ABSTRACT
Objective:To establish a double antibody sandwich ELISA assay for detection of human IL-37 in serum.Methods: Mouse anti-human IL-37 monoclonal antibody was used as capturing antibody,rabbit anti-human IL-37 polyclonal antibodies served as detection antibody,HRP labeled goat anti-rabbit IgG employed as second antibody and recombinant human IL-37 protein used as reference standard for the establishment of a doubleantibody sandwich ELISA.The working conditions were optimized,such as sensitivity,linear range,reproducibility and evaluated the serum IL-37 in patients with Dengue fever.Results: The sensitivity of the established ELISA method was 1.465 μg/L approximately.Likewise,the linearity range of this method was about (1.465-46.875) μg/L.Further,the co efficient of variation (CV) of inter-batch and intra-batch in this study were 6.6％ and 11.7％,respectively.Notably,this method could be used in the detection of IL-37 in serum of the patients with Dengue fever,showing that the level of IL-37 in Dengue fever patients was much higher than that in healthy controls.Conclusion: The double antibody sandwich ELISA assay for the detection of human IL-37 was successfully established,which can be apply to detect of human IL-37 in clinical samples.
ABSTRACT
We expressed B cell epitopes of dengue virus envelope protein and NS1 protein in prokaryotic cells,and purified and evaluated for its serological activities.A recombinant multi-epitope chimeric gene named rE including eight B cell epitopes was connected by linker peptide (EAAAK)2 and cloned into prokaryotic expression vector pET-28a(+),and transformed into E.coli BL21(DE3) cells for expression under induction of IPTG.The expressed recombinant protein was purified with 6× His purification media,and identified by SDS-PAGE and Western blot,and its antigenicity was analyzed by using an indirect ELISA assay.The recombinant expression vector pET28a-rE was constructed and expressed in BL21 (DE3) successfully,but the recombinant proteins mainly appeared as inclusion bodies.The target protein was obtained with high purity through the purification of affinity chromatography.SDS-PAGE and Western blot analysis showed that the molecular weight of fusion protein was in the expected line.The established indirect ELISA has high accuracy.This recombinant peptide antigen expressed in E.coli has good potential for serum testing.
ABSTRACT
Objective To establish uPA inducible expression system using recombinant retroviral system for the further construction of inducible uPA-SCID animal model .Methods The Inducible expression system need to construct two plasmids:pLNHXO1O2-Alb-GLUC-FMN2A -rtTA and pLNHXO5O6-TRE2-uPA-IRES-ZsGreen respectively. Both plasmids were based on retroviral vector pLNHX , Albumin promoter gene ( Alb) and rtTA gene or uPA gene and ZsGreen were obtained by PCR reaction and inserted into pLNHX .The Gaussia enzyme fluorescent element ( GLUC) was used to monitor rtTA expression in pLNHXO1O2-Alb-GLUC-FMN2A-rtTA, and the ZsGreen for uPA expression monitoring in pLNHXO5O6-TRE2-uPA-IRES-ZsGreen.The correct constructed plasmids were transfected into packaging cell line GP 2-293 to gain recombinant viral particles .NIH/3T3 cells were infected with these viral particles and selected with G 418.Gene expression in the surviving cells was confirmed by the PCR method .Results The recombinant retroviral vectors harbouring target genes were successfully cloned .The rtTA gene in pLNHXO1O2-Alb-GLUC-FMN2A-rtTA was expressed, and uPA can be induced to express in pLNHXO5O6-TRE2-uPA-IRES-ZsGreen by doxycycline (Dox) when the plasmid transfected into the HepG-Tet-on cell.The constructed recombinant two retroviral vectors were transfected into GP 2-293 packaging cells respectively to gain infectious viral particles .Then,NIH/3T3 cells were infected with these viral particles and single-cell clones which stably expressed the transgenes were successfully established .Conclusion This study primarily established uPA inducible expression system , it laid a foundation for the murine model of inducible liver damage , and provided a novel technical platform for further building the liver humanised murine models for viral hepatitis studying .
ABSTRACT
There has been no detailed reiport on the changes of the activity of serum glycylproline dipeptidyl aminopeptidase (GPDA) in patients with pancreatic career.Tn this paper,the serum level of GPDA was measured in 166 cases with various malignant and benign gastrointestinal diseases and 60 normal controls.It was found that the serum GPDA activity in patients with pancreatic cancer was 101,02?75.84u/L and significantly higher than that in the patients with pancreatitis and in the normal controls.There was a statistically significant difference of the GPDA activity value between the patients with malignant and benign pancreatic diseases.The elevation of GPDA activity was more marked in those patients with cancers in the head of the pancreas.In addition,the elevation of GPDA activity was dependent on the differentiation of cancer cells but not on the size of cancers.Significantly higher level of GPDA was also found in patients with hepatic carcinoma and carcinoma of ampulla of vater but significantly lower level in patients with gastric cancer.It is believed that the determination of serum GPDA activity might be helpful for the diagnosis of pancreatic cancer.