RESUMO
AIM: To construct the expression vector pET-28α-Trail(114-281); and find the optimal conditions for target gene expression, host bacteria lysis, and protein purification, and to detect the apoptosis function of the recombinant protein. METHODS: The functional domain of Trail(114-281); was amplified by PCR and cloned into the expression vector pET-28α(+). After confirmed by DNA sequencing, the Trail(114-281); was expressed in E.coli BL21 under the condition of different A(600);, IPTG concentration and temperature. Host bacteria were lysed using three different ways, including ultrasonication, osmotic shock and IP lysis, and the target protein was purified using Ni-NTA affinity chromatography or cutting-gel purification. The advantages and shortcomings of these methods were compared to find the most efficient ways for expression and purification of the recombinant protein. The immunocompetence of Trail protein from cutting-gel purification was analyzed by Western blotting, A549 cell apoptosis induced by purified protein from Ni-NTA chromatography was detected by flow cytometry. RESULTS: The 516 bp Trail(114-281); gene was cloned, and expressed in E.coli BL21. When A(600);=0.6, recombinant host bacteria were induced by 1.0 mmol/L IPTG at 37 DegreesCelsius for 4 h, which was the optimal condition for the expression of inclusion body, and the soluble protein was expressed stably on the condition of 25 DegreesCelsius, A(600);=1.0, IPTG1.0 mmol/L. Ultrasonication could get maximal protein compared to the other methods. The two purification ways both could purify taget protein successfully. Western blot analysis showed that the protein purified by cutting-gel has a good immunologic activity. Protein from Ni-NTA affinity chromatography caused cell apoptosis. CONCLUSION: The expression vector pET-28α-Trail(114-281); can be constructed and expressed in E.coli BL21 successfully. Temperature is a more important effect factor of Trail(114-281); expression in host bacteria compared with other factors. Cutting-gel protein has immunogenicity, and Ni-NTA protein could keep its function. These results provide a basis for the further functional research and application of Trail.
Assuntos
Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/isolamento & purificação , Linhagem Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Solubilidade , Ligante Indutor de Apoptose Relacionado a TNF/químicaRESUMO
Amphioxus, a cephalochordate, is the closest living relative to the vertebrates. In order to investigate the molecular mechanisms of the early embryogenesis of amphioxus, we constructed a neurula embryo cDNA library of Chinese amphioxus (Branchiostoma belcheri tsingtauense) and generated 5235 expressed sequenced tags in the present study. The initial ESTs consisted of 638 clusters and 1855 singletons, which revealed approximately 2493 unique genes in the data set. Of these sequences, 35.52% ESTs matched to known genes, 12.76% matched to other ESTs, and 51.71% had no match to any known sequences in GenBank. Interestingly we found homologous genes related to neural development and human disease. Bioinformatic analysis showed the direct evidence that the gene homologue found only in vertebrates in previous studies also exists in the amphioxus genome. This study provides a preliminary view of the gene information involved in the development of neurula embryos of Chinese amphioxus and helps our understanding of vertebrate evolution at gene level.
