人免疫缺陷病毒gp41基因在大腸桿菌中的表達.pdf

1、南開大學碩士學位論文人免疫缺陷病毒gp41基因在大腸桿菌中的表達姓名：劉坤申請學位級別：碩士專業(yè)：生物化學與分子生物學指導教師：陳啟民2000.5.1ABSTRACTGp41isthetransmembraneglycoproteinofHumanImmunodeficiencyVirustype1(HIV一1)IthasthemembranefusionructionanditisalsoanimportantHIVantigenFu

2、ll—lenghthofgp41geneCannotbeeffectivelyexpressedinY7RNApolymeraseexpressionsysteminEscherichiacoliOurFormerworkconstructedrecombinantplasmidcarryinggp41N—terminal31lbpThegp41proteinproductionwereverylowonly43％ofthetotalb

3、acterialproteinUsingthesoftwareDNAStar401，wepredictedthatthereweresixmainstem—loopsingp41mRNAsecondarystrycture：stemloop1(gp41113Int)，stem—loop2(gp414158nt)，stem—loop3(gp417389nt)，stemloop4(gp4198～128nt)，stem—loop5(gp414

4、63477nt)，stem—loop6(gp41482523nt)Keepingtheprimarysequenceuntouched，weintruducedsilencemutationsintothesixstemloopsandthusbrokethemAfterthestem—loop3andstem—loop4werebroken，theproteinproductionhighlyincreasedby48fold，fro

5、m43％tO207％ofthetotalbacterialproteinAfterdoingcontinuoussilencemutationstOstemloop1and2，stemloop5and6，theproteinexpressiononlyincreasedby80％and10％TheresultsshowedthattheN—terminalstemloop3and4greatlystalledthegp41geneexp

6、ressioninEcolisystemAndtheN—terminalstem—loop1and2，themiddlestem—loop5and6，seemednotveryimprotantforgp41expressionWefoundthatthereweresomeN—terminalfactorswhichcouldstalltheproteinexpressioninEcoliFurthermore，weimprovedt

7、heproteinexpressionmethod：wereplacedthehostcellwithEcoliBL21CodenPlusTM(DE3)RIL，andincreasedtheinductionOD600to08Aftertheseimprovements，theexpressionofgp41N—terminal31lbpWaSincreaSedfrom207％to286％AtlaStweclonedandexpress

8、edthegp41epitopesIandII，namedgp41E1E2，inF_coliItsexpressionwas486％ofthefullhostproteinTherecombinantproteinsgp41N—terminal31lbpandgp41E1E2wereallhighlyreactivewimHIVpositiveserulnandwellpurifiedbynickle—chelatingchromato