The Study On The Growth Of Insect Cells Affect By Bacteria Induction And The Activity Of Antimicrobial Proteins

In the early20th century infectious diseases were the leading cause of death worldwide. Then antibioticswere discovery and used to control the spread of infectious diseases, but which also lead to the bacteriaresistant. In order to find a substitute of antibiotics, lots of effort has been done to find a lot of possiblesubstitutes. As far as the current research status, antimicrobial protein is the most promising candidate to thenext generation of antimicrobial drugs.The heat inactivated Micrococcus luteus has been applied to challenge cell line NIH-SaPe-4, Sf9, Sf21inthis thesis. At the same time the three groups of insect cells non-induced by Micrococcus luteus were ascontrol. Then cell growth condition, the appearance time of antimicrobial proteins, the activity and stabilityof the antimicrobial proteins, antimicrobial proteins separation and purification were study. The main resultsare as follows:(1) The cells growth of NIH-SaPe-4, Sf21and Sf9response to the inductionCompared to non-induction, induction by inactivated Micrococcus luteus make cell numbers of recoveryphase changed little, but delay the recovery phase. Meantime entering into logarithmic phase is later thannon-induced, and the cell growth speed in the logarithmic phase less than non-induction group. The timeentering into the stable phase is also relatively later than non-induction group and advance into decline phasealso. Life cycle of the cells is shorter than the non-induction group. Micrococcus luteus makes the threekinds of cells extended the recovery phase, slowed the logarithmic phase, shorten the stable phase, advancedthe decline phase. In the other word the induction of Micrococcus luteus affect cells'growth.(2)The affect toAmps of induced by Micrococcus luteusIn normal, the cells of NIH-SaPe-4can produce one kind of antibacterial protein. This proteincan inhibit three kinds of Gram-positive bacteria (Micrococcus luteus, Staphylococcus aureus, Bacillussubtilis) strongly, but has no inhibitory to the other two kinds of Gram-negative bacteria (E.coli andgreen pus bacillus). The induction of the inactivated Micrococcus luteus make the time of producingantimicrobial protein advanced, and also enhanced in the amount. The test of difference shows: there aresignificant differences between the induction group an-d the non-induced on antibacterial protein activity. The induction of inactivation Mi-crococcus luteus makes NIH the-SaPe-4cell producing antimicrobialproteins ahead of the time, and the activity ofAmps strengthened.On the condition of normal culture, Sf21and Sf9cannot produce antibacterial proteins, but underinduction of inactivated Micrococcus luteus, they can produce antibacterial protein. The point of producingantimicrobial protein is between6-12h.(3)The stability ofAmpsThe antibacterial activity of Amps in the range of pH5~7and pH6~8have no significantly difference forthe NIH-SaPe-4and Sf21and Sf9cell lines respective, but the activity of the sample in the extremeenvironment of pH less than3and pH greater than11will be very a great impact, or even disappear. Boilingwater bath for2hs the Amps still maintain a high activity. In the other word, The Amps from the three celllines with the general nature of the antibacterial protein: acid-base relatively stability and high temperaturestability.(4) Separation and purificationThe crude extracts of induced and non-induced groups of NIH the-SaPe-4though gel column, thereare four same peaks, the third peak with antibacterial activity. Activity peak via reverse phase column, thereare three peaks obtained, the second peaks with antibacterial activity.The induction of inactivated Micrococcus luteus makes the supernatant of Sf21and Sf9culture gain theantibacterial activity. The supernatant though gel column, there are three peaks, the second peak withantibacterial activity. Then second peak was collected, and though reverse phase column respectively. Thereare6and7peaks obtained. Activity detected shows the third and fifth with antibacterial activity. In the otherword, the induction of inactivated Micrococcus luteus makes Sf21and Sf9cells gain antimicrobial proteins.(5) Detecting theAmps from NIH the-SaPe-4cells though Tricine-SDS-PAGEDetect the sample from gel filtration and reverse phase column though Tricine--SDS-PAGE, and getan antimicrobial protein with molecular weight of about60kDa. But strip from the group of induced byinactivated Micrococcus luteus wider and brighter than non-induction. In other word, the inactivatedMicrococcus luteus induc-tion make NIH-SaPe-4cells strengthen the antibacterial protein whose molecularweight is about60kDa.