【作者】 殷雪；

【导师】 刘世贵；

【作者基本信息】 四川大学 ， 遗传学， 2004， 硕士

【摘要】 本研究首先用DNA重组技术将已克隆的草鱼生长激素(CGH)基因cDNA编码区片断定向插入pGEX-4T-1质粒，转化大肠杆菌TOP10F’，用菌落PCR和质粒PCR方法筛选阳性克隆；以BamH Ⅰ和EcoRI酶切鉴定重组质粒。以IPTG诱导融合蛋白表达，并作RT-PCR及SDS-PAGE分析。从聚丙稀酰胺制备凝胶中回收正确表达的融合蛋白，以此免疫小鼠制备高免血清。得到高效表达草鱼GH的重组质粒pGCGH，RT-PCR分析确证重组质粒在大肠杆菌中能正确转录；SDS-PAGE分析证明诱导重组质粒菌株表达了融合蛋白，分子量约为50KD，与预期的26 KD的GST带和23.6KD的草鱼生长激素基因编码蛋白质构成的融合蛋白大小一致，以此免疫小鼠，制备得到滴度为1：12800的鼠高免血清。 在此基础上，完成了草鱼GH酵母表达菌株的构建与表达研究。设计合成了一对草鱼生长激素基因的特异性引物，上游引物加入EcoRI酶切位点；下游引物加入Not Ⅰ酶切位点，采用DNA聚合酶进行扩增，获得了草鱼生长激素基因表达片断。将该片断插入含有甲醇启动子及带有酵母相应信号肽的pPIC9K表达载体中，成功构建了pPIC9K—CGH酵母高效表达载体。选择pPIC9K作为草鱼生长激素基因的表达载体国内还未见报道。将已连接有草鱼生长激素基因CGH的表达载体pPIC9K—CGH经电击转化导入毕赤巴斯德酵母，经MD和MM培养基及G418抗性筛选和纯化，获得了高效表达草鱼生长激素的重组毕赤巴斯德酵母基因工程菌株。通过对所有筛选到的阳性菌株菌落PCR分析，证实了外源草鱼生长激素基因已经整合到了酵母菌中，并选出了4株高拷贝的重组毕赤巴斯德酵母基因工程菌株，对其培养液采用饱和硫酸氨沉淀纯化后，作SDS-PAGE分析，得到了分子量为23.6KD的蛋白带，与预期的四川大学硕士学位论文分子量相近。结果表明，草鱼生长激素基因得到了高效的胞外分泌表达。以免疫小鼠得到的鼠高免血清为一抗作W七stem印迹分析，证实了所产的胞外蛋白的特异性。 本研究将克隆到的草鱼生长激素基因CGH首次采用pPICgK表达载体构建了重组表达质粒，获得高拷贝的重组毕赤巴斯德酵母基因工程菌株，实现了CGH的高效表达。以原核表达产物为抗原，制备的鼠抗高免血清为一抗，Westem检测证实了毕赤巴斯德酵母基因工程菌株表达产物的特异性。本研究为进一步开展草鱼生长激素的应用和生物活性检测分析，以及研制新型调节鱼类生长的基因制剂的应用奠定了坚实的基础。关键词:草鱼生长激素基因高免血清毕赤巴斯德酵母高效 表达W七stern印迹更多还原

【Abstract】 Grass carp growth hormone (CGH) gene was correctly inserted into pGEX-4T-l plasmid in order to construct the expression plasmid pGCGH. The recombinant plasmid was transformed into E.coli TOP10F’ competent cells, the positive clone was screened by colony PCR and plasmid PCR and the recombinant plasmid was identified by BamH I and EcoRI digestion. Fusion protein expression was induced by IPTG and expression situation was assayed by RT-PCR and SDS-PAGE. The fusion protein was recovered from polyacrylamid gel and used to immunize the rabbit. The result of RT-PCR showed that the recombinant plasmid could be transcripted correctly in TOP 10F’ . A new protein band was found in SDS-PAGE with molecular mass of about 50 KDa which is consisted of a 23.6 KDa protein deduced from the CGH gene sequence and GST (26 KDa). The titer of rat anti-CGH serum from immunized rat was 1: 128,000.A new pair of primers of CGH gene was designed which contained aEcoRI site in sense primer and a Not I site in antisense primer, and a new CGH gene fragment was obtained by amplification which didn’t contain signal peptidc and intron. The new fragment was inserted into pPIC9K plasmid which contain methanol promoter and yeast signal peptide toconstruct expression plasmid pPIC9K - CGH, and the plasmid was transformed into Pichia Pastoris by electroporation. Four high copy positive clones were screened and purified by culturing on MD plate , MM plate and YPD-G418 plat and culture of the four clones was purified by saturation ammonium sulfate followed by SDS-PAGE assay, and a band of about 23.6KD was found which showed that CGH gene was expressed and secreted to the solution medium efficiently. Also the speciality of screted protein was conformed by Western-blot analysis with rat anti-CGH serum.CGH gene was inserted into pPIC9K plasmid and overexpression Pichia Pastoris yeast strains was constructed which realized efficient expression of CGH gene in yeast. Also the rat anti-CGH serum was obtained by immunizing rat with CGH Prokaryotic expressing product, and speciality of screted protein was conformed by the serum. This study sets a foundation for further research and application of recornbinant GH in Grass carp.更多还原