One of the aims of biotechnological pharmaceutics is to educate innovative highquality medical talents.The second classroom activities can be used as a complementation to ensure the teachiug effectiveness of the course.This study focuses on the experience of carrying out the second class of biotechnological pharmaceutics and tends to provide references for teachers who are going to carry out the second class.

During recent years, some new methods for phage display panning have been developed by using complex biological systems instead of purified antigens, antibodies or receptors as selector targets. These complex biological systems include living cells, viruses, mouse tissues and tumors etc. The peptides identified by using tumors as selector molecules can selectively home to blood vessels of experimental tumors and may have potential applications in targeted anti-tumor treatment.

Objective To develop the purification method of rhIL 18 expressed in E coli Methods The harvested cells were disrupted using lysozyme and sonication The rhIL 18 inclusion bodies were extracted by centrifugation Washed inclusion bodies were solubilized in 8 mol/L urea The solubilized inclusion bodies were first purified by ion exchange chromatography under denaturing conditions After renaturation, gel filtration chromatography was used for further purification Samples were analyzed by SDS PAGE, HPLC, Western blotting and N terminal amino acid sequencing The biological activity of purified rhIL 18 was also measured Results It was found that rhIL 18 was expressed intracellularly in E coli as insoluble inclusion bodies The purity of rhIL 18 in extracted inclusion bodies was above 80% The final purified rhIL 18 was of high purity and exhibited the mass of molecule 19?10 3 by SDS PAGE The sequence of 15 amino acid residues form NH2 terminus of the purified rhIL 18 was consistent with the predicted sequence The purified rhIL 18 also induced IFN ? production of Con A stimulated human PBMC Conclusion The established method for purifying rhIL 18 is simple and effective and might be useful in the development of the large scaled purification process of rhIL 18

0.05).The level of serum EGF in the tumor group(0.426?0.084 ng/ml～0.456?0.095 ng/ml)was significantly higher than that in the control group(0.243?0.086 ng/ml,P

Objective To study the character of transformation of H.pylori into coccoid form under oxidative stress, and the changes in the biological charasteristics after transformation. Methods The coccoid form of H.pylori was studied with bacteriological, enymological, and genetical methods to identify its differences from the original form. Animal experiment was conducted to determine its implantation capacity. Results Morphological transformation from spiral to coccoid form occurred with the prolongation of culture under oxygen stress. The metabolic activity was reduced gradually, finally maintained at a lower level. The activities of urease, alkaline phosphatase and acid phosphatase were decreased markedly. The mRNA levels of main virulence factors were declined. Both mRNA and activities of enzyme of Sod B and Kat retained high levels. A part of the coccoid forms showed reversion to helical form, and colonization in mice stomach could be found after being inoculated to mice. Conclusions The metabolism and virulence of H.pylori were all lowered under oxidative stress, but a part of the coccoid form may be viable. Sod B and Kat may play an important role in the oxygen metabolism of H.pylori.

Objective To investigate the fermentation conditions for the heat-labile enterotoxin B subunit (LTB) of genetic recombinant E.coli. Methods The effects of different kinds of media, the range of pH, the induction time, the glucose concentration, and the fed-batch on LTB level in 10-liter fermenter under the condition of cascade controlling dissolved oxygen content were analyzed. Results Under the established conditions, the cell output per liter was 40.5g, and the expression level was 30.6%. Conclusion The results indicate that the stable fermentation technology with high efficiency has been established.

Objective To clone hpaA gene of H.pylori and construct its eukaryotic expression plasmid. Methods Gene hpaA amplificated from genome of H.pylori 11637 strain by PCR was subcloned into pMD18-T vector. Then hpaA cut down from the vector with restriction enzyme was inserted to pTCAE, and the product confirmed by restriction enzyme digestion and sequence was transformed to E.coli DH5?. pT-hpaA was transfected into CHO cell by electroporation, and HpaA protein was detected by Western blot. Results hpaA gene was inserted into pTCAE and the immunological reaction band with anti-HpaA serum at MW 30 000 was detected by Western blot. Conclusion Eukaryotic expression plasmid of hpaA of H.pylori is successfully constructed. Western blot analysis demonstrates that the expression of HpaA protein can be detected in culture supernatants of transfeced CHO cells, which lays the foundation for the further study.

Objective To construct the recombinant adenovirus encoding human IL-24 gene for future gene therapy. Methods The human IL-24 gene fragment was cloned into the shuttle plasmid pAdTrack-CMV to form the transfer vector by the method of homogenous recombination in bacteria. Then the recombinant adenovirus was transfected into 293T cells using Lipofectine DOTAP. The target gene was detected by polymerase chain reaction (PCR). The titer and its infection rate were determined using the green fluorescent protein (GFP) expression in the shuttle plasmid. The expression of target protein was measured by the method of immunohistochemistry. Results Restriction endonuclease and PCR analysis confirmed that the human IL-24 gene was successfully inserted into the adenovirus vector. The titer of the recombinant adenovirus was 1.2?10 10 pfu/ml. The adenovirus has a strong effect on A549 cells and human IL-24 can express in it. Conclusion The recombinant adenovirus containing human IL-24 gene was successfully constructed by the method of homogenous recombination in bacteria.

Objective To construct the recombinant adenovirus encoding human p16 gene for future gene therapy.Methods The human p16 gene fragment was cloned into the shuttle plasmid pAdTrack-CMV to form the transfer vector by the method of homogenous recombination in bacteria.Then the recombinant adenovirus was transfected into 293T cells using Lipofectine DOTAP.The target gene was detected by polymerase chain reaction(PCR).The titer and its infection rate were determined using the green fluorescent protein(GFP) expression in the shuttle plasmid.Results Restriction endonuclease and PCR analysis confirmed that the human p16 gene was successfully inserted into the adenovirus vector.The titer of the recombinant adenovirus was 6.1?10~(10)pfu/ml.The adenovirus has a strong effect on human fibroblast cells.Conclusion The recombinant adenovirus containing human p16 gene was successfully constructed by the method of homogenous recombination in bacteria.

Objective To investigate and compare the humoral immune response induced by eukaryotic expression plasmid pTCAEureB (pTureB)and pTCAEhpaA (pThpaA) injected intramuscularly in mice. Methods The ureB gene fragment was inserted into a eukaryotic expression vector pTCAE, and pTCAEureB was constructed. A total of 60 BALB/c mice were randomly assigned to be immunized by intramuscular injection of blank plasmid pTCAE, plasmid pTureB and pThpaA (n=20 in each group). Titers of specific IgG antibody in serum of each group were detected through ELISA respectively on week 2, 4, 6 after immunization. Results Specific IgG were observed in pTureB and pThpaA immunized groups and titers of antibody increased as time prolonged. IgG level was significantly higher in pT-ureB immunized group than in pT-hpaA group on week 6 after immunization. Conclusion Plasmid pT-ureB possesses good immunogenicity and induces a higher immune response than pT-hpaA.