RESUMO
BACKGROUND: More than a dozen of fungal immunomodulatory proteins (FIPs) have been identified to date, most of which are from Ganoderma species. However, little is known about the similarities and differences between different Ganoderma FIPs' bioactivities. In the current study, two FIP genes termed FIP-gap1 and FIP-gap2 from G. applanatum, along with LZ-8 and FIP-gsi, another two representative Ganoderma FIP genes from G. lucidum and G. sinense were functionally expressed in Pichia. Subsequently, bioactivities of four recombinant Ganoderma FIPs were demonstrated and compared. RESULTS: All the four Ganoderma FIP genes could be effectively expressed in P. pastoris GS115 at expression levels ranging from 197.5 to 264.3 mg L- 1 and simply purified by one step chromatography using HisTrap™ FF prepack columns. Amino acid sequence analysis showed that they all possessed the FIP conserved fragments. The homologies of different Ganoderma FIPs were from 72.6 to 86.4%. In vitro haemagglutination exhibited that FIP-gap1, FIP-gsi and LZ-8 could agglutinate human, sheep and mouse red blood cells but FIP-gap2 agglutinated none. Besides, the immunomodulation activities of these Ganoderma FIPs were as: rFIP-gap2 > rFIP-gap1 > rLZ-8 and rFIP-gsi in terms of proliferation stimulation and cytokine induction on murine splenocytes. Additionally, the cytotoxic activity of different FIPs was: rFIP-gap1 > rLZ-8 > rFIP-gsi > rFIP-gap2, examined by their inhibition of three human carcinomas A549, Hela and MCF-7. CONCLUSIONS: Taken together, four typical Ganoderma FIP genes could be functionally expressed in P. pastoris, which might supply as feasible efficient resources for further study and application. Both similarities and differences were indeed observed between Ganoderma FIPs in their amino acid sequences and bioactivities. Comprehensively, rFIP-gaps from G. applanatum proved to be more effective in immunomodulation and cytotoxic assays in vitro than rLZ-8 (G. lucidum) and rFIP-gsi (G. sinense).
Assuntos
Proteínas Fúngicas/genética , Proteínas Fúngicas/farmacologia , Ganoderma/genética , Expressão Gênica , Fatores Imunológicos/genética , Fatores Imunológicos/farmacologia , Motivos de Aminoácidos , Animais , Linhagem Celular , Citocinas/genética , Citocinas/imunologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/fisiologia , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Ganoderma/química , Ganoderma/metabolismo , Testes de Hemaglutinação , Humanos , Fatores Imunológicos/isolamento & purificação , Fatores Imunológicos/metabolismo , Camundongos , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , OvinosRESUMO
BACKGROUND: To confirm if Puumala like viruses exist in China. METHODS: RNA was extracted from lungs of bank voles captured in the Northeast China, partial S segments genome of Puumala viruses were amplified and sequenced. RESULTS: 926 bp cDNA of S segments of Puumala like virus was amplified and sequenced. The phylogenetic analysis revealed that the Puumala like viruses found in China were most close to that found in Far East region of Russia. CONCLUSIONS: Puumala like virus does exist in Northeast China, and the nucleotides sequence of the viruses have high homolog to Puumala viruses found in Russia.
