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目的构建小鼠B/T淋巴细胞弱化因子(BTLA)胞外段的腺相关病毒载体,并感染中国仓鼠卵巢上皮细胞(CHO)进行真核表达。方法从C57BL/6小鼠脾脏中提取总RNA,经RT-PCR扩增BTLA胞外段,然后将其克隆到腺相关病毒载体的骨架质粒中,构建重组骨架质粒psBTLA。重组质粒经双酶切鉴定及测序正确后,与pHELP质粒以及pRC质粒3质粒共转染293T细胞,收获含病毒的上清后用氯仿-PEG8000的方法进行纯化浓缩,再用浓缩后的病毒液感染CHO细胞,并用SDS-PAGE和Western blot检测BTLA胞外段的表达。结果获得长度为502 bp的小鼠BT-LA胞外段基因,经测序证实其序列正确。含有BTLA胞外段基因的腺相关病毒载体感染CHO细胞后,SDS-PAGE和Western blot分析证实感染的CHO细胞可表达出约21 kD的BTLA胞外段蛋白。结论成功构建小鼠BTLA胞外段基因的腺相关病毒载体,并感染CHO细胞后表达出相应蛋白,为进一步研究BTLA胞外段的功能效应奠定实验基础。
Objective To construct adeno-associated virus vector of extracellular domain of mouse B / T lymphocyte attenuator (BTLA) and infect Chinese hamster ovary epithelial cells (CHO) for eukaryotic expression. Methods Total RNA was extracted from the spleens of C57BL / 6 mice. The extracellular domain of BTLA was amplified by RT-PCR and cloned into the adeno-associated virus vector backbone plasmid to construct the recombinant backbone plasmid psBTLA. 293T cells were co-transfected with pHELP plasmid and pRC plasmid 3 plasmid after the recombinant plasmid was identified by double restriction enzyme digestion and sequencing. The virus-containing supernatant was harvested and purified by concentration with chloroform-PEG8000, then concentrated virus solution CHO cells were infected and the expression of extracellular domain of BTLA was detected by SDS-PAGE and Western blot. Results The extracellular domain of mouse BT-LA with a length of 502 bp was obtained and its sequence was verified by sequencing. After infection of CHO cells with adeno-associated virus vector containing BTLA extracellular domain gene, the infected CHO cells could express about 21 kD BTLA extracellular domain protein by SDS-PAGE and Western blot analysis. Conclusion The adeno-associated virus vector encoding the extracellular domain of BTLA was successfully constructed and expressed in CHO cells, which provided experimental basis for further study on the functional effect of extracellular domain of BTLA.