摘要
重组汉逊酵母基因中外源基因拷贝数是影响目的基因表达水平和检测传代稳定性的重要因素,因此外源基因拷贝数的检测成为研究和分析重组基因的重要内容。利用快速、灵敏的荧光定量PCR法检测外源基因HBsAg拷贝数,以Mox基因为内源参照基因,通过梯度稀释法,建立了Mox基因和HBsAg基因的循环数(Ct值)与起始模板数的相关标准曲线,其相关系数分别达到0.9996和0.9982。通过比较目的基因HBsAg和内源参照基因Mox在同一荧光强度下出峰的循环数,获得了目的基因HBsAg在重组汉逊酵母中的拷贝数为39。发酵前后HBsAg基因在重组汉逊酵母中稳定存在,发酵前后拷贝数相差均小于6.2%。本方法快速、简便、准确,可以满足基因拷贝数检测的需要。
In recombinant hansenula polymorpha yeast, the recombinant gene copy is an important factor that can greatly influence the level of expression and genetic stabilityof the target gent. Estimating the recombinant gene copy number becomes a significant step in recombinant gene researsh, Rapid and sensitive fluorescence quantitative PCR was used to estimate the HBsAg copy number of recombinant Hansenula polymorpha yeast, and the Mox gene in Hansenula polymorpha yeast was used as endogenous reference gene. With a serial of dilutions, the standard curves of the threshold cycle（Ct） relative to the log of each initinal template copy of HBsAg gene and Mox gene were obtained, and the correlation coefficients were 0. 9996 and 0. 9982, respectively. The recombinant HBsAg gene copy number was 39 by comparing the threshold cycle betweeen HBsAg and Mox at the same quantitative fluorescence. The HBsAg gene was stability after fermentation, and the copy number difference between strains and fermentation liquid is less than6.2 %. This method detected the gene copy number of strains rapidly and conveniently.
出处
《生物技术通报》
CAS
CSCD
北大核心
2009年第10期178-180,193,共4页
Biotechnology Bulletin
基金
河南省重点科技攻关项目(082300450300)
华兰生物工程有限公司博士后科研基金
河南省动物学重点学科基金
