人乳头瘤病毒E6E7基因重组腺病毒的构建及鉴定

【关键词】人类乳头状瘤病毒，人；腺病毒科

constructionandidentificationofrecombinantadenovirusescarryinghpv16e6e7

【abstract】aim：toconstructthereplicationdeficientrecombinantadenovirusescarryinghumanpapillomavirus16（hpv16）e6e7andidentifythevectorwithwesternblot.methods：thehpv16e6e7genefragmentwasobtainedfromtheplasmidpet32a（+）e6e7byamplificationanddigestion，andtheshuttleplasmidpadtrackcmve6e7inwhichthee6e7wasinsertedintothedownstreamofcmvpromoterwasestablishedbyligation.thenthelinearizedshuttleplasmidwascotransformedintobj5183bacteriawithbackbonevectoradeasy1toobtaintherecombinantadenoviralplasmidpade6e7byhomologousrecombination.therecombinedadenovirusdnawastransfectedinto293cellsforpackingandtheamplificationproductofreplicationdeficientade6e7viruswaspurifiedbycscldensitygradientcentrifugation.theexpressionofe6e7wasverifiedbywesternblotin293cellafterinfectedwithade6e7.results：therecombinantplasmidpade6e7wasestablishedbyhomologousrecombinationandconfirmedbyrestrictionendonucleasedigestionandsequencing.gfpexpressioncouldbeobserved3dafterpackingofthelinearizedpade6e7in293cellsand6.9×1010pfu/ltiterofade6e7wasobtainedbycsclgradientpurification.the293cellwasinfectedwiththistiterofade6e7viruses，andthentotalproteinin293cellswasextracted.westernblotshowedthate6e7proteinwasexpressedobviously.conclusion：ade6e7expressingbothgfpande6e7simultaneouslycanbesimplyandrapidlygeneratedbyusingtheadeasysystem.

【keywords】papillomavirus，human;adenoviridae