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Designed experiment is a new experimental mode characterized by the student-centered and toacher-oriented teaching principle.To conform with the general trend of experimental teaching reform in medica university,Biochemistry Department of Third Military Medical University attemptod to adopt this teaching mode.It was indicated that designed experiment is favorable for the enhancement of comprehensive diathesis and will be an effective way to cultivate high-quality medical intellectuals.
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Through introducing and summarizing the application of multimedia technology in the process of biochemistry teaching,the article discusses the contribution of multimedia technology in teaching method,teaching organization,teaching content and so on and points out the importance of multimedia technology in biochemistry teaching.
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Objective To construct an expression vector pET32a(+)/SLC and express the fusion protein TrxA SLC. Methods Total RNA from human inflammatory tonsil was extracted and its cDNA was generated with reverse transcription. Mature secondary lymphoid tissue chemokine (SLC) gene was amplified by RT PCR and cloned into plasmid pET32a(+) with Nco Ⅰ and Eco RⅠ sites added to the 5′ and 3′ ends respectively. E. coli DH5? was transformed with the recombinant plasmid, and the grown clones were selected. The inserted DNA was verified by enzyme digestion and DNA sequencing. The 3 amino acids between enterokinase site and target gene were deleted with mutation and the new vector was verified by sequencing. Expression of SLC was analysed by SDS PAGE. The fusion protein was purified by metal affinity chromatography and weak cation exchange chromatography, which was analysed by SDS PAGE and Western blotting. Results Trx SLC fusion protein expression vector was successfully construced, and the fusion protein was expressed with solubility. The purified fusion protein displayed the ability of binding the goat anti human SLC polyclonal antibody. Conclusion The SLC fusion protein can be expressed with stability and solubility and primary purification is performed with metal affinity chromatography and weak cation exchange chromatography.
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Objective To clone the cDNA encoding human HMGB1, express it in E. coli, and identify its biological activity. Methods Human HMGB1 cDNA was amplified by RT-PCR and cloned into vector pUC19. After sequence analysis, the cDNA was ligated into prokaryotic expression vector pQE-80L and induced by IPTG to express HMGB1. The protein was purified with Ni~(2+)-NTA chromatography and polymyxin B affinity column. To identify the function of purified protein, the product was co-cultured with THP1 cells. Results Recombinant expression plasmid pQE-80L/HMGB1 was constructed successfully. After purification, the protein purity reached 96%. The recombinant HMGB1 stimulated THP1 to secrete TNF-? . Conclusion The highly purified HMGB1 was obtained successfully, which showed biological activity. These results lay the foundation for further research on the function of human HMGB1.
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Objective To construct, express, purify and screen immunosuppressive molecule against human soluble APRIL (a proliferation-inducing ligand). Methods The cDNA of soluble APRIL (sAPRIL) was mutated and TEL Th epitope was added. Mutants was expressed by pQE-80L/DH5? system, identified by SDS-PAGE and Western blotting, purified by Ni-NTA resin. Their activity on stimulating the proliferation of Raji cell was detected. Results Four sAPRIL mutants were constructed and expressed as follows: HEL Th epitope was added at C end (Ⅰ); HEL Th epitope was added at N end (Ⅱ); the last 45-bp DNA was deleted at C end and HEL Th epitope was added (Ⅲ); the first 45-bp DNA was deleted at N end and HEL Th epitope was added (Ⅳ). Specific protein bands according to mutant Ⅰ-Ⅳ were detected by SDS-PAGE and Western blotting. Mutant protein was purified by Ni-NTA successfully. Mutants Ⅰand Ⅱ promoted cell proliferation remarkably, and mutant Ⅲand Ⅳnot. Conclusion Four sAPRIL mutants was constructed and expressed successfully. Immunosuppressive molecule against sAPRIL that can not promote cell proliferation was screened out, and it laid a foundation for further study on their immunosuppressive function.
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Objective To observe the pathological changes of liver cells in the early periods of burn injury combined with endotoxemia and to explore its mechanisms preliminarily. Methods Rats were inflicted with 20% TBSA Ⅲ degree burn combined with intraperitoneal injection of lipopolysaccharide (burn injury combined with endotoxemia, combined group) or simply burned or simply injected. Each group included 25 rats, with 5 rats left for control. Rats in the former 3 groups were sacrificed and sampled at 1, 3, 6, 12 and 24 h after injury, 5 rats for each time point. The results were observed by HE staining, TUNEL and DNA gel electrophoresis, and the concentrations of plasma MDA and the activity of plasma SOD were assessed and analyzed. Results Apoptosis of liver cells was found in each of the 3 groups but its regularity was different from one another. Much more apoptotic cells in the combined group were found than the other 2 groups and apoptosis happened earlier with its maximum at 6 h postinjury and declined thereafter. Much less apoptotic cells were found in the simply burned group and reached the maximum at 12 h postburn. There were only a few apoptotic cells at 6 h postinjection in the simply injected group in which there were the fewest apoptotic cells in the three groups. The concentration of plasma MDA and the activity of SOD always moved in the opposite way at the same time. In the combined group, the maximum of MDA concentration and the minimum of SOD activity happened at the same time point, which was just before the one of the maximum of liver cell apoptosis. Conclusion During the early period of burn injury combined with endotoxemia, rat liver cells die mainly in the apoptotic way, with lots of apoptotic cells at 6 h and 12 h postinjury, combined with necrosis at 24 h. Lipid peroxidation may partly account for the apoptosis of liver cells.
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Objective:To generate phage displayed anti idiotypic antibody single chain variable fragments(anti Id ScFv)to monoclonal antibody MC3 directed against colorectal carcinoma.Methods:Balb/c mice were immunized i.p. with MC3(McAb against colorectal Carcinoma) conjugated with KLH,and mRNA was isolated from the spleens of the immunized mice.VH and VL DNAs of the antibody were amplified separately and assembled into ScFv DNAs with a linker DNA by PCR.The ScFv DNAs were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E.coli TG1.The transformed cells were infected with M13KO7 helper phage to yield phage antibody ScFv library.After four rounds of panning to the library with MC3,the MC3 positive clones were selected by ELISA from the preselected phages.Results:The VH,VL and ScFv DNAs were about 340,320 and 750 bp respectively.After four rounds of panning to the antibody library,15 MC3 positive phage clones displayed anti Id ScFv were selected from 50 enriched phage clones.Conclusion:The phage displayed anti Id ScFv to MC3 were successfully selected by phage antibody library technology,which might provide new putative candidate molecules for developing recombinant anti Id vaccine of colorectal carcinoma.
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Objective:To express hTRAIL_ 41～281 protein using E.coli and refold it into a functional form.Methods:The expression of protein was induced by IPTG,protein was purified by Ni-NTA chromatography column,and refolded by dialysis ,protein purified was determined by SDS-PAGE and Western blot.Antitumor activity of hTRAIL_ 41～281 protein was measured by DNA fragmentation electropherogram.Results:Results of SDS-PAGE and Western blot proved that the 30.5 kD protein purified were hTRAIL_ 41～281 protein,the purity of protein was more than 90%.After refolding,DNA fragmentation electropherogram showed that hTRAIL_ 41～281 protein had good antitumor activity.Conclusion:hTRAIL_ 41～281 protein with antitumor activity was successfully expressed with E.coli and purified by Ni-NTA chromatography column,refolded by dialysis.
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Objective To investigate the relationship of hyperglycemia to trauma score,infection,MODS and survival time of the dead patients caused by trauma.Methods A total of 455 cases of dead trauma patients selected randomly from our hospital,were divided into two groups with normal blood glucose(n=57) or hyperglycemia(n=298).The RTC,GCS and the cases of infection and MODS as well as the survival time of two groups were recorded,and the coefficients of relationship between the blood glucose and the indexes of MODS in the dead trauma patients were calculated.Results The levels of RTC,GCS in the group with hyperglycemia were higher than that with normal blood glucose(P