Microarray analysis of Escherichia coli O157:H7.

AIM: To establish the rapid, specific, and sensitive method for detecting O157:H7 with DNA microchips. METHODS: Specific oligonucleotide probes (26-28 nt) of bacterial antigenic and virulent genes of E. coli O157:H7 and other related pathogen genes were pre-synthesized and immobilized on a solid support to make microchips. The four genes encoding O157 somatic antigen (rfbE), H7 flagellar antigen (fliC) and toxins (SLT1, SLT2) were monitored by multiplex PCR with four pairs of specific primers. Fluorescence-Cy3 labeled samples for hybridization were generated by PCR with Cy3-labeled single prime. Hybridization was performed for 60 min at 45 degrees. Microchip images were taken using a confocal fluorescent scanner. RESULTS: Twelve different bacterial strains were detected with various combinations of four virulent genes. All the O157:H7 strains yielded positive results by multiplex PCR. The size of the PCR products generated with these primers varied from 210 to 678 bp. All the rfbE/fliC/SLT1/SLT2 probes specifically recognized Cy3-labeled fluorescent samples from O157:H7 strains, or strains containing O157 and H7 genes. No cross hybridization of O157:H7 fluorescent samples occurred in other probes. Non-O157:H7 pathogens failed to yield any signal under comparable conditions. If the Cy3-labeled fluorescent product of O157 single PCR was diluted 50-fold, no signal was found in agarose gel electrophoresis, but a positive signal was found in microarray hybridization. CONCLUSION: Microarray analysis of O157:H7 is a rapid, specific, and efficient method for identification and detection of bacterial pathogens.

[Detection of virulence-associated factors of Streptococcus suis by multiplex PCR assay].

OBJECTIVE: To rapidly and sensitively detect the four virulence-associated factors of Streptococcus suis, a multiplex PCR was developed. METHODS: In the process of this reaction, four distinct DNA targets were amplified. One target was based on the serotype 2 (and 1/2) specific cps gene and the others were based on Streptococcus suis mrp, epf (epf*) and sly gene, encoding the MRP, EF(EF*) and Sly proteins of Streptococcus suis. 72 isolates, which including 48 strains of Streptococcus suis and 24 strains of negative control, and 49 clinical specimens were detected by the multiplex PCR assay. RESULTS: All PCR products were detected by electrophoresis on 1.2% agarose gels. With the 48 Streptococcus suis strains, the positive detection rates of cps2+, mrp+, epf+, epf*+ and sly+ were 16/48, 14/48, 12/48, 3/48 and 26/48,respectively. The results were confirmed by bacteriological examination. There were no specific amplification products including 49 clinical specimens and 24 negative control strains. CONCLUSION: The results demonstrated that multiplex PCR was a highly specific and sensitive diagnostic tool for the detection of virulence-associated factors of streptococcus suis.

[Prevalence of hemorrhagic fever with renal syndrome virus in domestic pigs: an epidemiological investigation in Shandong province].

OBJECTIVE: To investigate the epidemiological significance of hemorrhagic fever with renal syndrome virus (HFRSV) infection in domestic pigs in Shandong province, and study the role of domestic pigs in the prevalence of HFRS. METHODS: Epidemiological investigation was performed in 4 cities of Shandong province. Reversed passive hemagglutination assay (RPHA), reversed passive hemagglutination inhibition (RPHI), HPR-SPA, immunofluorescent antibody (IFA), and reverse transcriptional PCR (RT-PCR) were used to detect antigen and antibody of HFRSV. RESULTS: HFRSV antigen and antibody were detected in the heart, liver, lung, spleen, kidney, blood, urine, and stool of domestic pigs as well as in the sewage of the pigpen facilities. The positivity rate of HFRSV antigen ranged from 3.33% to 5.00% in the organ of pigs, and HFRSV positivity rate in the blood, urine, stool, and sewage was 3.67%, 7.04%, 2.51%, and 5.56%, respectively, with a total serum antibody positivity rate of 1.96%. The virus was isolated from the HFRSV antigen-positive samples, and could infect many organs after artificial infection of the pigs. HFRSV antigen can be detected in suckling rat brain and histopathological examination suggested transient pathological changes in such organs as the liver, lung and kidney. HFRSV may proliferate in pigs and was discharged through multiple routes. CONCLUSION: Domestic pigs can be the host of HFRSV for the viral transmission.

[Screening and identification of mimic epitopes of monoclonal antibodies against hantaan virus using phage display technique].

AIM: To obtain mimic epitopes of monoclonal antibody (mAb) against Hantaan virus, and identify preliminarily their immunological characteristics. METHODS: Group-specific mAbs F3 and B11 were used as selective molecules for biopanning. After biopanning, the positive phages were identified by ELISA and DNA sequencing. Among the positive phages, F3-8, B11-18, B11-20 and B11-24 were selected to immunize BALB/c mice three times, respectively. The antiserum was then titered and confirmed by sandwich ELISA and competition ELISA. RESULTS: After 3 to 4 rounds of effective screening, the majority of the selected clones were found able to react to mAb F3 or B11 in a dose-dependent manner, but not to BSA, rHBsAg or other unrelated mAbs. The amino acid sequences of the clones binding to mAb F3 contained an identical sequence MHGPTKNQMWHT, which had higher homology to 750-759 amino acids between a pair of cysteines within glycoprotein G2 of HTNV/SEOV, while those of clones binding to mAb B11 had no evident homologous regions within HTNV/SEOV proteins. The specific antibodies increased significantly following immunization with phage peptides, which indicated that the phage-displayed peptides had not only good antigenicity, but also strong immunoreactivity. CONCLUSION: The phage-displayed peptides could mimic the epitope of HFRSV antigen, which would provide the potential for preparing more effective epitope-based vaccines and specific diagnostic reagents.

[Detection of Schistosomia japonicum 5D gene by polymerase chain reaction and genechip technique].

OBJECTIVE: In order to develop the diagnostic genechip for specific detection of Schistosoma japonicum (Chinese mainland strain). METHODS: Probe and primers were designed based on the Schistosoma japonicum 5D gene encoding an immunogenic miracidial antigen. The probe for the conservative and specific gene sequence was spotted onto the specially treated glass slides by pin-based spotting robot Pixsys 5500 and was employed to make genechips. A polymerase chain reaction (PCR) protocol was designed to effectively amplify the 5D gene fragment containing the probe sequence from cercaria, egg, adult worm and infected Oncomelania DNA as well as other flukes DNA, respectively. After 35 cycles by PCR, the products were then labeled with fluorescent Cy3-labeled primer, using dissymmetrical PCR. The labeled PCR products of the target genes were hybridized to the diagnostic genechips for detection of Schistosoma japonicum and a fluorescent scanner (ScanArray 3000) was used to observe and record the hybridization signals. RESULTS: The result obtained from the study showed that a 262 bp DNA fragment was amplified from cercaria, egg and adult worm with the designed primers and enable the genechip be applied to detect a single cercaria, egg and adult worm. When the genechip was used to detect Clonorchis sinensis, Fasciolopsis busk, and Paragonimus westermani DNA, the results showed negative, indicating that the genechip had good specificity. CONCLUSION: The genchip technique for detection of Schistosoma japonicum was established successfully and having the characteristics of high sensitivity and specificity.

Induction of SARS-nucleoprotein-specific immune response by use of DNA vaccine.

Induction of effective cytotoxic T lymphocyte (CTL) and/or a specific antibody against conserved viral proteins may be essential to the development of a safe and effective severe acute respiratory syndrome coronavirus (SARS-Cov) vaccine. DNA vaccination represents a new strategy for induction of humoral and cellular immune response. To determine the ability of SARS-Cov nucleoprotein (N protein) to induce antiviral immunity, in this report, we established a stable C2C12 line expressing SARS-Cov N protein, which was used as a target for specific CTL assay. We also expressed recombinant N proteins in Escherichia coli and prepared N protein-specific polyclonal antibodies. C3H/He mice were immunized with N protein-expressible pcDN-fn vector by intramuscular injections. We found that the DNA vaccination induced both N protein-specific antibody and specific CTL activity to the target. When C3H/He mice were immunized by three separate injections, high antibody titre (1:3200-1:6400, average titre is 1:4580) and high CTL activity (67.4+/-8.4% (E:T = 25:1), 69.6+/-6.7% (E:T = 50:1) and 71.8+/-6.2% (E:T = 100:1)) were observed. In the case of two vaccine injections, CTL activity was also high (56.6+/-12.7% (E:T = 25:1), 57.4+/-11.7% (E:T = 50:1) and 63.0+/-6.3% (E:T = 100:1)) However, antibody titres were much lower (1:200-1:3200, average titre is 1:980). Our results suggest that SARS-Cov nucleocapsid gene might be a candidate gene for SARS DNA vaccination.

[Expression, purification and serological reactivity of a chimeric antigen of GRA6 with P30 from Toxoplasma gondii].

Major surface protein (p30) and Dense Granule Antigen GRA6 of Toxoplasma gondii have good antigenicity, and could be used for detection of IgM against Toxoplasma gondii. GRA6 may complement P30 to reach more high sensitivity for detection of antibodies to Toxoplasma gondii, so, we try to express the chimeric protein of GRA6 and P30 by genetic engineering, identify its antignenicity and use for developing diagnosis reagent. Antigenic domains of p30 and GRA6 of Toxoplasma gondii were screened by analyzing their sequences using the software ANTHEWIN. Two DNA fragments encoding respectively antigenic domains of p30 and GRA6 were cloned, they were inserted into the same expression vector pET28a( + ) and expressed as a chimeric protein in Escherichia coli. BL21(DE3), the expressed chimeric protein of p30 with GRA6 in a form of inclusion body was about 25% of total proteins of E. coli. BL21(DE3). The inclusion body was washed once with 0.5% Triton X-100 and dissolved with 0.5% SKL, after renaturation by gradient dialysis, the recombinant protein was purified by DEAE-Sepharose FF cation column and then detected with 12% SDS-PAGE, it exists mainly in the eluted peak with 300 mmol/L NaCl and has high purity. By using enzyme-linked immunosorbent assay (ELISA), the recombinant protein was examined for reactivity with immunoglobulin M (IgM) antibodies in 6 sera from patients infected with Toxoplasma gondii ., it was reactive with all the 6 sera but not with sera from normal people, these results showed that the recombinant chimeric antigen has good antigenicity and specificity and could be used for detection of IgM against Toxoplasma gondii. The expressed chimeric protein could be used for epidemic investigation of Toxoplasma gondii, blood donor screening, especially for detection of pregnant women, and is of great significance in prevention of Toxoplasma gondii infection.

[Expression of TPO mimetic peptide chimeric proteins with human IgG1 Fc fragments and their biological characters].

Many antineoplastic agents can cause myelosuppression and thrombocytopenia. Thrombopoietin (TPO) is believed to be the major cytokine affecting the proliferation and maturation of megakaryocytes and increasing circulating platelet levels. We have designed and synthesized a TPO mimetic peptide, it can increase circulating platelet levels in vivo. For increasing half-life and forming dimer, the peptide was expressed as chimeric proteins with human IgG1 Fc fragments. The cDNA of TPO mimetic peptide was synthesized chemically and linked respectively to 5' terminus of human IgG1 Fc cDNA fragments in various length (Fc1: Fc 5' 648 bp; Fc2: Fc 5' 270 bp; Fc3: Fc 5' 267 bp; Fc4: Fc 5' 90 bp), and cloned into expression plasmid pET28a (+) for constructing four recombinant plasmids. By transforming the four recombinant plasmids into E. coli. BL21 (DE3) respectively, we got 3 kinds of engineered E. coli which express TPO + Fc chimeric proteins(28 kD TPO + Fc1, 12 kD TPO + Fc2 and 12 kD TPO + Fc3) at high level respectively, the expressed proteins were purified with DEAE-Sepharose FF and S-Sepharose FF column. The bioactivities of the expressed chimeric proteins(TPO + Fc1, TPO + Fc2 and TPO + Fc3), TPO mimetic peptide, and PEG4000 coupled TPO mimetic peptide were evaluated with Ba/F3-mp1 in vitro and with carboplatin-induced thrombocytopenia mice in vivo, the expressed chimeric proteins have higher activity than TPO mimetic peptide both in vitro and in vivo, the EC50 on Ba/F3-mp1 cells were 13, 10, 10, 50, and 25 nmol/L respectively, all of them can increase circulating platelet counts. Their imol/Lunogenicity were valuated in mice, none of them can elicit mice to produce antibodies to TPO mimetic peptide, meanwhile three TPO + Fc chimeric proteins can elicit mice to produce antibodies to human IgG1 Fc. These studies have laid basis for production of TPO mimetic peptide by genetic engineering.