiotic marker-free strain. Notwithstanding extensive proteolytic degradation of the hybrid protein SLHs-PA, quantitative ELISA revealed that approximately 8.1 × 10(6) molecules of SLHs-PA were gained from each Bacillus cell. Moreover, electron microscopy assay indicated that the typical S-layer structures could be clearly observed from the recombinant strain micrographs.

Construction of a novel Shigella live-vector strain co-expressing CS3 and LTB/STm of enterotoxigenic E.coli.

AIM: To construct and evaluate a polyvalent recombinant vaccine strain Shigella flexneri 2a T32 against enterotoxigenic E.coli (ETEC). METHODS: By using a host-plasmid balanced lethal system based on asd gene, a polyvalent recombinant strain was constructed to highly express CS3 and regularly express fusion enterotoxin of LTB subunit and mutant ST (LTB/STm) in a vaccine strain Shigella flexneri 2a T32 with specific deletion of asd gene. Fimbria CS3 was observed by immunofluorescence and electron microscopy assay. The security of LTB/STm was examined by ileal loop assay and suckling mouse assay. To evaluate this new candidate vaccine, it was compared with a previous vaccine strain in plasmid and protein level, growth assay and immunogenicity in Balb/c mice. RESULTS: The newly constructed vaccine expressed CS3 and grew better than the previously constructed vaccine except for the lower expression of LTB/STm. Serum IgG and mucosal IgA against CS3, LTB, ST, and host lipopolysaccharide (LPS) were produced after immunization of Balb/c mice by oral route with the new strain. The titers were not significantly different from the Balb/c mice with the previous strain. CONCLUSION: This novel candidate diarrheal vaccine can effectively induce serum and mucosal antibody responses against ETEC and Shigella.

Expression of Helicobacter pylori AlpA protein and its immunogenicity.

AIM: To construct a recombinant strain which expresses adhesin AlpA of Helicobacter pylori (H pylori) and to study the immunogenicity of adhesin AlpA. METHODS: Gene Ab, which was amplified from H pylori chromosomal DNA by PCR technique, was sequenced and the biological information was analyzed, and inserted into the Nco I and Not I restriction fragments of the expression vector pET-22b(+) using T4 DNA ligase. The resulting plasmid pET-AlpA was transformed into competent E.coli BL21(DE3) cells using ampicillin resistance for selection. Recombinant strains were incubated in 5 mL LB with 100 mug/mL ampicillin overnight at 37 degrees. Sonication of BL21(DE3)pET-22b(+)/AlpA was analyzed by Western blot to detect AlpA immunogenicity. RESULTS: The gene encoding AlpA protein was amplified by PCR with chromosomal DNA of H pylori Sydney strain (SS1) as templates. It revealed that AlpA DNA fragment amplified by PCR had approximately 1 500 nucleotides, compatible with the previous reports. The recombinant plasmid pET-22b(+)/AB was successfully constructed. DNA sequencing showed one open reading frame with the length of 588 bp. It encoded seven conservative regions that showed good antigenicity and hydrophobicity by Parker and Welling method. Furthermore, INTERNET EXPASY, NNPREDICT and ISREC predicted that it was a porin-like structure consisting of beta-pleated sheets that were embedded in the outer membrane. BLAST analyzed 836 767 protein sequences and found that the similar sequences were all belonging to H pylori OMP sequences. SDS-PAGE and scan analysis showed that the molecular weight of AB was 22.5 ku and recombinant protein amounted to 29% of the total bacterial protein, among which dissolved expression amounted to 21.9% of sonicated supernatant. The rAB purity amounted to 96% through affinity chromatography. Western blot analysis of rAB confirmed that it could be specially recognized by serum form rabbit immunized with AlpA and H pylori infected. CONCLUSION: Adhesin AlpA recombinant protein may be a potential vaccine for control and treatment of H pylori infection.

[Progress on the vaccine for anthrax].

Bacillus anthracis is the causative organism of the potentially fatal disease anthrax, and the used vaccines have some disadvantages. There are new developments appeared for the Bacillus anthracis in recent years, such as anti-PA antibody kills the spore of Bacillus anthracis, mucosal immunization induces immune responses in both systemic and secretory immune compartments, Poly (gamma-D-PGA) protein induce IgG antibodies to the vegetative bacteria, new pathogens were found by genomic analysis. The DNA vaccine and live vector vaccine will be the next generation vaccines for anthrax. It will have a shorter immunization schedule and will be greater protective efficacy than before.

Antigen epitope of Helicobacter pylori vacuolating cytotoxin A.

AIM: To construct and select antigen epitopes of vacuolating cytotoxin A (VacA) for nontoxic VacA vaccine against Helicobacter pylori (H pylori ) infection. METHODS: Eleven VacA epitopes were predicted according to VacA antigenic bioinformatics. Three candidates of VacA epitope were constructed through different combined epitopes. The candidate was linked with E. coli heat-labile enterotoxin B (LTB) by a linker of 7 amino acids, and cloned into plasmid pQE-60 in which fusion LTB-VacA epitope was efficiently expressed. To test the antigencity of the candidate, 6 BALB/c mice were treated with the fusion LTB-VacA epitope through intraperitoneal injection. To explore the ability of inhibiting the toxicity of VacA,cantiserum against the candidate was used to counteract VacA that induced HeLa cells to produce cell vacuoles in vitro. RESULTS: Serum IgG against the candidate was induced in the BALB/c mice. In vitro, the three antisera against the candidate efficiently counteracted the toxicity of VacA, and decreased the number of cell vacuoles by 14.17%, 20.20% and 30.41% respectively. CONCLUSION: Two of the three candidates, LZ-VacA1and LZ-VacA2, can be used to further study the mechanism of vacuolating toxicity of VacA, and to construct nontoxic VacA vaccine against H pylori infection.

Construction of attenuated Salmonella typhimurium Strain expressing Helicobacter pylori conservative region of adhesin antigen and its immunogenicity.

AIM: To construct a non-resistant and attenuated Salmonella typhimurium (S. typhimurium) strain which expresses conservative region of adhesion AB of Helicobacter pylori (H pylori) and evaluate its immunogenicity. METHODS: The AB gene amplified by PCR was inserted into the expression vector pYA248 containing asd gene and through two transformations introduced into the delta Cya, delta Crp, delta Asd attenuated Salmonella typhimurium strain, constructing balanced lethal attenuated Salmonella typhimurium strains X4072 (pYA248-AB). Bridged ELISA method was used to measure the expression of AB antigen in sonicate and culture supernatant. According to the method described by Meacock, stability of the recombinant was evaluated. Semi-lethal capacity test was used to evaluate the safety of recombinant. The immunogenicity of recombinant was evaluated with animal experiments. RESULTS: The attenuated S. typhimurium X4072 (pYA248-AB) which expresses AB was successfully constructed. Furthermore, bridged ELISA assay showed that the content of AB in recombinant X4072 (pYA248- AB) culture supernatant was higher than that was in thallus lytic liquor. And after recombinant X4072 (pYA248- AB) was cultured for 100 generations without selection pressure, the entire recombinant bacteria selected randomly could grow, and the AB antigen was defected positive by ELISA. The growth curve of the recombinant bacteria showed that the growth states of X4072 (pYA248) and X4072 (pYA248-AB) were basically consistent. The survival rate of C57BL/6 was still 100%, at 30 d after mice taking X4072 (pYA248-AB) 1.0 x 10(10) cfu orally. Oral immunization of mice with X4072 (pYA248-AB) induced a specific immune response. CONCLUSION: In vitro recombinant plasmid appears to be stable and experiments on animals showed that the recombinant strains were safe and immunogenic in vitro, which providing a new live oral vaccine candidate for protection and care of H pylori infection.

Cloning and expression and immunogenicity of Helicobacter pylori BabA2 gene.

AIM: To construct a recombinant strain which expresses BabA of Helicobacter pylori (H pylori) and to study the immunogenicity of BabA. METHODS: BabA2 DNA was amplified by PCR and inserted into the prokaryotie expression vector pET-22b (+) and expressed in the BL21 (DE3) E.coli strain. Furthermore, BabA immunogenicity was studied by animal test. RESULTS: DNA sequence analysis showed the sequence of BabA2 DNA was the same as the one published by GenBank. The BabA recombinant protein accounted for 34.8% of the total bacterial protein. The serum from H pylori infected patients and Balb/c miced immunized with BabA itself could recognize rBabA. CONCLUSION: BabA recombinant protein may be an potential vaccine for control and treatment of H pylori infection.

Expression of Helicobacter pylori Hsp60 protein and its immunogenicity.

AIM: To express Hsp60 protein of H pylori by a constructed vector and to evaluate its immunogenicity. METHODS: Hsp60 DNA was amplified by PCR and inserted into the prokaryote expression vector pET-22b (+), which was transformed into BL21 (DE3) E.coli strain to express recombinant protein. Immunogenicity of expressed Hsp60 protein was evaluated with animal experiments. RESULTS: DNA sequence analysis showed Hsp60 DNA was the same as GenBank's research. Hsp60 recombinant protein accounted for 27.2% of the total bacterial protein, and could be recognized by the serum from H pylori infected patients and Balb/c mice immunized with Hsp60 itself. CONCLUSION: Hsp60 recombinant protein might become a potential vaccine for controlling and treating H pylori infection.

Recombinant Helicobacter pylori catalase.

AIM: To construct a recombinant strain which highly expresses catalase of Helicobacter pylori (H. pylori) and assay the activity of H. pylori catalase. METHODS: The catalase DNA was amplified from H. pylori chromosomal DNA with PCR techniques and inserted into the prokaryotie expression vector pET-22b (+), and then was transformed into the BL21 (DE3) E.coli strain which expressed catalase recombinant protein. The activity of H. pylori catalase was assayed by the Beers and Sizers. RESULTS: DNA sequence analysis showed that the sequence of catalase DNA was the same as GenBank's research. The catalase recombinant protein amounted to 24.4 % of the total bacterial protein after induced with IPTG for 3 hours at 37 degrees and the activity of H. pylori catalase was high in the BL21 (DE3) E.coli strain. CONCLUSION: A clone expressing high activity H. pylori catalase is obtained, laying a good foundation for further studies.

Expression of Colonization Factor Antigen I of Enterotoxigenic Escherichia coli in Shigella flexneri 2a T32.

A host-plasmid lethal balancing system was constructed based on asd gene in an avirulent strain of S.flexneri to express colonization factor antigen I(CFA-I) of enterotoxigenic Escherichia coli.The results of Western blotting suggested that avirulant strain of S.flexneri Fwl01 expressed CFA-I steadily. Examination of negatively-stained preparation of cultures by electron microscopy showed that S.flexneri Fwl01 carrying the plasmid pZHY21 had thick pili on its surface. Antibodies against CFA-I were decteted in sera of mice immunized with recombinant bacteria either orogastrically (o.g.) or intranasally(i.n.); simultaneously sIgA against CFA-I was also decteted in the intestine. This work is helpful for constructing multivalent reombinant vaccine for prevention of bacterial diarrhea.

Cloning and Expression of alpha-Bungarotoxin Gene.

The genetic engineering methods to produce the snake neurotoxin obviously have potential advantages over the traditional methods of extracting neurotoxin from snakes. This work was to study the expression and the feasibility of scaled production of snake neurotoxin in the routine expression systems such as E.coli with the -bungarotoxin as an example. First, on the basis of the reported amino acid sequence of alpha-bungarotoxin, DNA sequence of -bungarotoxin was deduced and four partially complementary oligonucleotide fragments were designed. The coding region of -bungarotoxin was obtained by renaturing the DNA fragments, nick filling-in, ligation and PCR. The coding region of -bungarotoxin was cloned into plasmids pGEX-2T, named pDZ04, and transformed E.coli BL21 in order to study the expression of alpha-bungarotoxin gene. The results of SDS-PAGE analysis showed that the recombinant plasmid pDZ04 could express efficiently in BL21, and the fusion protein took up about 30%-40% of total bacterial protein. Finally, The biological activity of the recombinant alpha-bungarotoxin and the fusion protein was studied with the natural alpha-bungarotoxin purified from the snake venom as control, ELISA results showed that they had similar antigenicity.

Cloning and Secretion Expression of Heat-shock Protein 70 Gene of Helicobacter pylori.

Heat-shock protein 70 gene (hsp70)was obtained by PCR method from Helicobacter pylori chromosomal DNA. Sequencing analysis exhibited that the hsp70 gene isolated from Hp Y(2) was highly homologous with the gene encoded in Helicobacter pylori 26695 and J99, which had been sequenced for complete genome. The hsp70 gene was recombined in vitro with fusion secretion expression vector pMAL-p2 and was transformed into E.coli cells. The E.coli strains, containing hsp70 recombinant plasmid, expressed a 113 kD fusion protein which accounted for 19.4% of the total bacterial periplasm protein after the induction with IPTG for 5 h at 30 degrees. The expressed fusion protein could react specifically with anti-Helicobacter pylori rabbit IgG, as proved by Western blot method.

[Co-expression of CFA/I and CS6 of Enterotoxigenic Escherichia coli ( ETEC ) in Shigella flexneri 2a T32 Derivative Strain FWL01].

Among the known colonization factors of enterotoxigenic Escherichia coli (ETEC), CFA/I and CS6 (the common antigen in the CFA/IV fimbrial antigens ) are two of the most prevalent fimbriae found in clinical isolates but are never expressed by the same wild-type strains. In this study, CFA/I and CS6 of ETEC were co-expressed in Shigella flexneri 2a T32 derivative strain FWL01 by using a host-plasmid lethal balancing system based on asd gene. The results indicate that the recombinant plasmid carrying CFA/I and CS6 could be stably integrated in FWL01. Expression of the two antigens did not interfere the host growth. The results of immunofluorescence analysis showed that CFA/I and CS6 were localized on the surface of the strain FWL01. In Balb/c mice orally immunized with the recombinant strain, the immune responses against CFA/I and CS6 were observed. Those observations show the feasibility of a multivalent vaccine expressing different fimbrial antigens in attenuated Shigella flexneri.

[Simultaneous expression of CS3 colonization factor antigen and LT-B/ST fusion enterotoxin antigen of enterotoxigenic Escherichia coli by attenuated Shigella flexneri 2a].

Enterotoxigenic Escherichia coli (ETEC) causes watery dehydrating diarrhea in infants in developing countries, and is the most common cause of travelers diarrhea. It has been known that the colonazition factor antigens (CFAs) and enterotoxins are important virulence factors of ETEC, and these two kinds of proteins should be included in any effective vaccine against ETEC. In this study, a host-plasmid lethal balancing system was constructed based on asd gene in an avirulent strain of S.flexneri to express CS3 antigens and the fusion LT-B/ST enterotoxins of Escherichia coli. Both of these antigens were expressed steadily in the S. flexneri vector without any antibiotic markers. Antibodies against CS3, LT, ST and LPS of Shigella were detected in sera of mice that were immunized with recombinant bacteria either oragastrically (o.g.) or intranasally (i.n.). SIgA against CS3 and enterotoxins were detected simultaneously in feces of mice. This work is helpful for constructing multivalent recombinant vaccine for prevention of bacterial diarrhea.

Simultneous Expression of CS3 Colonization Factor Antigen and Cholera Toxin B Subunit in Salmonella typhi.

A host-plasmid balancing system was established based on asd gene in an avirulent strain of Salmonella typhi to express enterotoxigenic E.coli surface antigen CS3 and V.cholerae toxin subunit B(CTB). The plasmid can be stably maintained in the host and can express CS3 and CTB in the host cell without any antibiotic selection, although expression level and growth characteristics of the recombinant strain expressing either CS3 or CTB are superior to that of the recombinant strain which expresses both of the antigens. Antibo-dies against CS3 and CTB can be detected in sera of mice immunized with recombinant bacteria either orally or subcutaneously, and mice immunized subcutaneously can be protected from challenging with virulent strain of Salmonella typhi. This work may be helpful in constructing multivalent recombinant vaccines for prevention of bacterial diarrhea.

Simultaneous expression of CS3 colonization factor antigen and LT-B/ST fusion enterotoxin antigen of enterotoxigenic Escherichia coli by attenuated Salmonella typhimurium.

LT and ST are the main enterotoxins of enterotoxigenic Escherichia coli (ETEC) found in clinical isolates, and CS3 (the common antigen in the CFA/II family of fimbrial antigens) is one of the most prevalent antigens of colonization factors. The genetic determinants encoding CS3 and LT-B/ST fusion toxin were manipulated so that these important antigens are expressed simultaneously in attenuated Salmonella typhimurium oral vaccine strain X4072. These antigens produced by X4072 (pXZL88) could be recognized with monospecific CS3, LT or ST antibodies respectively. The specific antibodies against CS3, LT and ST could be detected. In the sera of immunized mice via oral route with the live bacteria. Significantly, the antibody to ST was able to neutralize the biological activity of native ST. This prototype construct may be proved to be useful in investigating the live vector approach to immunoprophylaxis of ETEC diarrhea disease.

[Cloning and immunogenicity of conservative region of adhesin gene of Helicobacter pylori].

OBJECTIVE: To construct a candidate strain of Helicobacter pylori (Hp) that expresses the proteins of the conservative region of 4 adhesins (BabA2, AlpA, AlpB, and HopZ) and study its immunogenicity. METHODS: The DNA of Hp was extracted. Primers were designed according to the C-terminal structural gene sequence (called CB) of AlpA. The CB gene was amplified by PCR and inserted into the prokaryotic expression vector pET-22b (+) and expressed in BL21 (DE3) strain of Escherichia coli. The product of expression, CB, was purified by affinity chromatography and identified by Western blot analysis. ELISA assay was used to measure the CB-specific antibody in the specimens of serum of 55 Hp infected patients. Rapid urease test (RUT) was used on biopsy specimens collected by gastroscopy as parallel control. RESULTS: A recombinant plasmid pET-22b (+)/CB was constructed with the conservative region of the 4 adhesins. DNA sequencing showed one open reading frame of 588 bp encoding a polypeptide of 195 amino acids. The recombinant CB (rCB) protein, with a molecular weight of 22.5KD, amounted to 29% of the total bacterial protein. The purity of purified rCB was 96%. Western blot analysis showed that the rCB protein could be specifically recognized by the serum from Hp infected patients. The kappa coefficient was 0.76 for evaluation by ELISA and RUT results. CONCLUSION: CB has the potential to be used as a vaccine against Hp infection.

[Construction of the non-resistant attenuated Salmonella typhimurium strain expressing Helicobacter pylori catalase].

OBJECTIVE: To construct a non-resistant attenuated Salmonella typhimurium (S.typhimurium) strain capable of expressing Helicobacter pylori (Hp) catalase. METHODS: After PCR amplification, the gene fragment encoding Hp catalase was inserted into the expression vector pYA248 containing asd gene, and the recombinant vector was then introduced into the host S.typhimurium strain X4072 depleted of genes encoding adenylate cyclase (delta cya), cyclic adenosine monophosphate receptor protein (delta crp) and aspartate-beta-semialdehyde dehydrogenase (delta asd). Bridged enzyme-linked immunosorbent assay (ELISA) was employed to measure the antigenicity of the catalase expressed in the sonicate and culture supernatant. According to Meacock's method and with the assistance of the growth curve, the stability of the recombinant strain was evaluated. A half lethal oral dose test was conducted to evaluate the safety of recombinant strain. RESULTS: S.typhimurium X4072 (pYA248-CAT) with expected capacity was successfully constructed, and bridged ELISA demonstrated higher catalase levels in the culture supernatant than in the sonicate of the recombinant strain X4072 (pYA248-CAT). After the strain was passaged for 100 generations without selection pressure, all the randomly selected colony of the recombinant strain grew well with positive catalase antigenicity as identified by ELISA. The growth curve of the recombinant strain showed comparable growth status of the 2 strains X4072 (pYA248) and X4072 (pYA248-CAT). The survival rate of C57BL/6 mice was 100% 30 d after oral administration of 1.0x10(10) cfu X4072 (pYA248-CAT). CONCLUSION: Non-resistant S. typhimurium vaccine X4072 (pYA248- CAT) is constructed successfully, which is stable in vitro and safe as confirmed by animal experiment. This vaccine provides a new candidate for viable oral vaccine against Hp infection.

Conservative region of the genes encoding four adhesins of Helicobacter pylori: cloning, sequence analysis and biological information analysis.

OBJECTIVE: To clone the conserved regions of the genes encoding the 4 adhesins (BabA, AlpA, AlpB and HopZ) of Helicobacter pylori (H. pylori) and analyze their sequences and biological information, thus facilitating further research in the molecular mechanism and immunogenicity of H. pylori adhesins. METHODS: Common conserved region (designated as CB) was identified from the confirmed sequences (by ANTHEPROT V4.3c software package) of the 4 adhesin proteins. Their DNA sequences were deduced, according to which primers specific to CB were designed for subsequent PCR, and the products were inserted directionally into pET-22b(+) vector to construct recombinant clones of the conserved region. The DNA sequences were determined with the basic local alignment sequence tool (BLAST) and the biological properties analyzed with ANTHEPROT V4.3c software package. RESULTS: The recombinant plasmid containing the CB sequence was constructed. DNA sequencing showed an open reading frame of 588 bp in length, encoding 195 amino acids. The homogencity of conservative region of the 4 adhesion genes was above 50%. The corresponding protein possessed a relative molecular mass (Mr) of 22 500 as predicted by ANTHEPROT V4.3c software prediction, with excellent antigenicity and hydrophobicity. There were 836 767 sequences analyzed with BLAST, in which those with homogencity of 40% with the identified CB sequence were categorized into H. pylori sequences. CONCLUSION: There are conservative regions in the 4 adhesin genes with similar homogencity, suggesting similar molecular basis for adhesion of the adhesins. Biological information analysis indicates that CB has excellent immunogenicity and strict species specificity.

[Study on the cloning, expression and the immunogenicity of helicobacter pylori heat shock protein 60 gene].

OBJECTIVE: To construct a recombinant strain of bacteria expressing heat shock protein of (Hsp) Helicobacter pylori (Hp) and study the immunogenicity of Hsp60. METHODS: PCR amplification of Hsp60 DNA was performed before it was inserted into the prokaryotic expression vector pET-22b(+) to transform BL21(DE3) E.coli strain. Hsp60 expressed by the recombinant E.coli was collected and purified for immunogenicity assessment in mice. RESULTS: DNA sequence analysis showed identical DNA sequence of Hsp60 thus produced to that published in Genbank. Accounting for a ratio of 27.2% among the total protein production in the bacterium, recombinant Hsp60 protein was recognized by the serum from Hp-infected patients and produced corresponding antibody in Balb/c mice in response to immunization. CONCLUSION: Recombinant Hsp60 protein can be used potentially as a vaccine for controlling and treating Hp infection.