Construction And Application Of Recombinant Baculovirus With Ice Nucleation Active Gene

Ice nucleation active bacteria that have the ability to catalyze supercooled water to fine smooth and rule ice-crystal under the low temperature, had widely applied in many fields such as insect pest control, high sensitive detection of pathogenic microorganisms, resident breeding of crops, plant frost protection, cold storage and freezer concentration of food. Wild INA and engineering INA are difficult to prevent and control the farm overwintering pest,though wild INA could considerably raise the supercooling point of freeze-intolerance pest and increase the death rate of insect pest control.This paper constructed the recombined baculovirus AcMNPV-ice with inserting the high ice nucleation active gene iceA,that was PCR amplified from ice nucleation active bacteria Erwinia ananas110, into downstream of polh promotor of baculovirus AcMNPV in combination with the advantages of insect baculovirus as good safety, harmless to human and animal,don’t pollute the enviroment,high specificity,little damage to target creature,to cause epidemic spread,strong and long effect, resistant pest less,etc. The results are as follows.(1)Acquisition of high ice nucleation active gene iceA.Recombinant expression plasmid pET-23a(+)-ice were constructed by cloning the iceA gene,that were amplified by PCR amplified from Erwinia ananas110,into the expression plasmid pET-23a(+).Inducing by IPTQ analysis of SDS-PAGE and detection of ice nucleation activity of expression of protein indicated that ice nucleation active protein with the molecular weigh of about180kD and inclusion bodies was successfully expressed and freezen drop rate of recombinant BL21/pET-ice and INA all were100%under the temperature-5、-4、-3℃,but0%under-2℃.The results showed that There was no difference between recombinant BL21(DE3)pLysS/pET-ice and wild INA,in addition,ice nucleation active protein of expression from BL21(DE3)pLysS was type A of ice nucleation active.(2)Construction and application of recombinant baculovirus AcV/polh-ice. Recombinant shuttle vector Bacmid/polh-ice was extracted and purified after the recombinant transfer plasmid pFASTBacTMual-polh-ice that gene iceA and polh were cloned into pFASTBacTMual transformed into E.coli DH10Bac which contains shuttle vector Bacmid and assistant plasmid,and the detection of PCR amplification testified gene iceA and polh to the correct position of Bacmid.The detection of SDS-PAGE electrophoresis after Bacmid/polh-ice transfected into the sf9cells showed that polyhedrin protein with the molecular weigh about29KD,consistent with the prediction was successfully expressed. In addition,twenty of Spodoptera exiguas. that injected surpernatant of AcV/polh-ice were all liquefaction of death,but other twenty of normal were still successive propagation.