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1、D-天冬氨酸受体2B亚基基因重组腺病毒镇痛疫苗的构建中华麻醉学杂志2007年8月第27卷第8期ChinJAnesthesiol,August2007,Vol27,No.8699?疼痛诊疗与研究?N一甲基一D一天冬氨酸受体2B亚基基因重组腺病毒镇痛疫苗的构建王公明陈建平杨少兵田学愎高峰杨辉安珂田玉科【摘要】目的构建携带N一甲基一D一天冬氨酸受体2B亚基(NR2B)基因的重组腺病毒镇痛疫苗.方法以携带NR2B基因的腺病毒穿梭质粒pDC515一NR2B和腺病毒骨架质粒pBHGfrt(de1)E1,3FLP共转染腺病毒包装细胞293
2、细胞,连续观察细胞的形态学.取形成病毒空斑的293细胞,分别采用PCR,RT.PCR及Westemblo【ling的方法从基因水平,转录水平和蛋白水平对病毒空斑筛选和鉴定,对鉴定正确的病毒空斑进行扩增和纯化.结果转染后12d可观察到细胞病变效应,15d后形成病毒空斑.NR2B基因不仅正确插入重组腺病毒载体,而且可在293细胞正确表达NR2B蛋白.鉴定正确的病毒空斑命名为NR2B基因重组腺病毒5型镇痛疫苗,滴度为5×10"VP/ml,纯度100%.结论成功地构建了NR2B基因重组腺病毒镇痛疫苗,可用于疫苗镇痛的研究.【关键词】
3、受体,N一甲基一D天冬氨酸;腺病毒科;疫苗;镇痛ConstructionofanalgesicvaccinewithrecombinantadenovirusencodingNMDAreceptor2BgeneWANGGong-ming,CHENJian-ping,YANGShao-bing,eta1.DepartmentofAnesthesiology,ron~iHospitalofron~iMedicalCollege,HuazhongUniversityofScienceandTechnology,Wuhan43003
4、0,ChinaCorrespondingauthor:T1ANYu-ke(E-mail:Yiketian@tjh.tjmu.edu.com)【Abstract】ObjectiveToconstructtherecombinantadenovirusanalgesicvaccineencodingNR2Bgene.MethodsTheshuttleplasmidpDC515一NR2BwascotransfectedwithanAdenovirusgenomicplasmidpBHGfrt(de1)E1,3FLPintoE1co
5、mplementingcellline293cells.Thetransfected293cellswereobservedcontinuouslyforthedetectionofcytopathiceffectandtheviralplaques.Astheformationoftheviralplaques,someviralplaqueswerepickedandwereidentifiedusingPCR,RT—PCRandWesternblotting.Theamplificationandpurificatio
6、noftheidentifiedviralplaquesweredoneandthetiterandpurityoftheviruswereassayed.ResultsCytopathiceffectwereobservedonposttransfectionalday12andwell—definedplaqueswereobservedonposttransfectionalday15.Thepresenceandexpressionof~ansgeneNR2Bintherecombinantadenovirusvec
7、torwereconfirmedusingPCR,RT—PCRandWesternblotting.Theidentifiedviralplaquesweredesignatedastherecombinantadenovirusserotype5analgesicvaccineencodingNR2B(rAd5/NR2B).ThetiterofrAd5/NR2Bwas5×10"VP/mlandthepuritywas100%.ConclusionTherecombinantadenovirusanalgesicvaccin
8、eencodingNR2Bareconstructedsuccessfullyandcanbeputintoresearchingaboutvaccineanalgesia.【Keywords】Receptors,N—methyl—D—aspartate;Adenoviridae;Vacc
