RESUMO
Gene engineering is the main course of biological engineering. It should be adapted to the demand of innovation spirit, practice ability and comprehensive quality of students. Educational reform of gene engineering conducted by constructing system of theory and practice, optimizing course teaching content, strengthening practice teaching content, using modern teaching technology, strengthening web course construction and improving teaching methods. We pay attention to impart specialty knowledge and learning methods to students. Its aim was to increase teaching effects and meet the demands of bioengineering specialty and qualified personal training in 21 century.
RESUMO
K88ac genes, heat-stable enterotoxin I (ST1) mutant genes and heat-labile enterotoxin B subunit (LTB) genes from plasmids of Escherichia coli C83902 were amplified by PCR. The recombinant expression plasmid pXKST3LT5 containing K88ac-ST1-LTB fusion gene was constructed by recombinant DNA technique and then transformed into Escherichia coli BL21(DE3). The K88ac-ST1-LTB fusion protein was highly expressed in recombinant strain BL21 (DE3)(pXKST3LT5) and the expression level of the K88ac-ST1-LTB fusion protein was about 75.53% of total cellular protein by SDS-PAGE and thin-layer gel scanning analysis. More importantly, mice immunized with crude preparation containing the fusion protein inclusion bodies or inactivated recombinant strain produced antibodies that were able to recognize ST1 in vitro. These sera antibodies were able to neutralize the biological activity of native ST1 in the suckling mouse assay. Hence the fusion protein was nontoxic and immunogenic, the constructed recombinant strain could be used as a candidate of vaccine strain.
Assuntos
Animais , Camundongos , Toxinas Bacterianas , Genética , Alergia e Imunologia , Eletroforese em Gel de Poliacrilamida , Métodos , Enterotoxinas , Genética , Alergia e Imunologia , Ensaio de Imunoadsorção Enzimática , Métodos , Escherichia coli , Genética , Proteínas de Escherichia coli , Expressão Gênica , Engenharia Genética , Proteínas Recombinantes de Fusão , Genética , Alergia e Imunologia , Recombinação Genética , Dodecilsulfato de SódioRESUMO
The ScFv gene containing Nco I and Bam HI was amplified by PCR, and inserted into the prokaryotic expression vector pHOG21 with Nco I and Bam HI digestion. After screening, high-level expression recombinant XL1-Blue (pHOG2E3) were obtained, and the different expression levels of ScFv were detected by SDS-PAGE and EUSA. When the strain was induced by 0.5 mmol/LIPTG and 0.4mol/L sucrose in the culture medium LB at 37℃ for 6 hours, the high level production of ScFv protein was obtained. The molecular weight of the expressed ScFv is 31, 000 D. The ScFv was mainly in the form of inclusion body, but it could also be in the culture medium and soluble periplasmic content. Above all, the ScFv protein could neutralize the phospholipase C activities of alpha-toxin of Gostridium perfrigens type A.