乳腺特异性基因转基因山羊表达特性的研究

STUDYONSPECIALEXPRESSIONFEATUREOFMAMMARYGLANDINTRANSGENICGOATS

采用胚胎显微注射的方法，将牛乳酪蛋白（αｓｌｃａｓｅｉｎ）基因作为乳腺定位表达调控序列与人乙型肝炎表面抗原（ＨＢｓＡｇ／ａｙｗ）基因组成的两个融合构件（λ１０６和λ２０７）导入山羊原核期受精卵。产出的Ｇ０代羊用ＨＢｘ和ｃａｓｅｉｎ８００两个探针进行ＰＣＲ＋Ｓｏｕｔｈｅｒｎ整合检测，分别获得λ１０６和λ２０７构件整合阳性羊１６和２４只。用ＥＬＩＳＡ／ＨＢｓＡｇ试剂盒检测转基因羊乳汁中的重组人乙型肝炎表面抗原（ｒＨＢｓＡｇ），在Ｇ０代转基因羊中，λ１０６乳腺表达率是４７２％（１７／３６）；λ２０７是６８６％（２４／３５）。Ｇ０代转基因母羊与正常公羊交配，所产的４１只Ｇ１代羊中有１３只整合阳性（３１７％），其中６只Ｇ１代整合羊中，乳腺表达率为６６６７％（４／６）。表达阳性羊乳免疫注射６只成年兔，结果在它们的血清中全部检测到ＨＢｓＡｂ（６／６）。试验表明，由αｓｌｃａｓｅｉｎ和ＨＢｓＡｇ／ａｙｗ基因组成的λ１０６和λ２０７两个融合基因构件导入山羊原核期受精卵后均可获得乳腺特异性表达转基因羊，最高表达量达１３０７μｇ／ｌｉｔｅｒ，由此证明通过牛αｓｌ酪蛋白调控序列作为定位基因来指导ＨＢｓＡｇ／ａｙｗ基?

Thefusiongenesof106and207linkedbovineslcaseinflankingsequencewithHepatitissurfaceantigen(HBsAg/ayw)genewereintroducedintopronuclearzygotesfromgoatsbymicroinjection.TransgenicgoatswereidentifiedbyusingPCRsouthernanalysis.Thetransgenesweredetectedin16and24offoundergoatsintroduced106and207construct,respectively.13ofthe41F1(firstgeneration)wereincorporatedthe106and207fusiongeneintogenomeExpressiongoatsintroduced106and207transgenewereobservedwithHBsAg/ELISAkitinthemilkofthefoundergoatsduringlactation17outof36(472%106)and24outof35(686%207)founders,and4outof6(667%106)transgenicF1showedrHBsAgpositiveintheirmilkAntiHBsAgantibodywasdetectedinseraofsixrabbitsthatweresubjectedtosubcutaneousinjectionofrHBsAgpositivemilkOurresultssuggestthatbovineslcaseinconstructisefficientatdirectingtheexpressionofHBsAgingoatsmammaryglandduringthelactationThefusiongenesnotonlycanbetransmittedtonextgeneration,butalsoexpressrHBsAginthemilkofG1goats