-
[show abstract]
[hide abstract]
ABSTRACT: This study was conducted to potentiate the expression of outer membrane protein OmpL17 of the strong virulent L. interrogans serovar Lai and investigate its immunogenicity in rabbits. The OmpL17 was cloned into prokaryotic expression vector pGEX-1lambdaT. The recombination expression plasmid pGEX-OmpL17 was transformed into E. Coli JM109. The GST fused protein GST-OmpL17 was expressed after induction by IPTG, then GST-tag was by thrombin and purified using Bulk GST purification Modules. SDS-PAGE and Western blotting analysis indicated that the molecular weight of GST-OmpL17 and OmpL17 was about 54 KDa and 28 KDa respectively. The outer membrane protein OmpL17 was subcutaneously injected into rabbits and high titre anti-OmpL17 antibody was obtained (1:4896) which could conjugate specifical with OmpL17. In conclusion, OmpL17 and specifical anti-OmpL17 antibody were obtained, which provided an experimental basis for researching pathogenic effect and immunity functions of OmpL17.
Sheng wu yi xue gong cheng xue za zhi = Journal of biomedical engineering = Shengwu yixue gongchengxue zazhi 05/2005; 22(2):250-3.
-
[show abstract]
[hide abstract]
ABSTRACT: To construct anti-human AFP single chain fragment variable (ScFv) gene, transform it into BL-21 (DE3) E. coli for expression, and identify its bioactivity.
VH and VL genes of anti-human AFP monoclonal antibody were cloned by RT-PCR from hybridoma. The ScFv gene was spliced by sequence overlap extending (SOE) PCR, and then it was ligated into pGEM-T vector to be identified by endonuclease digestion, PCR and sequencing. ScFv gene was cloned into pET32 (a+) vector and transformed into BL-21 (DE3) E. coli. The positive clones were screened out by IPTG induction, and the ScFv antibody was purified to be identified by SDS-PAGE and competitive inhibition ELISA test.
The VH DNA consisted of 339 bases, coming from the mouse IgG gamma chain. The VL DNA consisted of 312 bases, coming from the mouse IgG kappa chain. The VH and VL genes were spliced by 45 bases coding a (G4S)3 flexible linker. The ScFv gene consisted of 696 bases. The ScFv antibody expressed by BL-21 (DE3) fused with TrxA tag protein and formed inclusions. The relative molecular mass of TrxA-ScFv fusion protein is about 40 x 10(3) and that of ScFv is about 24 x 10(3). The ScFv antibody has excellent activity tested by competitive inhibition ELISA, the TrxA-ScFv could inhibit about 41% of the McAb to bind antigen and ScFv could inhibit about 53%.
We have successfully constructed an anti-human AFP ScFv gene with 696 bases; it can express in BL-21 with high activity.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 02/2005; 36(1):20-3.
-
[show abstract]
[hide abstract]
ABSTRACT: To clone the variable region genes of the monoclonal antibody (McAb) against human heterogeneous nuclear ribonucleoprotein A2/B1 (HnRNPA2/B1), ligate them to assemble single chain Fv (ScFv) gene and express in Escherichia coli.
The specificity of the anti-HnRNPA2/B1 McAb 3E8 to synthetic HnRNPA2/B1 peptide, HnRNPA2/B1 protein in lung cancer cells were examined by dot-immunobinding assay, Western blot and immunohistochemistry. The variable region genes of heavy chain (VH) and light chain (VL) were amplified from hybridoma cell by reverse transcription-polymerase chain reaction(RT-PCR), and then were linked by a linker peptide using SOE-PCR (splicing by overlap extension-PCR) to construct recombination ScFv gene. The latter was cloned into the expression vector pET28 (a+) and expressed in E coli BL21. The expressed product was identified by SDS-PAGE and competitive ELISA inhibition test.
It was shown that the McAb combined specifically with synthetic HnRNPA2/B1 peptide and HnRNPA2/B1 protein in three lung cancer cells. The cloned VH gene and VL gene were 345 bp and 309 bp respectively and were linked successfully to obtain ScFv gene. The ScFv protein was expressed in the form of inclusion body, with molecular weight of 28,000 and immunoreactivity to HnRNPA2/B1.
VH gene, VL gene and ScFv gene of anti-HnRNPA2/B1 antibody were cloned, constructed and functionally expressed in E coli. These results provide the experimental basis for elucidating the role of HnRNPA2/B1 in lung cancer.
Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 01/2005; 21(6):548-51.
-
[show abstract]
[hide abstract]
ABSTRACT: To prepare adrimycin-loaded human serum albumin microspheres(ADR-HSA-MS) and to study its pharmaceutic properties and in vitro cytotoxicity.
ADR-HSA-MS, consisting of ADR and HSA, were prepared by emulsion heating solidification method and its morphology was observed by optical microscopy and scanning electron microscopy. Pepsin digestion method was used for determining drug loading of ADR-HSA-MS and membrane diffusion technique for detection of in vitro release of ADR from the microspheres. MTT colorimetric assay was used to determine cytotoxicity of ADR-HSA-MS for human hepatoma SMMC-7721 cell line.
ADR-HSA-MS looked like smooth red spheres with an average diameter of 1.2 microns and had an apparent drug-loading of 2.73% or an effective drug-loading of 1.69%. Its peak of the accumulated drug-releasing fraction was 65%, and 50% of ADR releasing from the MS would take five days, which suggested a slow release of ADR from the albumin microspheres. ADR-HSA-MS exerted remarkable cytotoxicity to human hepatoma SMMC-7721 cells with dose-dependence at a range of ADR concentration from 0.3 microgram/ml to 2.5 micrograms/ml when incubated with the target cells for four days. Its killing curve was similar to that of free ADR.
Emulsion heating solidification method could be used for preparation of adrimycin-loaded human serum albumin microspheres with sustained release property.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 02/2004; 35(1):107-9, 137.
-
[show abstract]
[hide abstract]
ABSTRACT: To observe if antibody-targeted immunonanoparticles could internalize into sensitive and multidrug resistance (MDR) cells of human hepatoma; and to study if the immunonanoparticles could reverse the MDR.
Human hepatoma-specific adriamycin-loaded human serum albumin immunonanoparticles (HAb18-ADR-HSA-NP) were incubated with human hepatoma sensitive cell line (SMMC-7721) or MDR cell line (SMMC-7721/MDR+) and the internalization of immunonanoparticles were observed by laser confocus microscopy, scanning electron microscopy and transmission electron microscopy; MTT colorimetric assay was used for assaying in vitro cytotoxicities of HAb18-ADR-HSA-NP to the resistant variant cells. Then based on these data, IC50 value of the immunonanoparticles and RF (Resistant Factor) of MDR cells were calculated.
Laser confocus microscopy showed that many fluorescent particles (labeled immunonanoparticles) tightly adsorbed to SMMC-7721 cells and were also seen in cytoplasm of SMMC-7721 cells. When incubated with immunonanoparticles at 37 degrees C, many of immunonanoparticles were visualized in cytoplasm of SMCC-7721 or SMCC-7721/MDR+. These immunonanoparticles-contained cells exhibited damaged ultrastructures and the damage degree depended on incubation time. When the human hepatoma cells were pretreated with HAb18 antibody and incubated with immunonanoparticles, few immunonanoparticles were seen in cytoplasm of the cells, suggesting antibody-specific internalization of the immunonanoparticles. Scanning electron microscopy demonstrated specific binding of the immunonanoparticles to the resistant variant cells. That immunonanoparticles exerted enhanced cytotoxicity to the resistant variant cells was demonstrated by a decrease of RF value of MDR cells, compared with free ADR (4.4 vs. 2.1).
Human hepatoma-specific adriamycin-loaded human serum albumin immunonanoparticles could specifically internalize into sensitive or multidrug resistance cells of human hepatoma via antibody direction. The immunonanoparticles could enhance the sensitivity of MDR cells to ADR cytotoxicity, suggesting its reverse effect on MDR.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 08/2003; 34(3):431-4.
