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目的 :应用 p1 6(MTS1 )反义寡核苷酸抑制转染p1 6逆转录病毒载体后外源性 p1 6基因的表达 ,减弱p1 6表达对包装细胞的生长抑制作用。方法 :合成p1 6cDNA起始密码子区的反义寡核苷酸和正义寡核苷酸 ,加入筛选的转染p1 6逆转录病毒载体的包装细胞培养液中 ,记录各孔中出现细胞克隆的时间和数量 ,测量各形成细胞克隆的病毒上清滴度。Westernblot检测反义寡核苷酸对外源性P1 6蛋白表达的影响。MTT法检测加入反义寡核苷酸后对转染的包装细胞生长的影响。结果 :p1 6(MTS1 )反义寡核苷酸可以抑制外源性 p1 6基因的表达。加入反义寡核苷酸的包装细胞形成细胞克隆的时间提前 ,克隆数量和滴度都高于对照组。结论 :p1 6(MTS1 )反义寡核苷酸可以抑制外源性 p1 6基因的表达 ,促进p1 6逆转录病毒载体转染的包装细胞的生长并提高病毒滴度。
OBJECTIVE: To study the inhibitory effects of p16 (MTS1) antisense oligonucleotide on the expression of exogenous p1 6 gene after transfection with p1 6 retroviral vector and the inhibition of the growth of packaging cells induced by p1 6. METHODS: Antisense oligonucleotides and sense oligonucleotides of the initiation codon region of p1 6 cDNA were synthesized and added to the culture medium of the packaging cells transfected with p1 6 retroviral vector, and the cell clones in each well were recorded Time and amount of virus supernatant titers were measured for each cell clone. Effect of antisense oligonucleotide on the expression of exogenous P1 6 protein by Western blot. The effect of antisense oligodeoxynucleotide on the growth of transfection packaging cells was detected by MTT assay. Results: p16 (MTS1) antisense oligonucleotide can inhibit the expression of exogenous p1 6 gene. Antisense oligodeoxynucleotide packaging cells to form cell clones ahead of time, the number of clones and titers were higher than the control group. CONCLUSION: The antisense oligonucleotide p16 (MTS1) can inhibit the expression of exogenous p1 6 gene, promote the growth of packaging cells transfected with p1 6 retroviral vector and increase the virus titer.