Vero细胞LTR基因的克隆及其DNA定量PCR检测方法的建立

Cloning of Long Tandem Repeat Sequence of Vero Cells and Its Application in Quantitative PCR for DNA

目的克隆Vero细胞基因组DNA的长串联重复序列(Long tandem repeats,LTR),并建立Vero细胞DNA定量PCR检测方法。方法提取Vero细胞基因组DNA,经HindⅢ酶切后,克隆至pET-30a(+)载体上,酶切及测序鉴定,并与Gen-Bank中登录的序列进行同源性比较。以172 bp序列为靶基因设计定量PCR引物和Taq探针,建立Vero细胞DNA定量PCR检测方法,并进行特异性验证。结果经酶切及测序鉴定,共获得8个阳性克隆质粒pET-30a-172,与GenBank中登录的基因序列同源性在98.3%～99.4%之间;建立的Vero细胞DNA定量PCR检测方法的灵敏度可达1×10-6 ng/μl,线性范围为1 ng/μl～1×10-6 ng/μl;以建立的方法检测鸡胚细胞、CHO细胞和地鼠肾细胞DNA,均未见扩增曲线,仅人二倍体细胞(MRC-5)DNA有明显的扩增曲线,表明该法特异性良好。结论已成功建立了以172 bp LTR为靶基因的Vero细胞DNA定量PCR检测方法,可用于疫苗中Vero细胞DNA残留量的检测。

Objective To clone the long tandem repeat sequence（LTR） in genomic DNA of Vero cells and develop a quantitative PCR method for Vero cell DNA.Methods To clone the long tandem repeat sequence（LTR） in genomic DNA of Vero cells and develop a quantitative PCR method for Vero cell DNA.Results Eight positive clones of pET-30a-172 were obtained by restriction analysis and sequencing,with homologies of 98.3% ～ 99.4% to the gene sequence reported in GenBank.The sensitivity and liner range of the developed quantitative PCR method were 1 × 10-6 ng / μl and 1 ng / μl ～ 1 × 10-6 ng / μl respectively.By the developed PCR method,amplification curve was only obtained from the DNA of human diploid MRC-5 cells but not from that of chick embryo cells,CHO cells or hamster kidney cells,indicating high specificity.Conclusion A 172 bp LTR-based quantitative PCR method for Vero cell DNA was successfully developed,which might be used for residual Vero cell DNA content in vaccines.