RESUMO
AIM: To construct a prokaryotic recombinant vector of Epstein-Barr virus (EBV) membrane protein gp85, to express the protein in E.coli and characterize the antigenicity of this non-glycosylated protein. METHODS: The BXLF2 gene coding 5'-terminal truncated of EBV gp85 was amplified from the EBV strain B95-8 cell line with specific primers. After identification by the restriction digestion with Hind III and Xho I, the PCR product was inserted into the prokaryotic expression plasmid pGEX-5T and confirmed by sequencing. The constructed prokaryotic expression vector pGEX5T-85N was transformed into the competent E.coli BL21. The expressed recombinant protein gp85N was purified by affinity chromatography, characterized by Western blot, and used immunize BALB/c mice. The titer of antiserum from the immunized mice was detected by ELISA. RESULTS: Sequencing analysis revealed that the obtained truncated BXLF2 gene was identical to that published in GenBank and successfully cloned into pGEX-5T. SDS-PAGE showed that the expressed recombinant protein was partially soluble with a relative molecular mass of 45,000. ELISA results indicated that the expressed gp85N was recognized by that the anti-gp85 mAb and contiserum with high titer was obtained from the immunized mice. CONCLUSION: The obtained recombinant gp85N with an excellent antigenicity should provide preliminary data for characterization of the antibody produced by the immunized mice.
Assuntos
Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Animais , Anticorpos/imunologia , Western Blotting , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Vetores Genéticos/genética , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
OBJECTIVE: To identify the mutational genotype of three Chinese families with X-linked adrenoleukodystrophy (X-ALD: MIM#300100). METHODS: Total RNA was extracted from the peripheral blood leukocytes of patients 1, 2 and the mother of patient 3, using RNA blood Mini kit (QIAGEN). After reverse transcription, cDNA was amplified in four overlapping segments. The PCR products were purified and directly sequenced. To confirm the mutations, the genomic DNA was isolated from the patients and their family members using DNA blood isolation kit (MO-BIO) and analyzed by PCR-restrictive digestion or amplification refractory mutation system. RESULTS: Three distinct mutations were detected in the ABCD1 gene of the three pedigrees. A mutation of CCC-->CGC was detected at codon 534 of the ABCD1 gene from patient 1, resulting in the arginine for proline substitution. A change of GGG-->AGG was found at codon 266 of the second patient's gene, accompanied with the replacement of glycine by arginine. A mutation of CGC-->GGC was found at codon 617 in one ABCD1 allele of the third patient's mother, leading to the glycine for arginine substitution. The three mutations were confirmed through restriction analysis or amplification refractory mutation system. CONCLUSION: Three ABCD1 gene missense mutations were detected in three unrelated Chinese families with X-linked adrenoleukodystrophy, one of which, the mutation (P534R), is novel in Chinese with ALD, and the other two G266R and R617G mutations, have been reported outside China.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Adrenoleucodistrofia/genética , Mutação , Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP , Criança , Pré-Escolar , Humanos , Masculino , LinhagemRESUMO
AIM: To express Japanese encephalitis virus (JEV) E protein in methylotrophic yeast Pichia pastoris. METHODS: The gene coding for JEV E protein was sub-cloned into vector pPIC9k. The constructed plasmid was transformed into yeast SMD1168 by electroporation. The recombinant transformants with a high copy number of the plasmid were selected by using MD plate and G418. The expression of E protein in yeast was induced by the addition of methanol and analyzed by SDS-PAGE and Western blot. RESULTS: The protein was produced at a yield of 50 mg per litre of culture. Owing to heterogeneous glycosylation, relative molecular mass (M(r)) of the expressed E protein was sized about 113 000 and 78 000. CONCLUSION: JEV E protein was expressed successfully in yeast Pichia pastoris, which should be useful for the production of diagnostic reagents and genetically engineered vaccine of JEV.
