重组人骨形态发生蛋白-2的制备及成骨活性表征 .pdfVIP

重组人骨形态发生蛋白-2的制备及成骨活性表征

李贵龙;王靖;刘昌胜

【摘要】Bonemorphogeneticprotein-2(BMP-2),oneoftheimportant

osteoinductivegrowthfactors,isaneffectiveandcrucialcytokineforclinic

boneregenerationaswellasbioactivityimprovementofbonesubstitute.

However,extractionofBMP-2fromanimalscanhardlymeettheclinic

requirementsduetoitsverylowcontent.Thisworkstudiedthe

preparationofBMP-2tomeettheclinicalneeds,andevaluatedits

biologicalactivity.Firstly,DNAsequenceofoptimizedhBMP-2genewas

obtainedtopreparerhBMP-2strainsofE.coliusingcodonoptimization

methodwithfurtherreplacementofthepartialnucleotidecoding.Then,

throughfermentationandprocessoptimizationtheBMPinclusionbody

wasacquired.Afterseparation,purificationandrenaturation,rhBMP-2of

highpuritywasprepared.Invitrocellexperiments,theactivityofalkaline

phosphatase(ALP)inC2C12cellswasmeasuredtocharacterizethe

osteogenicactivityofhaploidanddiploidBMP-2.Itwasobservedthatthe

osteogenicactivityofdiploidrhBMP-2wassignificantlyhigherthanthatof

haploidrhBMP-2,andtheactivityofalkalinephosphataseincreasedwith

theincreaseoftheBMP-2concentration.Invivoectopicboneexperiment,

afterrhBMP-2wasimplantedintothemuscleofmicefor3weeks,the

ectopicbonegranuleswerenbsp;brightandfresh,andthebonestructure

wascomplete.HEandMasson’strichromestainingsshowedgood

ectopicboneformation.ItcanbeconcludedthattherhBMP-2prepared

usingthismethodcanwellinduceosteogenicdifferentiation,andcanbe

usedforbonetissuerepairtomeettheclinicalneeds.%骨形态发生蛋白-2

（BMP-2）是重要的骨诱导生长因子，是提高骨修复材料活性和临床骨修复效果

的有效手段和关键物质。由于BMP-2在体内含量低，依靠从动物体内提取难以满

足临床需求。本文研究满足临床需求的BMP-2的制备方法，并评价其生物活性。

采用密码子优化方法，并通过进一步更换其中部分核苷酸编码，得到优化的

hBMP-2基因的DNA序列，制备大肠杆菌rhBMP-2菌株，通过发酵及工艺优化，

获得BMP包涵体，经分离纯化与复性，制备出高纯度的rhBMP-2。测定C2C1