摘要
为构建烟台黑猪SLA-2基因(SLA-2-YT)的真核表达载体SLA-2-YT/pCDH并将其在真核细胞中表达,根据SLA-2-YT编码区基因序列设计1对引物,以SLA-2-YT/pMD18-T全基因克隆表达载体为模板进行PCR扩增获得SLA-2-YT编码区基因片段,在上下游引物的5'端分别添加限制性内切酶Xba I和Not I的酶切位点,将目的基因经pMD 19-T Simple Vector TA克隆后与pCDH-CMV-MCS-EF1-Puro真核表达载体连接,获得的重组质粒转化至大肠杆菌Stbl 3感受态细胞,对扩大培养的单克隆菌株抽提质粒,使用双酶切和测序验证插入序列。对真核表达载体构建正确的菌株抽提无内毒素质粒,通过慢病毒包装和感染将质粒转染至sT2细胞,通过Western Blotting检测sT2细胞中SLA-2-YT基因的表达情况。结果显示,成功构建了SLA-2-YT/pCDH重组真核表达载体。重组质粒进行慢病毒包装和感染sT2细胞,经嘌呤霉素筛选后,成功获得阳性细胞克隆。Western Blotting检测显示SLA-2-YT/pCDH在sT2细胞中得到了优势表达,其融合蛋白的分子量大小为45 kD,与理论设计值相符。该实验成功构建了SLA-2-YT编码区基因的真核表达载体,并证实了SLA-2-YT编码区基因能够在sT2细胞中优势表达,为下一步开展SLA-2-YT递呈CTL表位的研究提供了材料。
In order to construct the recombinant eukaryotic expression vector SLA-2-YT/pCDH from a Yantai black pig’s SLA-2 gene(SLA-2-YT)and to express it in eukaryotic cells,a pair of primers were designed according to SLA-2-YT coding region gene sequence,and the SLA-2-YT coding region gene fragment was amplified by PCR using SLA-2-YT/pMD18-T whole-gene cloning expression vector.The restriction enzyme Xba I and Not I were added to the 5'end of the forward and lower primers,respectively.The target gene was firstly cloned into pMD 19-T Simple Vector TA and was ligated to the pCDH-CMV-MCS-EF1-Puro(pCDH)eukaryotic expression vector.The recombinant plasmids were transformed into Escherichia coli Stbl 3 competent cells.The recombinant plasmids were extracted from the expanded culturing monoclonal strain and the sequences were verified by double enzyme digestion and sequencing.A large quantity of endotoxin-free plasmids was extracted from the recombinant eukaryotic expression vector with correct inserting sequence,and then they were transfected into sT2 cells through lentivirus packaging and infection.The expression of SLA-2-YT gene in sT2 cells was detected by Western Blotting.The results showed that the recombinant eukaryotic expression vector SLA-2-YT/pCDH was successfully constructed.After packaging by lentivirus,the recombinant plasmids infected sT2 cells followed by puromycin screening,and then a positive cell clone was successfully obtained.Western Blotting detection showed that SLA-2-YT/pCDH was predominantly expressed in sT2 cells with the molecular weight of 45 kD,which was consistent with the theoretical designing value.In this experiment,the SLA-2-YT coding region gene was successfully constructed into the eukaryotic expression vector,and predominantly expressed in sT2 cells,which will supply good materials for studying CTL epitoes presented by SLA-2-YT in future.
作者
胡晓
王宝宝
窦少华
姜南
付常振
金行
高凤山
HU Xiao;WANG Bao-bao;DOU Shao-hua;JIANG Nan;FU Chang-zhen;JIN Hang;GAO Feng-shan(College of Life Science and Technology,Dalian University,Dalian 116622)
出处
《生物技术通报》
CAS
CSCD
北大核心
2021年第10期143-151,共9页
Biotechnology Bulletin
基金
国家自然科学基金项目(31672525)
大连大学创新团队项目(XLJ202005)。
关键词
烟台黑猪
SLA-2基因
真核表达载体
慢病毒包装
转染
Yantai black pig
SLA-2 gene
eukaryotic expression vector
lentivirus packaging
transfection
