Effect Of Increasing Regular Repetitive Sequence On The Antifreeze Activity Of Antifreeze Protein ApAFP914 From Anatolica Polita

Anatolica polita is belong to Tenebrionidae family and be found in Guerbantonggute Desert.We have found several antifreeze genes form Anatolica polita's body in previously research,ApAFP914 have 7 regular repetitive sequence in wild type,the regular sequence have Thr-X-Thr motif which can be bound to ice surface.But the relationship between TXT motif number change and antifreeze activity of ApAFP914 has not been understand.This experiment use the antifreeze protein gene ApAFP914(derive form Anatolica polita)as a cornerstone to obtain other mutants which have diverse coils.In order to compare the difference of ApAFP914 antifreeze ability and protein structure,which have diverse coils,we have designed and synthesized ApAFP914-S,ApAFP914-C,ApAFP914-T which have 5,8 and 10 coils respectively.1.It's has been construct on the expressive plasmids pET32 a which have diverse coils ApAFP914 and has been identified by sequence identification.We have trans the mix-plasmids to BL21(DE3)and got ApAFP914 which have diverse coils after induced by IPTG.After appraise via SDS-PAGE,the protein has been induced in correctly which have diverse coils.2.Thermal hysteresis activity(THA)of ApAFP914 and its mutants was measured with differential scanning calorimetry(DSC).The result showed that TrxA and BSA not have THA,by comparing the thermal hysteresis activity(THA)of 3 kinds of mutants and ApAFP914,the sequence is ApAFP914-C > ApAFP914= ApAFP914-T > ApAFP914-S.3.It's have beneficial effect on the Escherchia coli DH5? in frost temperature which add have diverse coils ApAFP914 and livability has been improved.On the concentration 50?g/m L,the protective effect coresponding to the result of THA measurement.4.On the experiment vitrification of mouse oocytes,we addition 5?g/mL TrxA-ApAFP914,TrxA-ApAFP914-C and TrxA as final concentration.Theresult showed that: addition ApAFP914 and ApAFP914-C can improve mouse oocytes survival rate.We also detected the mouse oocytes gene expression after vitrification,the result showed that: the relative expression of sod1,mad2 and eg5 have significant improve.