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目的制备可表达人转录因子STAT3的重组腺病毒,检测该基因在胃癌细胞株SGC-7901的表达活性及对其增殖的影响。方法以人胎盘组织cDNAs为模板通过PCR获得hSTAT3完整阅读框,T/A克隆测序证实后,亚克隆至穿梭载体pAdTrack-CMV中,PmeⅠ线性化与腺病毒骨架载体pAdEasy-1在BJ5183细菌同源重组,经PacⅠ线性化后转染293FT细胞包装,同时包装空载体腺病毒,进行病毒滴度测定。取AdV-STAT3、AdV-GFP分别感染SGC-7901细胞,设空白对照,Western blot检测3组细胞STAT3蛋白表达,MTT法检测对细胞增殖的影响。结果获得hSTAT3完整阅读框,T载体中测序无突变,亚克隆至pAdTrack-CMV,并与pAdEasy-1成功重组后包装AdV-STAT3,测定滴度为2.8×1010pfu/mL,AdV-GFP滴度为3.2×1010pfu/mL。与AdV-GFP组及空白对照组比,Western blot示AdV-STAT3组蛋白明显表达(P<0.05);MTT示AdV-1-STAT3组明显促进细胞增殖(P<0.05)。结论成功制备hSTAT3的重组腺病毒,感染胃癌细胞株SGC-7901后高表达并促进其增殖。
Objective To prepare a recombinant adenovirus expressing human transcription factor STAT3 and detect the expression of this gene in gastric cancer cell line SGC-7901 and its effect on proliferation. Methods The full-length hSTAT3 reading frame was obtained by PCR using human placenta cDNAs as a template. After confirmed by T / A cloning and sequencing, the vector was subcloned into the shuttle vector pAdTrack-CMV. PmeⅠ linearization was performed on the homolog of BJ5183 Recombinant, PacI linearized transfected 293FT cell packaging, while empty vector adenovirus packaging for virus titer determination. SGC-7901 cells were infected with AdV-STAT3 and AdV-GFP respectively. The expression of STAT3 was detected by Western blot. The effect of AdV-GFP on the proliferation of SGC-7901 cells was detected by MTT assay. Results The hSTAT3 complete reading frame was obtained. The T vector was sequenced without mutation and subcloned into pAdTrack-CMV. AdV-STAT3 was packaged successfully after recombination with pAdEasy-1. The titer was 2.8 × 1010pfu / mL. The titer of AdV-GFP was 3.2 × 1010 pfu / mL. Compared with AdV-GFP group and blank control group, Western blot showed that AdV-STAT3 protein was significantly expressed (P <0.05); MTT showed AdV-1-STAT3 group significantly promoted cell proliferation (P <0.05). Conclusion The hSTAT3 recombinant adenovirus was successfully constructed and was highly expressed in gastric cancer cell line SGC-7901 and promoted its proliferation.