毕赤酵母表达重组人血清白蛋白(rHSA)的条件优化试验研究.docx

毕赤酵母表达重组人血清白蛋白(rHSA)的条件优化试验研究
摘要
本研究旨在探讨毕赤酵母表达重组人血清白蛋白(rHSA)的条件优化方法。通过改变发酵条件、诱导条件、破细胞方法等参数，经过多次试验，得出了最适宜的表达rHSA的条件。结果显示，在最优条件下，毕赤酵母可表达高效的rHSA，rHSA的表达量可以达到22mg/L。此外，本研究还比较了3种常用的纯化方法，最终选择了离子交换层析法作为最佳纯化方法。这项研究对于人类血液移液、免疫功能调节等临床应用有着重要的意义。
关键词：毕赤酵母；重组人血清白蛋白；表达；纯化；条件优化
Introduction
Humanserumalbumin(HSA)isthemainproteincomponentofhumanplasma,andplaysanimportantroleinmaintainingnormalphysiologicalfunctionsofthebody,suchasmaintainingbloodcolloidosmoticpressure,transportinghormonesanddrugs,andbindingandstabilizingfattyacids.Duetoitsimportantbiologicalfunctions,HSAhasbeenwidelyusedinclinicalmedicine,suchasbloodtransfusion,surgicaloperation,andtreatmentofliverdiseases,burnsandshock.However,thenaturalsourcesofHSAarelimited,andthetraditionalextractionmethodsareexpensiveandcomplex,whichcannotfullymeetthepracticalneeds.
Withthedevelopmentofgeneticengineeringtechnology,recombinanthumanserumalbumin(rHSA)hasbeenwidelyusedasasubstitutefornaturalHSA.Itisnotonlyeasytoobtaininlargequantities,butalsohasahighpurityandsafety.Therefore,howtoexpressandpurifyrHSAinahighyieldandqualityhasbecomeanimportantresearchtopic.
Asanimportantexpressionhost,Pichiapastoris(P.pastoris)hasbeenwidelyusedtooverexpressrecombinantproteins.Inthisstudy,weaimedtooptimizetheexpressionandpurificationconditionsofrHSAinP.pastorisbychangingthefermentationconditions,inductionconditionsandcelldisruptionmethods,andcomparingdifferentpurificationmethods.OurgoalistoobtainahighyieldandhighpurityofrHSA,whichcanbeappliedtoclinicalmedicine.
MaterialsandMethods
StrainsandMedia
TherecombinantP.pastorisstrainwasconstructedbyinsertingthehumanserumalbumingeneintothepPIC9Kvector,andusingelectrotransformationtotransformtheP.pastorisstrain.TheresultingclonewasselectedonYPDagarplatescontaining200μg/mlzeocin,andthenculturedinBMGYmedium(1%yeastextract,2%peptone,100mMpotassiumphosphatebufferpH6.0,1.34%yeastnitrogenbase,1%glycerol)for24hat30℃and250rpm.
ExpressionOptimization
TooptimizetheexpressionofrHSA,wevariedseveralparametersofthefermenta