远红荧光蛋白HcRed基因的原核和真核表达及鉴定

1、远红荧光蛋白HcRed基因的原核和真核表达及鉴定         11-03-23 15:48:00     编辑：studa20         作者：何志强 薛渊 石燕 杨恒 赵银霞 王楷文周成林 王胜军 邵启祥 焦志军 许化溪【摘要】  目的： 分别构建携带HcRed基因的PET28a(+)原核和PIRES真核载体，并分别在大肠埃希菌和鼠树突状细胞系(DC2.4)中表达、鉴定,为后

2、续基因及蛋白的转染提供基础。方法： 参照基因库中HcRed1基因序列，设计HcRed CDS全长引物，以含有HcRed的质粒为模板，通过PCR获得目的基因，并分别连接于PET28a(+)原核载体和PIRES真核载体。经PCR、酶切鉴定后，原核载体转化大肠埃希菌，经IPTG诱导表达;真核载体用脂质体转染DC2.4，G418筛选出克隆后，应用RTPCR和流式细胞仪进行检测。结果： 扩增出687 bp的HcRed CDS区序列，构建了原核和真核表达载体PET28a(+)/HcRed和PIRES/HcRed，分别于大肠埃希菌和DC2.4细胞系中成功表达。结论： 成功构建HcRed基因的原核、真核表达载

3、体，并分别在工程菌和细胞系中成功表达，为后续基因及融合蛋白的筛选、示踪和体内观测奠定了基础。 【关键词】  远红荧光蛋白HcRed; 树突状细胞系2.4; 原核表达Abstract Objective: In order to use the farred fluorescent protein HcRed, we amplified HcRed gene and cloned into PET28a(+) and PIRES plasmids. Then, the HcRed was expressed and identified in E.coli and Dendritic

4、cell line, which provided the basis for further research.Methods： The primers were designed with HcRed gene in GenBank. Using standard PCR and cloning techniques, the HcRed gene was amplified and cloned into the PET28a(+) and PIRES vectors successfully. The PET28a(+)/HcRed and PIRES/HcRed plasmids w

5、ere identified by PCR amplification and restriction digestion. The PET28a(+)/HcRed plasmid was transformed into Rosetta and the red fluorescent protein HcRed was expressed in E.coli upon IPTG induction. At the same time, the PIRES/HcRed vector was transfected into Dendritic cell line by liposomes. T

6、he HcRed gene was identified by RTPCR. The expressed HcRed protein in DC2.4 was detected by FCM. Results： The 687 bp HcRed gene was amplified. PET28a(+)/HcRed and PIRES/HcRed vectors were constructed successfully and expressed in E.coli and DC2.4. Conclusion： PET28a(+)/HcRed and PIRES/HcRed plasmids

7、 were constructed successfully and expressed in E.coli and DC2.4, respectively, which brought a foundation for further research on screening，tracing and observing in vivo of the gene and protein.Key words farred fluorescent protein HcRed; dendritic cell line 2.4; prokaryotic express红荧光蛋白HcRed的前体最早是G

8、urskaya等1从紫点海葵(Heteractis Crispa)中分离的一种类似于绿色荧光蛋白(GFP)的色蛋白，通过定点突变和随机突变改造而来。改造后的HcRed最大激发和发射波长分别为592 nm和645 nm，距离其他常用荧光标记蛋白(EGFP，ECFP，EYFP等)的激发和发射波长相距甚远，因此无论单独或是联合其他荧光蛋白使用都可以方便检测。本研究旨在通过建立HcRed基因的原核和真核表达载体，并在大肠埃希菌和树突状细胞系DC2.4中成功表达，来建立应用红色荧光蛋白HcRed示踪的方法，为进一步开展融合蛋白的细胞内定位和目的基因转染示踪提供了前提，同时也为建立HcRed标记的树突状细

9、胞系进行体内研究奠定基础。1 材料与方法1.1 材 料大肠埃希菌DH5，Rosetta，质粒PET28a(+)，PIRES,细胞系DC2.4均为本实验室保存;含有HcRed质粒模板由本校寄生虫教研室提供;试剂：LIPO2000，Trizol购自Invitrogen公司，去内毒素质粒大量制备试剂盒购自Axygen公司;rTaq酶，核酸内切酶，T4连接酶均为Fermentas产品;ReverTraAceaKit 购自ToYoBo公司;质粒小量制备试剂盒，胶回收试剂盒和PCR产物纯化试剂盒购自上海捷瑞公司。1.2 方 法TATGGATCCATGGTGAGCGGCCTG3，5ACGTCTAGAATGG

10、TGAGCGGCCTGC3,下划线处分别为BamH和Xba 酶切位点;下游序列为：5GGCGTCGACT33CAGTTGGCCTTCTC3,下划线处为Sal 酶切位点。预计扩增片段全长为705 bp。引物由上海生工生物技术服务有限公司合成。25 l PCR反应体系：0.25 l rTaq酶(5 U/l )，2.5 l 10×KCl 缓冲液，2.5 l MgCl2(25 mmol/L)，上下游引物各0.25 l(10 mmol/L)，1.5 l 模板cDNA，18 l H2O。PCR反应条件：94预变性5 min, 94变性30 s, 58退火30 s, 72延伸60 s,共进行30次循环, 再72延伸5 min。取5 l PCR扩增产物于10 g/L琼脂糖中电泳后, EB染色, 分析鉴定。2 结 果2.1 HcRed PCR扩增结果应用HcRed编码区特异性引物PCR扩增获得705 bp片段，与预期结果一致(图1)。2.2 PET28a(+)