含野生型p53cDNA重组逆转录病毒的制备

目的：制备野生型p53cDNA重组体的复制缺陷型逆转录病毒。方法：设计引物PCR扩增野生型p53cDNA，用T－A克隆的方法克隆入质粒载体pGEM-T,转化JM109，提取pGEM-T/p53重组体，双酶切得到野生型p53cDNA,然后再克隆人逆转录病毒表达载体pLXSN,转化感受态大肠杆菌 JM109，筛选pL(p53cDNA)SN重组体，脂质体法转染包装细胞AM12，G－418（geneticin)筛选，收集病毒液。结果：通过PCR和重组质粒酶切筛选出重组阳性克隆，收集含pL(p53cDNA)SN重组体的逆转录病毒。结论：含pL(p53cDNA)SN重组体的复制缺陷逆转录病毒可用于转染真核细胞等进一步研究。

Preparation of Recombinant Retrovirus Containing Wild type p53cDNA

Objective:To prepare the recombinant retrovirus containing wild type p53cDNA. Methods:The wild type p53cDNA was amplified by PCR and put into pGEM T vector by T A strategy. The recombinant pGEM T/p53 was extracted and the wild type p53cDNA was obtained by two restriction endonucleases. Then it was cloned into retroviral expression vector and transformed E.coli. The recombinant clones were picked out and transfected retroviral packing cell lines AM12 using clonfectin transfection. The transfected clones were selected with G 418, and the supernatants from these clones were collected and used for further experiments. Results:The recombinant clones were picked out by the methods of PCR test and restriction endonucleases,and the retrovirals with pL(p53cDNA)SN recombination were collected. Conclusion:The retrovirals with pL(p53cDNA)SN recombination may be used in further studies.