RESUMO
To construct and express Hsp70-HSV2gD fusion protein. Genes of Hsp70 and HSV-2gD were subcloned into vectors pGEX-4T-1 respectively. After confirmed by DNA sequence analysis, the recombinant plasmids pGEX-4T-HSP-gD was transformed into E. coli DH5alpha and induced to express with IPTG. The expressed protein was characterized by SDS-PAGE and Western blot after purified. BALB/c mice were immunized with fusion proteins respectively via intra-m uscular injection. The proliferation of spleen lymphocytes, the level of y-IFN in culture and anti-HSV-2gD IgG antibody in serum was detected was detected. The expressed protein was analyzed by SDS-PAGE after induced with IPTG, which showed a new band with an apparent molecular mass corresponding to the predicted size (118 kD). Western Blotting analysis demonstrates that the purified Hsp70-HSV2gD fusion protein had specific binding activity. The stimulation indexes of spleen lymphocytes, the level of gamma-IFN in culture and anti-HSV-2gD IgG antibody in serum of GST-Hsp70-gD group was obviously higher than that of other groups (P < 0.05 respectively). The successful expression of the Hsp70-HSV2gD fusion protein, which can induce immune responses, laid a solid foundation for its further research.
Assuntos
Proteínas de Choque Térmico HSP70/genética , Proteínas Recombinantes de Fusão/genética , Proteínas do Envelope Viral/genética , Animais , Western Blotting , Proliferação de Células , Expressão Gênica , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico HSP70/imunologia , Proteínas de Choque Térmico HSP70/isolamento & purificação , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Interferon gama/sangue , Camundongos , Plasmídeos/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Baço/citologia , Baço/imunologia , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/isolamento & purificaçãoRESUMO
AIM: To express human anti-keratin Fab in Pichia pastoris secretively and optimize the expression condition. METHODS: Genes of kappa chain and Fd fragment of anti-keratin antibody from the plasmids p3MH/Fab were subcloned into vectors pPIC9K and pPICZalphaA respectively. After confirmed by DNA sequence analysis, the recombinant plasmids pPIC9K/L and pPICZalphaA/Fd were transducted into the genome of GS115 Pichia pastoris using two-step integrating technology. Mut(+) multiple insert transformants were screened by G418 and Zeocin. The pH value and methanol concentration was adjusted to optimize the expression condition. RESULTS: Under the optimized expression condition, the Fab of anti-keratin antibody was efficiently secreted into the medium. Western blot assay proved that the expressed protein had specific keratin binding activity. CONCLUSION: The successful expression of the anti-keratin Fab in Pichia pastoris has laid a solid foundation for its further application.
Assuntos
Anticorpos/genética , Anticorpos/imunologia , Expressão Gênica , Fragmentos Fab das Imunoglobulinas/biossíntese , Fragmentos Fab das Imunoglobulinas/imunologia , Queratinas/imunologia , Pichia/genética , Animais , Anticorpos/metabolismo , Especificidade de Anticorpos , DNA Fúngico/análise , DNA Fúngico/genética , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Expressão Gênica/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Fragmentos Fab das Imunoglobulinas/genética , Metanol/farmacologia , Plasmídeos/genética , Reação em Cadeia da PolimeraseRESUMO
AIM: To construct an eukaryotic expression vector containing the gene of anti-keratin antibody and express it in CHO(dhfr(-)) cells. METHODS: Both the V(L) and V(H) genes of an anti-keratin Fab from a phage display library were amplified by PCR. Using PCR product as the template, the V(kappa) and V(H) genes with the leader sequences (named L(kappa) and L(H) respectively) were amplified by overlapping PCR. After digested with endonuclease Xba I/BamH I and Xho I/Hind III, L(kappa) and L(H) genes were inserted into pWD digested with Xba I/BamH I and Xho I/Hind III, respectively to construct pWDkappaH. After PCR and sequencing, the expression plasmid pWDkappaH was transfected into CHO (dhfr(-)) cells. The culture supernatant of the transfected cells was collected and assayed for IgG activity. RESULTS: The eukaryotic expression vector pWDkappaH was constructed successfully and expressed in CHO(dhfr(-)) cells. The expression of intact IgG against keratin was identified by ELISA and RT-PCR. CONCLUSION: The successful expression of intact IgG against keratin lays the foundation for its clinical application.
Assuntos
Células Eucarióticas/metabolismo , Fragmentos Fab das Imunoglobulinas/genética , Imunoglobulina G/genética , Queratinas/genética , Animais , Células CHO/metabolismo , Cricetinae , Cricetulus , Vetores Genéticos , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Imunoglobulina G/metabolismo , Queratinas/imunologia , Queratinas/metabolismo , Biblioteca de Peptídeos , Plasmídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , TransfecçãoRESUMO
AIM: To express Fd fragment and L chain of human anti-keratin Fab in E.coli BL21(DE3) respectively and obtain human anti-keratin Fab by renaturation in-vitro. METHODS: Genes of L chain and Fd fragment of anti-keratin antibody from the plasmids p3MH/ Fab were subcloned into vector pET32a respectively. The recombinant plasmids pETL and pETFd were transformed into E.coli BL21(DE3) and induced to express with IPTG. Fd fragment and L chain inclusion bodies were solubilized and combined in equal molar ratio in the refolding solution. The renatured Fab was characterized by SDS-PAGE, Western blot and ELISA. RESULTS: Fd fragment and L chain of human anti-keratin Fab were efficiently expressed. ELISA and Western blot showed that the renatured Fab could bind with human keratin. CONCLUSION: The successfully prepared anti-keratin Fab with binding activity to human keratin laid a solid foundation for its further application.
