豬圓環(huán)病毒2型Cap和豬IFNa融合蛋白重組腺病毒的構(gòu)建與鑒定.pdf

1、摘要豬圓環(huán)病毒2型Cap和豬IFNO【融和蛋白重組腺病毒的構(gòu)建與鑒定摘要豬圓環(huán)病毒2型(PorcineCircuvirus2)是引起斷奶仔豬多系統(tǒng)衰竭綜合征(Postweaningmultisystemicwastingsyndrome，PMWS)的主要病原PCV2包含兩個(gè)開放式閱讀框ORFl和ORF2，其分別編碼病毒復(fù)制酶相關(guān)蛋白(Rep)和病毒衣殼蛋白(Cap)，其中Cap蛋白含有病毒主要抗原表位，可誘導(dǎo)豬體產(chǎn)生免疫保護(hù)作用干擾素是

2、一類抗病毒蛋白，它可阻止另一種病毒增殖其中IFN一僅具有廣譜的抗病毒作用，表現(xiàn)在抑制病毒復(fù)制、減緩病毒傳播速度以及降低感染細(xì)胞損傷程度1PCV2Cap蛋白于大腸桿茵系統(tǒng)中的表達(dá)，純化與抗原性分析本研究通過(guò)PCR方法獲得PCV2的ORF2全長(zhǎng)基因，通i_EcoRIINotl酶切選取3’羧基端片段，插入原核表達(dá)載@pGEX6ptEcoRFNotI酶切位點(diǎn)，轉(zhuǎn)化大腸桿菌DH5a，涂布胺芐平板，挑選單個(gè)茵落，提取質(zhì)粒進(jìn)行酶切鑒定及序列分析，獲得

3、Cap蛋白羧基端片段重組質(zhì)粒再將陽(yáng)性質(zhì)粒轉(zhuǎn)化大腸桿茵B(yǎng)L21，對(duì)誘導(dǎo)表達(dá)務(wù)件進(jìn)行優(yōu)化，結(jié)果為加入終濃度為03mMIPTG、30“C誘導(dǎo)表達(dá)6小時(shí)，可以有效表達(dá)重組蛋白，大小約為40kD，具有PCV2抗原性收集菌體并進(jìn)行超聲裂解后證實(shí)，表達(dá)產(chǎn)物以可溶蛋白與包涵體兩種形式存在分別利用GST親和柱以及包含體洗滌尿素變性方法分別提取純化可溶蛋白與包涵體，用已知PCv2陽(yáng)性血清進(jìn)行ELISA試驗(yàn)鑒定重組蛋白具有PCV2抗原性，結(jié)果表明兩種方法提純

4、的重!gCap蛋白的抗原性無(wú)明顯差異2PCV2CapIFNa融合蛋白重組腺病毒的構(gòu)建與鑒定用已構(gòu)建的PCV2ORF2和PRRSVORF5重組質(zhì)粒rShuttleORF2ORF5中經(jīng)跏I酶切獲得ORF2段基因，同時(shí)將已經(jīng)含有豬IFNc【基因的重組穿梭質(zhì)粒pShuttleCMVIFNa用KenI線性化，將目的基因克隆至上述重組質(zhì)粒中，轉(zhuǎn)化大腸桿菌DH5o【，涂布卡那平板，挑選單個(gè)茵落，提取質(zhì)粒進(jìn)行PCR及勛^I酶切鑒定，成功構(gòu)建質(zhì)粒pShu

5、ttleCMVORF2IFNa，然后將該重組質(zhì)粒用PmeI線性化后與骨架載體pAdEasy1共轉(zhuǎn)化大腸桿菌BJ5183，進(jìn)行同源重組，經(jīng)PCR及PacI酶切鑒定，挑選陽(yáng)性重組質(zhì)粒pAdORF2IFNa，轉(zhuǎn)染HEK293A細(xì)胞，荻得重組腺病毒tAdCapIFNa經(jīng)RTPCR及Westernblot證明融合蛋白得到正確表達(dá)，大小約為49kD將4周齡的BALB／c健康小鼠分為4個(gè)組，每組5ABSTRACTCONSTRU(mONANDIDENT

6、IFICAnON0FRECOMBINANTADENOVIRUSEXPRESSINGFUSl0NPROTEIN0FPCV2(1氣PANDPORCINEIFNaABSTRACTPorcinecircovirns2(Pcv2)iscausalagentofpostweaningmultisystemicwastingsyndrome(PMWS)PCV2genomecontainsatleasttwolargeopenreadingframes

7、namedORFlandORF2，whichencodetheRepproteininvolvedinreplicationandmainstructureCapproteinrespectivelyItwasfoundthatthemainepitopesexistonCapproteinwhichcouldinduceimmunoprotectionagainstPCV2infectioninswineInterferoas(IFN

8、)belongtothefamilyofantivirusproteins，whichCallinhibitsomevirusesproliferation1ENahasabroad—spectrumantiviruseffect，whichdisplayoninhibitingvirusreplication，degradingconveyinganddecreasingthelesionofcells1Theexpressionan

9、dpurificationandimmunogenicityofPCV2CapproteininEcoliTheORF2geneofPCV2wasamplifiedbyPCR，andtheCterminalgenewasdigestedwithEcoR～NonwhichwasclonedintoaprokaryoticexpressionplasmidpGEX6p1Afteridentificationwithenzymedigesti

10、onandsequenceanalysis，therecombinantvectorwastransformedintoEcoliBL21strainByoptimizationforinducecondition，thefusionproteinofabout40kDwasexpressedcorrectlywithIPTGatconcentrationof03mmoI／Landinductionat30℃for6hoursThere

11、sultsofwestemblotshowedthattheexpressionproteinhasagoodimmunityofPCV2，anditcouldexistasinclusion—bodyandsolubleproteinThustheproteinwwaspurifiedwithGSTaffinitycolumnandwashingaftercarbamidedenaturation，respeetlyandtheimm

12、unogenicitywasdetectedinELISAwithPCV2positiveserumTheresultsindicatedthattherewerenoobviouslydifferencesbetweentheSolubleproteinandinclusionbodyItcouldbeused笛a(bǔ)ntigeninELISAfordetectyantigentoPCV22Constructionandidentific

13、ationofrecombinantadenovirusexpressingCapandIFNaproteinInthisstudyCapproteingeneinrecombinantplasmidrShuttleORF2ORF5wasdigestedwithKenIandclonedintoatransfervectorpShuttle—CMVIFNathatconteinIFNageneArecombinantplasmid，ca