揚(yáng)州鵝α--干擾素的克隆表達(dá)及其生物學(xué)活性研究.pdf

1、學(xué)位論文獨(dú)創(chuàng)性聲明本人鄭重聲明：所提交的學(xué)位論文是本人在導(dǎo)師指導(dǎo)下進(jìn)行的研究工作和取得的研究成果。本論文中除引文外，所有實(shí)驗(yàn)、數(shù)據(jù)和有關(guān)材料均是真實(shí)的。本論文中除引文和致謝的內(nèi)容外，不包含其他人或其它機(jī)構(gòu)己經(jīng)發(fā)表或撰寫過的研究成果。其他同志對本研究所做的貢獻(xiàn)均己在論文中作了聲明并表示了謝意。學(xué)位論文作者簽名：王卷日期：如降耳r竅0裔學(xué)位論文使用授權(quán)聲明研究生在校攻讀學(xué)位期間論文工作的知識產(chǎn)權(quán)單位屬南京師范大學(xué)。學(xué)校有權(quán)保存本學(xué)位論文的電

2、子和紙質(zhì)文檔，可以借閱或上網(wǎng)公布本學(xué)位論文的部分或全部內(nèi)容，可以采用影印、復(fù)印等手段保存、匯編本學(xué)位論文。學(xué)?？梢韵驀矣嘘P(guān)機(jī)關(guān)或機(jī)構(gòu)送交論文的電子和紙質(zhì)文檔，允許論文被查閱和借閱。(保密論文在解密后遵守此規(guī)定)保密論文注釋：本學(xué)位論文屬于保密論文，密級：——保密期限為——年。學(xué)位論文作者簽名：互參指導(dǎo)教師簽名：多危％日期：沁脖年皇裔力a日期：如／中年≤融力QABSTRACTABSTRACTInterferonisakindofimpo

3、rtantcytokines，whichhasbroad—spectrumantiviralactivityexcellentinhibitionofcelldivisionandantitumoractivityefficientimmuneregulationandotherfunctionsRecently，aseriesofthestudiesofpoultryinterferonhavebeenreported，especia

4、llyaboutwhichofchickenandduck，butrarereportsofgooseinterferonhavebeenpublishedThisexperimentchosetostudytypeIinterferon——theinterferon—alphabecauseitsantiviralactivityismuchstronger，andaccordingtothepublishedintheGenBank

5、ofgooseinterferonalphagenesequences(HQl15583)designedtwopairsofprimers，andchosethelocalvarietiesofYangzhougooseasexperimentalanimalsBythemethodofRTPCRsuccessfullyamplifiedtheYangzhougooseinterferonalpha’Sbothwholegeneand

6、maturegene，theirsizesare576bpand486bprespectivelyTheIFN—uwholegenewasclonedintopGEMTcarrierandthentherecombinantplasmidpGEM—TGolFN—ifwassenttothesequencingcompany。YangzhouGolFNc‘a(chǎn)ndHQ115583twosequences’homologyatthenucle

7、otidelevelwasrevealed996％pET32amGoIFN一位wereconstructedforIFN僅maturegeneexpressionTherecombinantplasmidwastransformedintoEcoliDH5a(DE3)，andwasinducedwithIPTGformGolFN—ageneexpressionTheexpressedproductwasidentifiedbySDS—P

8、AGE，afusionproteinabout363kDawasfoundAfterthepurificationofthisinclusionbodycanalmostgetasinglespecificband，andusingWesternBlottingmethodtodetecttheprotein，theresultwasthesameastheexpected，UsedtherecombinantproteinpET32a

9、mGolFNatoimmuneBALB／cmice，thenobtainedtheratantimGolFN0【polyclonalantibodymeasuredtheantibodytiterbyELISAmethod，theresultwas1：16000Thehyperimmuneseltlm’sspecificitywastestedbyWesternBlotting，theresultswasinthelocationoft

10、he363kDaappearedaspecificband，thissuggestingthattheserumhasgoodspecificityandreactionogenicityYangzhougooseIFN—c【wholegenecloningtopcDNA3。l()carrierthussuccessfullyconstructedtheeukaryoticexpressionsystempcDNA31一GolFN0La