Kozak序列引导的人p53基因重组智能腺病毒载体的构建与表达

Construction and Expression of Recombinant High IQ Adenovirus Vector Containing Human p53 Gene Leaded by Kozak Sequence

目的构建Kozak序列引导的人p53(khp53)基因重组智能腺病毒载体rAdMH-khp53,并检测p53基因的表达。方法将带有Kozak序列的p53基因克隆到重组腺病毒穿梭载体中,再利用AdMaxTMHi-IQ系统,获得重组腺病毒,检测病毒滴度,并进一步感染Saos-2细胞,利用RT-PCR及Western blot检测p53基因的表达。结果khp53基因成功克隆到腺病毒载体中,重组腺病毒滴度为1.63×108IU/ml,RT-PCR方法检测到p53基因的转录产物,Western blot检测到p53基因在Saos-2细胞中的高水平表达。结论已成功构建了带有Kozak序列的重组腺病毒载体rAdMH-khp53,并高效表达人p53基因。

Objective To construct a recombinant high IQ adenovirus verctor containing p53 gene leaded by Kozak sequence.Methotis The p53 gene with Kozak sequence was cloned to recombinant adenovirus shuttle plasmid pDC15（io）, and recombinant adenovirus rAdMH-khp53 was obtained by using AdMaxTM Hi-IQ system,determined for titer and used for infection of Saos-2 cells.The expression of p53 gene was identified by RT-PCR and Western blot. Results khp53 gene was successfully cloned to adenovirus vector, and the recombinant adenovims reached a liter of 1.63 ×10^10IU/ml. RT-PCR proved the transcription, and Western blot proved the high expression of p53 gene in Saos-2 cells. Conclusion The recombinant high IQ adenovirus vector rAdMH-khp53 containing p53 gene leaded by Kozak sequence was successfully constructed, and human p53 gene was highly expressed.