【作者】 宁方勇；

【导师】 安铁洙；

【作者基本信息】 东北林业大学 ， 发育生物学， 2013， 博士

【摘要】 蜘蛛丝无与伦比的生物学特性决定了其在应用领域具有广阔的应用前景。以生物学方法获取具有蜘蛛丝特性的活性蛋白,亦或是通过转基因的方法直接获得类似蜘蛛丝的动物纤维,一直以来是许多科学研究者的奋斗目标。本研究利用蜘蛛拖丝蛋白基因四聚体4S、pcDNA3.1和pIRES2-EGFP载体,在构建pcDNA3.1-4S和pIRES2-EGFP-4S表达载体的基础上,以脂质体转染法将蜘蛛拖丝蛋白(Spidroin1)基因转染细胞并获得转基因阳性细胞株；以转基因阳性细胞为核供体,采用体细胞核移植的方法获得转蜘蛛Spidroinl基因绵羊克隆胚；采用PCR方法检测转基因克隆胚的蜘蛛拖丝蛋白(Spidroinl)基因整合状况。本研究的结果如下：1. pcDNA3.1-4S及pIRES2-EGFP-4S表达载体的构建利用pcDNA3.1载体和蜘蛛拖丝蛋白基因四聚体4S构建了pcDNA3.1-4S表达载体,BgtlⅡ, BamHI双酶切及PCR鉴定显示,蜘蛛拖丝蛋白基因4S已连接到pcDNA3.1-4S中；利用pIRES2-EGFP载体和蜘蛛拖丝蛋白基因4S构建了pIRES2-EGFP-4S, XhoⅠ、BglⅡ双酶切及PCR鉴定显示,蜘蛛拖丝蛋白基因4S连接到pIRES2-EGFP中。2.转蜘蛛Spidroin1基因绵羊成纤维细胞系的建立将0.44g/mL经线性化的pcDNA3.1-4S和pIRES2-EGFP-4S,通过脂质体法分别转染利用常规方法经原代和传代培养的绵羊成纤维细胞,用含有抗生素和含400μg/mL G418的培养基进行筛选,获得转pcDNA3.1-4S和pIRES2-EGFP-4S的阳性细胞；对获得的转基因阳性细胞进行形态学观察、生长曲线、倍增时间等生物学特性检测结果,符合正常成纤维细胞的特性。3.绵羊卵母细胞获取方法的筛选通过比较抽吸、切割、抽吸结合切割的方法对绵羊的获卵效果,其结果,与抽吸和切割法相比,划线法辅助的切割法,能够获得最多的A、B级卵母细胞(约8个/每个卵巢)。4.绵羊卵母细胞体外成熟将A、B级卵母细胞以TCM199-HCO3+2.2g/mL NaHCO3+5μg/mL FSH+1IU/mL LH+1μg/mL雌二醇+50μg/mL庆大霉素+10%FBS+0.38mmol/L丙酮酸钠+10mmol/L Hepes为培养液,对绵羊卵母细胞进行体外成熟培养,可以获得83.4%的最高成熟率。表明该成熟液适合用于绵羊卵母细胞的体外成熟。5.成熟卵母细胞孤雌激活体外成熟绵羊卵母细胞用5μmol IA23187激活5min后,再用2mmol/L的6-DMAP培养4h,能够获得最高91.5%的激活率,激活后的孤雌胚经培养后84.7%胚胎发育至桑椹胚。本研究结果显示,IA23187联合6-DMAP的激活方法可用于绵羊体外成熟卵母细胞的孤雌激活。6.转基因阳性细胞的体细胞核移植利用上述研究中建立的体细胞核移植程序,分别以绵羊成纤维细胞和转蜘蛛拖丝蛋白基因细胞进行体细胞核移植,其结果,正常绵羊体细胞融合率(72.7%),与其相比,转基因细胞的融合率(70.7%),两者差异不显著,表明外源基因的转入对早期核移植重构胚的发育能力没有显著影响。7.转蜘蛛拖丝蛋白基因阳性细胞的体细胞重构胚的发育能力将采用体细胞核移植方法获得的转蜘蛛拖丝蛋白基因重构胚进行体外培养,其结果与正常的绵羊成纤维细胞通过体外发育桑椹胚的比率(13.8%)相比,转基因阳性细胞的体细胞的核移植的体外发育至桑椹胚的比率(11.6%),两者之间的差异不显著；将获得的转蜘蛛拖丝蛋白基因融合重构胚直接移入未妊娠的受体绵羊输卵管中,观察到受体妊娠,但未能获得转蜘蛛拖丝蛋白基因的绵羊后代。综上所述,本研究利用所构建的蜘蛛拖丝蛋白基因表达载体pcDNA3.1-4S和pIRES2-EGFP-4S,获得转基因阳性细胞；利用转基因阳性细胞,采用体细胞核移植方法建立了获取转蜘蛛拖丝蛋白基因重构胚的方法；获得的重构胚具有体外发育至桑椹胚的能力。本研究为进一步开展被毛中表达蜘蛛拖丝蛋白的转基因绵羊相关研究奠定了坚实的基础。更多还原

【Abstract】 The unapproachable biological characteristics of spider silk determines it has broad application in many fields. To obtain the active protein with spider silk characteristics using biological method, or animal fibers like spider silk using transgenic approach,is some scientific researchers’goal all these years.This study utilized the spider dragline silk protein gene4S、 pcDNA3.1and pIRES2-EGFP vector to contruct pcDNA3.1-4S and pIRES2-EGFP-4S expression vector,utilized the liposomal and linearized spider dragline silk protein gene4S to transfect sheep fibroblasts,then obtained the positive cell line;used the positive cell line as donor nuclear to obtain trans-Spidroinl gene sheep reconstruceted embryos by nuclear transfer; identified whether the the spider dragline silk protein gene integrated into the reconstruceted embryos.The results of this study were showed as follows.1. Construction of pcDNA3.1-4S and pIRES2-EGFP-4S expression vectorThe pcDNA3.1-4S expression vector was constructed by pcDNA3.1vector and spider dragline silk protein gene4S, the result showed that the spider dragline silk protein gene4S had integrated into the pcDNA3.1-4S expression vector after BglⅡ、BamHI double enzymes cutted and PCR identificated;the pIRES2-EGFP-4S vector was constructed by spider dragline silk protein gene4S and pIRES2-EGFP vector, the result showed that the spider dragline silk protein gene4S had integrated into the pIRES2-EGFP-4S vector after XhoⅠ, BglⅡ double enzymes cutted and PCR identificated.2. Establishment of trans-Spidroninl gene sheep fibroblasts cell lineUtilized the liposomal to transfect the sheep primary and passage fibroblast with0.4μg/mL linearized pcDNA3.1-4S and pIRES2-EGFP-4S,screened the sheep fibroblasts by culture media with400μg/mL G418and antibiotics, and finally obtained trans-pcDNA3.1-4S and pIRES2-EGFP-4S cell line; observed the morphology,drawed the growth curve,calculated the proliferation time of the transgene cell,all the results were accord to the normal fibroblast feature.3. Screened the method of sheep oocytes collectingCompared the effect of sheep oocytes collecting method by suction,cut and suction combined with cut, the results showed that the cut assisted with lineation can obtain about8oocytes (including A and B levels) each ovary.4. Vitro maturation of sheep oocytesUsed the media:TCM199-HCO3+2.2g/mL NaHCO3+5μg/mL FSH+1IU/mL LH+1μg/mL E2+50μg/mL Gentamicin+10%FBS+0.38mmol/L sodium pyruvate+10mmol/L Hepes as culture media to culture A and B levels of sheep oocytes,obtained the highest83.4%maturation rate,it showed that this culture media were fit for sheep oocytes vitro maturation.5. Parthenogenetic activation of sheep matured oocytesThe vitro matured sheep oocytes were firstly activated with5μmol IA23187,then cultured in2mmol/L6-DMAP for4h,it could obtain91.5%activation rate,after cultured84.7%of the parthenogenetic activation embryos were developed into morula. it showed that IA23187combined with6-DMAP were fit for parthenogenetic activation of matured sheep oocytes.6. Somatic cells nuclear transfer of transgene cellUsed the sheep fibroblasts and trans-spider dragline silk protein gene cell as the donor nuclear,after somatic cells nuclear transfer operation which were founded before,the results showed that the abnormal cell melt rate was72.7%,the melt rate of transgene cell was70.7%,there were no difference,it indicated that there were no influence of the exogenous gene integrated into the reconstruceted embryos on it’s development ability.7. Development ability of trans-spider dragline silk protein gene reconstruceted embryos by nuclear transferCultured the trans-spider dragline silk protein gene reconstruceted embryos by nuclear transfer,the morula rate of transgene cell as donor nuclear was11.6%,moreover the morula of abnormal cell as donor nuclear wasl3.8%,there were no difference between them transferred the trans-spider dragline silk protein gene reconstruceted embryos into the oviduct of the donor sheep,pregnance was observered,but no trans-spider dragline silk protein gene lamb obtained.In summary,this study used the pcDNA3.1-4S and pIRES2-EGFP-4S expression vector were constructed to obtain the transgene cell line;used the transgene cell as donor nuclear to obtain trans-spider dragline silk protein gene reconstruceted embryos, the reconstruceted embryos obtained had the ability developing into morula.this study built the foundation for further research of obtaining trans-spider dragline silk protein gene sheep in hair.更多还原