摘要
目的 构建人共刺激分子CD80 (B7 1)、T细胞识别的鳞状细胞癌抗原 3(Sart3)融合基因真核表达载体 ,并分别在原核和人成纤维细胞中表达。方法 采用RT PCR技术 ,从人胎盘组织抽提的总RNA中克隆Sart3基因的部分cDNA序列 (第 1～ 12 81bp) ,此段序列包括已报道的能诱导HLA限制性细胞毒性T淋巴细胞的抗原表位。然后将不含终止密码子的B7 1cDNA和Sart3cDNA共克隆入真核表达载体 pEGFP N1,测定核苷酸序列确定重组成功后 ,分别转染大肠杆菌和人成纤维细胞 ,利用流式细胞仪和Westernblot检测B7和Sart3的表达。结果 构建人B7、Sart3融合基因真核表达载体B7Sart3/pEGFPN。测序结果与GenBank的B7、Sart3相应序列 (NM_0 0 5 191、NM_0 14 70 6 )一致 ,阅读框无改变。流式细胞仪测定发现在转染的人成纤维细胞表面有明显的荧光增强 ,免疫印迹发现融合蛋白可以与抗B7抗体反应 ,融合蛋白分子量与B7 Sart3 EGFP的预测分子量相当。结论 成功构建真核表达载体B7Sart3/pEGFPN ,并在人成纤维细胞中表达。为下一步研究将SART3蛋白作为肿瘤疫苗应用于骨肉瘤的生物治疗打下基础。
Objective To clone partial cDNA of Sart3 gene and construct a recombinant eukaryotic expression vector containing a fusion gene B7 1/Sart3.Methods Partial cDNA of Sart3 gene was cloned by reverse transcription from human placenta tissue. The fragment was cloned into a plasmid vector pEGFP N1 to construct a recombinant eukaryotic expression vector Sart3/pEGFPN. Then B7 1 gene without terminator codon was cloned into recombinant vector Sart3/pEGFPN. The sequence of the cloned cDNA was confirmed by DNA sequencing, PCR and restriction enzymes analysis. The expression of the fusion protein was detected using Western blot and FACS. Results The results of sequence analysis corresponded to the data from GenBank (NM_005191, NM_014706). The expected B7 Sart3 fusion protein was detected by Western blot. FACS analysis confirmed the expression of B7 Sart3 on fibroblast cell membrane. Conclusion The successful construction and expression of the B7 Sart3 gene might provide materials for our further research on the antigenic protein Sart3 in immunotherapy of osteosarcoma.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2004年第2期165-168,共4页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
国家自然科学基金资助项目 (No.30 2 0 0 0 6 1)
