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目的构建表达融合基因TGF-βⅡ型受体(TβR-Ⅱ)胞外区及活化T细胞表达和分泌的调节因子(RANTES)重组腺病毒载体,并观察其抗肿瘤作用。方法RT-PCR扩增小鼠TβR-Ⅱ胞外区和RANTES基因,重叠PCR扩增融合基因TβR-Ⅱ胞外区-RANTES。采用adMax adenovjrus vector creation试剂盒构建表达融合基因重组腺病毒。体外感染小鼠肺腺痛LA795细胞系,绘制细胞生长曲线。采用Western blot法检测感染后细胞融合基因的表达;酶联免疫吸附试验(FLISA)法检测其培养上清的蛋白含量;Annexin V-FITC法检测感染后细胞的凋亡;并观察感染后细胞培养上清对小鼠脾细胞的趋化作用。将感染后的细胞(1×105个)接种于T739小鼠,观察成瘤时间和生存时间;1×1010pfu的重组腺病毒局部注射荷瘤小鼠,观察肿瘤的大小变化,并统计肿瘤的重量和计算抑瘤率。结果经测序证实,RT-PCR正确扩增小鼠TβR-Ⅱ胞外区和RANTFS基因,重叠PCR正确扩增融合基因TβR-Ⅱ胞外区-RANTES。重组质粒pDC316-融合基因经酶切鉴定正确,与pJM17双质粒共转染获得表达融合基因重组腺病毒,病毒滴度为8×1010pfu/ml。Western blot结果表明,感染后的LA795细胞有融合蛋白的特异性条带出现;培养上清中,游离TGF-β1的水平显菩减低,而RANTES的水平显著升高。感染融合基因重组腺病毒的细胞生长速度明显减低,凋亡率为16.9%;培养上清可趋化小鼠脾细胞,与对照组之间差异均有统计学意义。感染融合基因重组腺病毒组与对照组相比,体内成瘤时间和生存时间明显延长;局部注射融合基因重组腺病毒可显著抑制肿瘤的生长,抑瘤率为37.6%。结论成功构建表达融合基因TβR-Ⅱ胞外区-RANTES重组腺病毒可有效结合TGF-β1,显著逆转TGF-β介导的免疫抑制状态,高水平表达RANTES强化肿瘤局部的免疫功能,具有显著的抗肿瘤作用。
Objective To construct a recombinant adenovirus vector expressing the expression and secretion of TGF-βⅡ receptor (TβR-Ⅱ) extracellular domain and activated T cells (RANTES) and to observe its anti-tumor effect. Methods The extracellular domain of TβR-Ⅱ and RANTES gene were amplified by RT-PCR. The fusion gene TβR-Ⅱ extracellular domain-RANTES was amplified by overlap PCR. The recombinant adenovirus expressing fusion gene was constructed by adMax adenovjrus vector creation kit. In vitro infection of mouse lung adenocarcinoma LA795 cell line, the cell growth curve was drawn. The expression of fusion gene was detected by Western blot. The protein content of the culture supernatant was detected by enzyme-linked immunosorbent assay (FLISA). The apoptosis of infected cells was detected by Annexin V-FITC method. Chemotaxis of supernatants to mouse splenocytes. The infected cells (1 × 105) were inoculated into T739 mice to observe the tumorigenic time and survival time. 1 × 1010pfu recombinant adenovirus was injected into the tumor-bearing mice locally to observe the tumor size changes and to calculate the tumor weight And calculate the inhibition rate. Results The sequencing confirmed that RT-PCR correctly amplified the extracellular domain of TβR-Ⅱ and the RANTFS gene, and the overlap PCR amplified the extracellular domain-RANTES of fusion gene TβR-Ⅱ correctly. The recombinant plasmid pDC316-fusion gene was identified by restriction enzyme digestion and co-transfected with the pJM17 double plasmid to obtain a recombinant adenovirus expressing the fusion gene. The virus titer was 8 × 1010 pfu / ml. Western blot results showed that the specific bands of fusion protein appeared in the infected LA795 cells. In the culture supernatant, the level of free TGF-β1 was significantly reduced and the level of RANTES was significantly increased. The cell growth rate of the recombinant adenovirus infected with the fusion gene was significantly reduced, with a apoptotic rate of 16.9%. The culture supernatant of the transfected mouse splenocytes was significantly different from the control group. Infection with fusion gene recombinant adenovirus group compared with the control group, tumor formation time and survival time was significantly extended; local injection of fusion gene recombinant adenovirus can significantly inhibit tumor growth, inhibition rate was 37.6%. Conclusion The recombinant adenovirus expressing the fusion gene TβR-Ⅱ extracellular domain-RANTES could effectively bind to TGF-β1 and significantly reverse the immunosuppressive status induced by TGF-β. The high expression of RANTES enhanced the local immune function, Antitumor effect.