| This thesis mainly is consists of two parts.The first part is the optimization,recombination expression and validation of biological functions of FIP-gap.In order to study the active ingredients and biological functions of Ganoderma Applanatum,adopting the method of homologous cloning has cloned two kings of FIP-gap gene(FIP-gap1 and FIP-gap2)from the fruiting body genome of Ganoderma applanatum.According to pichia codon preference optimizing FIP-gap1/2 genes,adding histidine label sequence in the C end of the gene,and building the recombinant expression vector pPIC9-His-FIP-gapl/2.Using pichia GS115 to efficiently express recombinant protein FIP-gap1/2,SDS-PAGE and Western blot results found rFIP-gapl/2 had protein bands around 14 kDa and 17 kDa,this showed that recombinant proteins expressed correctly.Expression of rFIP-gapl/2 were highest in the fourth day,with yeild of 207.4 mg/L and 157.5 mg/L respectively,far higher than nonoptimized FIP-gap1/2 expression quantity(2.66 mg/L and 4.56 mg/L).Obtaining purified rFIP-gap1/2 after rFIP-gap1/2 were run HIS TrapTM FF column chromatography,dialysis and freeze drying,and detecting biological activity of purified rFIP-gapl/2.The results show that the rFIP-gap1 could agglutinate human,mice and sheep red blood cells with concentration greater than 1?g/mL.While rFIP-gap2 was able to agglutinate the three types of red blood cells when the concentration was more than 8 ?g/mL;rFIP-gapl/2 also could apparently promote the mice spleen lymphocyte proliferation,and the best concentrations were 4 ?g/mL;In addition,rFIP-gapl/2 also could obviously increase mice spleen lymphocytes to secrete level of IL-2 and IFN-y,and amount of the expression of IL-2 up to 622.5 and 416.5 pg/mL respectly,yet level of IFN-y up to 1381 and 1624 pg/mL,respectly;In vitro,MTT method was used to determine the growth inhibitory effect rFIP-gapl/2 twards A549,Hela and MCF-7 tumor cells,the results show that inhibition effect of the rFIP-gap1 on A549,Hela and MCF-7 cells was good,when the concentration of rFIP-gapl was 32 ?g/mL,the inhibition rate was 53.7%,82.9%and 82.5%respectively,IC50 alue was 29.89,8.34 and 12.19 ?g/mL respectively.At the same concentration condition,inhibition effect of rFIP-gap2 towards A549 and Hela was bad,the inhibition rate was 13.9%and 8.7%,respectively,IC50 alue was 60.92 and 41.05 ?g/mL respectively,while for the MCF-7 cell inhibitory effect no found.By P.pastoris GS115 efficiently expression,obtaining rFIP-gap1/2 with biological activity.This provides a theoretical basis for the further development and practical application of FIP-gap.The second part is comparative analysis of rFIP-gapl/2,rLZ-8 and rFIP-gsi.Synthesizing LZ-8 and FIP-gsi.Constructing recombinant expression vector pPIC9-His-LZ-8 and pPIC9-His-FIP-gsi.And transformed into P.pastoris GS115 to obtain recombinant protein LZ-8 and FIP-gsi.SDS-PAGE and Western blot results showed that LZ-8 and FIP-gsi successfully expressed in P.pastoris GS115 with a yield of 133.6 and 124.3 mg/L,respectly.Recombinant proteins by histidine gel column purification,dialysis and lyophilization process for purification rLZ-8 and rFIP-gsi,and detecting their biological activities,and prefoming comparative analysis of biological function with rFIP-gapl/2.Results showed that when the concentration of rFIP-gap1,rLZ-8 and rFIP-gsi was 5 ?g/mL,they could agglutinate human,mice and sheep red blood cells,yet rFIP-gap2 did not agglutinate the three types of blood cells;rFIP-gapl/2,rLZ-8 and rFIP-gsi(5 ?g/mL)also could obviously promote the mice spleen lymphocyte proliferation,but they had different function efficiency;rLZ-8 and rFIP-gsi(5 ?g/mL)could significantly enhance the mice spleen lymphocytes secreted cytokines IL-2 and IFN-?,rLZ-8 and rFIP-gsi could enhance the level of IL-2 realse up to 524.5 and 236.5 pg/mL,respectly.And enhancing the level of IFN-y realse was 606 and 1538 pg/mL respectively;MTT method was used to determine antitumor activity of rLZ-8 and rFIP-gsi on A549,Hela and MCF-7 cells,the results show that when the concentration of rLZ-8 was 16?g/mL,the inhibition rate of rLZ-8 towards A549,Hela and MCF-7 was 25.1,33.9 and 18.7%,respectively,IC50 alue was 21.65,17.53 and 31.38?g/mL respectively.The inhibition rate of rFIP-gsi towards Hela and MCF-7 was 25.1%,33.9%and 18.7%,when the concentration of rFIP-gapl was 16 ?g/mL,respectively,yet IC50 alue of rFIP-gsi on A549,Hela and MCF-7 cells was 21.65,17.53 and 31.38 ?g/mL respectively.The comparative analysis of biological activities of rFIP-gapl/2 with rLZ-8 and rFIP-gsi has important scientific significance for further comprehensively clarifing the biological function of FIP. |
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