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目的HVEM基因突变体腺病毒载体对大鼠混合淋巴细胞反应(MLR)的影响。方法RT-PCR获得人HVEM的cDNA,使其与LIGHT分子结合的表位点突变,保留与BTLA结合的关健位点。构建经修饰的HVEM基因的复制缺陷型腺病毒载体Adv-mHVEM-IRES2-EGFP并对其进行鉴定和转染效率测定。将该载体用于混合淋巴细胞反应中,观察对淋巴细胞增殖的影响。结果成功构建HVEM突变体和EGFP复制缺陷型腺病毒载体,经过测序与预期一致,体外培养细胞转染效率达到95%以上。该载体能够对大鼠MLR有明细的抑制作用。结论构建的腺病毒载体能够共表达目的基因的产物HVEM突变体和EGFP蛋白,对体外细胞具有高效转染能力。该载体能够对大鼠混合淋巴细胞反应有明显的抑制作用。
Objective To investigate the effect of HVEM gene mutant adenovirus vector on mixed lymphocyte reaction (MLR) in rats. Methods Human HVEM cDNA was obtained by RT-PCR. The site of binding to LIGHT molecule was mutated and the key sites of binding to BTLA were retained. The replication-deficient adenoviral vector Adv-mHVEM-IRES2-EGFP of the modified HVEM gene was constructed and identified and the transfection efficiency was assayed. The vector was used in a mixed lymphocyte reaction to observe the effect on lymphocyte proliferation. Results The HVEM mutant and EGFP replication-deficient adenovirus vector were successfully constructed. After sequencing, the transfection efficiency reached more than 95%. This vector can have a detailed inhibitory effect on rat MLR. Conclusion The constructed adenoviral vector co-expresses the HVEM mutant and EGFP protein of the target gene and has high transfection efficiency in vitro. The vector can significantly inhibit the mixed lymphocyte reaction in rats.