携带超抗原金黄色葡萄球菌肠毒素A基因低免疫原性病毒的构建

Construction of weak immunogenicity virus vector with super antigen staphylococcal enterotoxin Agene

目的构建具备表达超抗原金黄色葡萄球菌肠毒素A（SEA）基因的低免疫原性慢病毒载体，以期发挥高效激活T细胞抗肿瘤作用。方法应用聚合酶链反应（PCR）获取大小分别为851 bp的人端粒酶反转录酶（hTERT）-CMV启动子序列及774 bp的SEA目的基因，将目的基因构建到慢病毒载体质粒中，基因测序正确后，转入感受态细胞DH5α中扩增、收集。利用穿梭质粒和包装质粒共转染293FT细胞中包装病毒，超速离心浓缩法收集病毒上清液，荧光滴度法检测病毒滴度。感染复数（MOI）＝10病毒上清转染膀胱肿瘤细胞BIU-87细胞株，收集PCR产物测序目的基因，Western blot法检测SEA基因表达。结果载体质粒大小为10 458 bp，基因测序正确，无明显碱基突变。荧光滴度法检测病毒滴度为（4.30±2.00）×109 TU/ml。PCR产物测序目的基因证实目的基因正确，分光光度计检测24、48、96 h总RNA产物分别为742、803、726 mg/L。Western blot结果显示SEA基因成功表达。 结论 成功构建携带超抗原SEA基因低免疫原性病毒。

ObjectiveTo construct a weak immunogenicity lentiviral vector expressing the staphylococcal enterotoxin A （SEA） gene in order to exert a potent antitumor effect on activated T cells. MethodsThe human telomerase reverse transcriptase （hTERT）-CMV promoter sequence of 851 bp and the SEA gene of 774 bp were obtained by polymerase chain reaction （PCR）. The target genes were constructed into lentiviral vector plasmids. After sequencing, they were transferred into DH5α competent cells and amplified. Viral supernatants were collected by co-transfection of 293FT cells with shuttle plasmids and packaging plasmids, and virus titer was determined by fluorescence titer. Different concentrations of virus supernatant were transfected into BIU-87 cell line. The target gene was sequenced and the expression of SEA gene was detected by Western blotting. ResultsThe size of the vector was 10 458 bp, and the gene sequence was correct. No obvious mutation was found. The titer of the virus titer was （4.30±2.00）×109 TU/ml. PCR product sequencing of the target gene confirmed that the target gene was correct, the total RNA were 742、803、726 mg/L respectively by spectrophotometric assay. Western blotting revealed that SEA gene was successfully expressed.ConclusionThe weak immunogenicity virus carrying the superantigen SEA gene was successfully constructed.