長白山白眉蝮蛇磷脂酶a,2的基因工程研究

1、大連理工大學(xué)碩士學(xué)位論文長白山白眉蝮蛇磷脂酶A的基因工程研究姓名：卜鵬程申請學(xué)位級別：碩士專業(yè)：生物化工指導(dǎo)教師：包永明20040601簍旦生皂旦蝗蝗旦墊!!：!!：叁塑莖鬯王型塑塑一AbstractBasedonNterminalsequencingandpeptideprintsanalysisofthepurifiedphosphotipaseA：proteinfromAgkistvdonblomhoffiiglSsurensiS

2、，itseDNAwasobtainedbymeansofreversetranscriptionpolymerasechainreaction(RTPCR)andRapidAmplificationofeDNAEnds(RACE)。Theopenreadingframe(ORF)ofthematuregeneofGin49一PLA2consistsof366basesencoding122aminoacidsAminoacidssequ

3、enceanalysisandmolecularmodeloftheGln49PLAzshowthephospholipaseA2，namedGin49一PLA2，isanewphospholipaseA2withaGinatposition49GIn49PLA2isdevoidofphospholipaseAsactivitywhichisprobablybecauseofFailingtobindthecofactorCa2Comp

4、arisonwithotherPLA2s，theaminoacidsequenceoftheGln49一PLA2shOWShighersimilaritytoAsp49PLAzvariants(93％to61％)thanthatofLys49PLAovariants(60％to56％)，ThephylogenetictreeindicatesthattheAsp49PLA2variantsaremorecloselyrelatedtot

5、heGIn49PLA2thantheLys49PLA2variantsGln49一PLA2wasclonedintothevectorpET32a()。thentransformedinto曼c(diǎn)obstrainBL21(DE3)AfterinducedbyisopropylbDthiogalactoside(IPTG)，thegenewasexpressedintheformofsolubleproteinWesternblotconf

6、irmedthattheproteinspecificallyreactswithyolkIgYagainstthenativeGln49一PLA2fromAgkistrodonblomhoffiiussurensisItsexpressionlevelwasstudiedandtheoptimalcultureconditionsisobtainedasfollows：Harvestingcellsafter6hrofIPTGindu

7、ctionat30℃ByimmobilizedmetalaffinitychromatographyionexchangechromatographyandhydrophobieinteractionchromatographytherecombinantGin49PLA2waspurifiedwith91％purityTherecombinantGln49一PLA2hasapparentanticoagulantactivityinv