资源描述：
《小鼠fsh-occludin重组多价避孕疫苗的构建及表达》由会员上传分享，免费在线阅读，更多相关内容在教育资源-天天文库。
1、小鼠FSH-Occludin重组多价避孕疫苗的构建及表达作者：向延芳,何畏,梁志清,史常旭单位：第三军医大学西南医院妇产科,重庆400038提要:目的构建FSH与Occludin重组基因的毕赤酵母分泌型表达载体。方法 利用RT-PCR技术,从小鼠新鲜脑组织中提取总RNA扩增获得FSH-β亚单位,同法从小鼠新鲜睾丸组织中提取总RNA扩增Occludin,通过双重PCR扩增,获得含FSH-β亚单位特定基因序列和闭锁因子第2个胞外环序列的耦合片段,构建两种重组质粒pPIC9KFSH-Occlu-di
2、n;经酶切鉴定后电转化毕赤酵母菌株GS115,YPD-G418平板筛选高拷贝转化子,甲醇诱导后SDS-PAGE电泳鉴定。结果 通过双酶切分析、DNA测序鉴定明确特定的FSH-β亚单位和Occludin基因耦合片段序列与设计完全一致。目的蛋白相对分子质量分别约为14×103与12×103,与理论预测值相吻;Westernblot检测具有良好的免疫反应性。结论 耦合片段被成功克隆到巴氏毕赤酵母表达载体pPIC9K中并高效、正确的表达。 关键词:卵泡刺激素;闭锁因子;毕赤酵母 中图
3、法分类号:R169.41;R392.13;R394-33 文献标识码: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
