Construction and immunization of an attenuated vaccine candidate enteropathogenic Escherichia coli O45 / 生物工程学报

In order to obtain an attenuated vaccine candidate for enteropathogenic Escherichia coli (EPEC) O45, a ler deletion mutant of pig enteropathogenic E. coli (PEPEC) O45 was constructed by using the suicide vector pCVD442, termed as PEPEC O45(deltaler). The culture supernatant of PEPEC O45(deltaler) deletion mutant was inoculated in vero cell culture. PEPEC O45(deltaler) deletion mutant lost the toxigenicity to vero cell. Test group and control group of mice were orogstrically inoculated with the PEPEC O45(deltaler) deletion mutant and the virulent strain O45 respectively. Mice were observed daily for clinical signs and weight changes. Test group of mice inoculated with PEPEC O45(deltaler) gained weight normally and experienced no clinical signs. In contrast, control group of mice inoculated with virulent strain O45 exhibited weight loss and all died in four days. In another experiment, pregnant mice and pig were orally vaccinated by PEPEC O45(deltaler) twice at interval of 14 days respectively. Subsequently, the suckling mice and pig were orally challenged with O45 at 7 days of age respectively. The results showed that 80% of the sucking mice born by vaccinated mice and 75% of the sucking pig born by vaccinated pig were survival; 15% of the sucking mice born by non-vaccinated mice and 10% of the sucking pig born by non-vaccinated pig were survival. This study demonstrated that PEPEC O45(deltaler) deletion mutant lost the toxigenicity to vero cell and to be safety to mice and pig. Oral immunization can induce specific immune responses in mice and pig, and this mutant strain could be used as an attenuated vaccine candidate against PEPEC O45.

Cloning and expression of a hemolysin gene of Aeromonas hydrophila and the immunogenicity of the toxoid / 生物工程学报

According to the GenBank sequences (GenBank Accession No. AF539467), one pair of primers was designed to amplify hly gene of Aeromonas hydrophila by PCR. After sequencing, homology analysis indicated that a DNA fragment of 1485 bp was amplified from isolated DNA from Aeromonas hydrophila, and it shared more than 99% homology in nucleotide sequence compared with other reference strains in GenBank. The gene was cloned in pET-28a vector to construct a recombinant plasmid pET-28a-hly, which was transformed into Escherichia coli BL21 (DE3), and the recombinant strain BL21(DE3)(pET-28a-hly) was obtained. The hemolysin was highly expressed when the recombinant strain BL21 (DE3) (pET-28a-hly) was induced by IPTG. The expressed protein was 56 kD as estimated by 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The immunogenicity of the expressed Hly protein was confirmed by Western blotting. Mice were immunized with inactivated whole bacteria vaccine and the genetic engineering vaccines showing promise that all these vaccines have a high protective ability. The results showed that the recombinant strain BL21 (DE3)(pET-28a-hly) could be candidate of hemolysin toxoid vaccine to provide protective immunity against diseases caused by Aeromonas hydrophila.

Development and application of a mammlian one hybrid-based high-throughput screening model for Eralpha modulator / 生物工程学报

Estrogen Receptor (ERalpha) is a member of superfamily of ligand-activated transcription factors which play critical roles in many biological processes. To screen novel modulators of ERalpha for drug development and biological function research, we developed a mammalian one-hybrid-based high-throughput screening model for ERalpha modulator. We cloned the ERalpha LBD gene from the total mRNA of fat tissue by RT-PCR and fused it with the GAL4 DNA binding domain of pBIND-GAL4 plasmid to construct a chimara expression plasmid pBIND-GAL4-Eralpha(LBD). The L02 cells was cotransfected with pBIND-GAL4-ERalpha(LBD) and a GAL4-responsive luciferase reporter plasmid pGL3-GAL4, and following treatment with test compounds for 24 h, the activities of luciferase were detected to evaluate the transactivities of ERalpha modulators. After manner optimizations of transfection conditions, Estradiol, an agonist control, induced the expression of luciferase in a dose-dependent with EC50 of 0.17 micromol/L, the maximum folds of induction was about 28.1. Tamoxifen, an antagonist control, efficiently suppressed the estradiol-mediated luciferase induction with EC50 of 0.10 micromol/L. Using this screening model, we discovered four ERalpha agonists from 2000 natural and synthetic compounds.

Construction and immunogenicity of a genetic engineered strain expressing nontoxic ST1-LT(B)-alpha-beta fusion protein against diarrhea of piglet / 生物工程学报

We constructed a recombinant strain BL21 (DE3) (pETST3LTBalphabeta) including ST1-LT(B)-alpha-beta fusion gene via molecular technology. The SDS-PAGE and Western blotting indicated that the ST1-LT(B)-alpha-beta fusion protein was highly expressed in Escherichia coli and the molecular weight of the fusion protein was about 110 kD. The recombinant strain was induced in different concentrations of lactose and different aeration rate. The optimal culture conditions in 20 L fermentor were 1% inoculation (V/V), initial aeration 5 L/min, 0.03 mol/L lactose addition 3 hours after inoculation, and increased the aeration to 12.5 L/min for the following 6 hours. The fusion protein was about 38.53% of total cellular protein. It was nontoxic, immunogenic and protective against enterotoxigenic E. coli and Clostridium perfringens infection. The constructed recombinant strain BL21 (DE3) (pETST3LTBalphabeta) could serve as a candidate vaccine strain against diarrhea of piglet.

Cloning, expression and immunity of pilA gene and ompC gene from avian pathogenic Escherichia coli / 生物工程学报

In order to amplify pilA gene and ompC gene of avian pathogenic Escherichia coli (APEC) strain, two pairs of primers were designed according to the GenBank sequences, and a 549 bp pilA gene and a 1104 bp ompC gene were obtained by PCR separately. Sequence analysis indicated that the homology of the nucleotide sequence of AEPC strain to those other reference strains was 98.18% of the pilA gene and 97.28% of the ompC gene. Two expression plasmids pETpilA and pETompC were constructed by inserting pilA gene and ompC gene into the prokaryotic expression vector pET-28a. The two plasmids were transformated into E. coli BL21 separately and two recombinant strains BL21 (pETpilA) and BL21 (pETompC) were obtained. The type 1 fimbraie and the out membrane protein were highly expressed when the recombinant strain BL21 (pETpilA) and BL21 (pETompC) were induced by IPTG Two specific proteins were detected by SDS-PAGE and immunogenicity of the expressed protein was confirmed by Western blotting and ELISA. The expressed fimbraie and OmpC were transformed into vaccine. The protective immune response was proved after the mice were immunized with the two vaccines. The results showed that the recombinant strain BL21 (pETpilA) and BL21 (pETompC) could be as candidate vaccine to provide protective immune response against AEPC infection.

A Study on the Relationship Between Post-stroke Depression and Coping Styleand Social Sustian / 中国临床心理学杂志

Objective: To explore the relationship between post-stroke depression and coping style and social sustian for guiding psychological intervention.Methods: A control study was done in 78 patients with post-stroke depression and 65 patients with non-post-stroke depression by using Medical Coping Modes Questionnaire and Social Sustian Scale.Results: The score of confrontation was lower and that of resignation was significantly higher in depression group than in non-depression group(P