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目的构建NT4-SAC-HA2-TAT融合肽cDNA克隆,探讨前列腺凋亡反应基因-4(Par-4)核心区凋亡肽(SAC)作为目的基因对前列腺癌的治疗作用。方法应用互为引物模板法合成两端具有NaeI和KpnI酶识别位点的SACcDNA片段。将所得SAC和已有HA2-TAT片段克隆到具有相应酶切位点的pBV220-NT4质粒,得到pBV220-NT4-SAC-HA2-TAT重组质粒。PCR扩增NT4-SAC-HA2-TATcDNA片段,并将其克隆到pGEM-T easy中,转化细菌筛选阳性克隆,酶切鉴定,序列测定分析。结果经DNA测序、限制性内切酶酶切等证实,PCR获得了编码NaeI和KpnI酶切位点的SAC cDNA片段,并将NT4、SAC、HA2-TAT亚克隆于pBV220内。结论成功构建了NT4-SAC-HA2-TAT融合基因的原核表达载体pBV220-NT4-SAC-HA2-TAT。
Objective To construct a cDNA clone of NT4-SAC-HA2-TAT fusion peptide and investigate the therapeutic effect of prostate-specific apoptosis-inducing factor-4 (Par-4) apoptosis-targeting peptide (SAC) Methods SAC cDNA fragments with NaeI and KpnI recognition sites at both ends were synthesized by primer-template method. The obtained SAC and the existing HA2-TAT fragment were cloned into the pBV220-NT4 plasmid with the corresponding restriction sites to obtain the recombinant plasmid pBV220-NT4-SAC-HA2-TAT. NT4-SAC-HA2-TAT cDNA fragment was amplified by PCR and cloned into pGEM-T easy. The positive clones were screened by transformed bacteria, identified by restriction enzyme digestion and sequenced. Results After DNA sequencing and restriction endonuclease digestion, the SAC cDNA fragments encoding NaeI and KpnI sites were obtained by PCR and the subclones of NT4, SAC and HA2-TAT were subcloned into pBV220. Conclusion The prokaryotic expression vector pBV220-NT4-SAC-HA2-TAT of NT4-SAC-HA2-TAT fusion gene was successfully constructed.