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1、小鼠FSH-Occludin重組多價避孕疫苗的構(gòu)建及表達(dá)作者：向延芳,何畏,梁志清,史常旭單位：第三軍醫(yī)大學(xué)西南醫(yī)院婦產(chǎn)科,重慶400038提要:目的構(gòu)建FSH與Occludin重組基因的畢赤酵母分泌型表達(dá)載體。方法 利用RT-PCR技術(shù),從小鼠新鮮腦組織中提取總RNA擴增獲得FSH-β亞單位,同法從小鼠新鮮睪丸組織中提取總RNA擴增Occludin,通過雙重PCR擴增,獲得含F(xiàn)SH-β亞單位特定基因序列和閉鎖因子第2個胞外環(huán)序列的耦合片段,構(gòu)建兩種重組質(zhì)粒pPIC9KFSH-Occlu-di
2、n;經(jīng)酶切鑒定后電轉(zhuǎn)化畢赤酵母菌株GS115,YPD-G418平板篩選高拷貝轉(zhuǎn)化子,甲醇誘導(dǎo)后SDS-PAGE電泳鑒定。結(jié)果 通過雙酶切分析、DNA測序鑒定明確特定的FSH-β亞單位和Occludin基因耦合片段序列與設(shè)計完全一致。目的蛋白相對分子質(zhì)量分別約為14×103與12×103,與理論預(yù)測值相吻;Westernblot檢測具有良好的免疫反應(yīng)性。結(jié)論 耦合片段被成功克隆到巴氏畢赤酵母表達(dá)載體pPIC9K中并高效、正確的表達(dá)?！ ￡P(guān)鍵詞:卵泡刺激素;閉鎖因子;畢赤酵母 中圖
3、法分類號:R169.41;R392.13;R394-33 文獻(xiàn)標(biāo)識碼:AConstructionofamousemultivalentcontraceptivevaccinebasedonfolliclestimulatinghormoneandoccludingenesXIANGYan-fang,HE-Wei,LIANGZhi-qing,SHIChang-xu(DepartmentofObstetricsandGynecology,SouthwestHospital,ThirdMilitaryMedical
4、University,Chongqing400038,China) Abstract:Objectives ToconstructtherecombinantplasmidpPIC9KFSH-Occludinforpreparingamultivalentcontraceptivevaccine,whichinterferingthefunctionofSertolicellsinthemousetestis.Methods AfterisolatedthetotalRNAfrommousebrainandte
5、stistissues,thespecificregionoffolliclestimulatinghormone-b(FSH-b)subunitandthesecondaryextracellulardomainofoccludinwereamplifiedbyRT-PCR,respectively.ThecatenationfragmentsofFSH-occludinwereobtainedthroughdoublePCRamplificationandclonedintotheexpressionvect
6、orPPIC9KofP.pastoristoconstruct2differentrecombinantplasmids(pPIC9KFSH-occludin).TherecombinantvectorwasintroducedintoP.pastorisstrainGS115byelectroporationandse-lectedwithG418forFSH-occludingeneintegration1.SuperG418resistantcloneswereselectedandscreenedforF
7、SH-occludingeneexpression.TheFSH-occludingenewassuccessfullyclonedintopPIC9K.Results Therecombinantplasmids(pPIC9KFSH-occludin)wereidentifiedbyDNAsequencinganddoubleenzymaticdigestionwithEcoRⅠandNotⅠ.Itwasconfirmedthatthereconstructplasmidscontainingthesamese
8、quencesasthetheoreticaldesigning.TheresultsofSDS-PAGEshowedthatthemolecularweightoftheexpressedprod-uctwas1.4×103and1.2×103.Conclusion Theresultsindicatesuccessfulconstruction
