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中文摘要：
目的构建人C反应蛋白（CRP）真核表达载体,并观察其在NIH3T3细胞中的表达定位情况。方法利用带HA反向互补序列碱基的特异性引物,从p91023/CRP载体上将编码人CRP的基因序列亚克隆到真核表达载体pcDNA3上;对阳性克隆进行PCR、酶切和测序鉴定,利用脂质体转染方法转染NIH3T3细胞;并使用Westernblot和细胞免疫化学技术对重组质粒pcDNA3/HA-CRP在NIH3T3细胞中的表达及其蛋白定位进行分析。结果PCR、双酶切和测序鉴定表明,pcDNA3/HA-CRP真核表达质粒构建正确;转染实验发现,该质粒能够在NIH3T3细胞中表达,表达产物主要定位在细胞膜和核周边内质网的区域。结论成功构建了带HA标签的人CRP真核表达载体,该载体能够在哺乳动物细胞NIH3T3中表达,为进一步研究CRP的细胞内生物学功能提供了一个重要的工具。
英文摘要：
Objective To construct the eukaryotic expression vector of C-reactive protein （CRP）, and detect the vector＇s expression and localization in NIH3T3 cells. Methods With a specific primer beating HA reverse complementary sequence, the CRP gene sequence was amplified with the vector p91023 as template by PCR, and inserted into vector pcDNA3 to construct the eukaryotic expression vector pcDNA3/HA-CRP. The NIH3T3 cells were transfected with the vector pcDNA3/HA-CRP, which had been identified by PCR and double digestion with restriction endonudease, followed by sequencing. The transfected cells were then cultured for a certain time, and lyzed to the extracted protein, which were then loaded for SDS-PAGE followed by Western blotting for detecting the expression of HA-CRP, Some of the cells were fixed and labeled with antibody against HA and fluorescence conjugated second antibody to detect the localization of HACRP with immunocytochemical method. Results The results of identification by PCR, digestion with restriction endonuclease and sequencing indicated that the vector pcDNA3/HA-CRP was correctly constructed the transfection experiments and Western blotting suggested that the HA-CRP gene carried by vector pcDNA3/HA-CRP could express in mammalian cell line NIH3T3, and the protein HA-CRP mainly localized on the membranes and in the area of endocytoplasmic reticulum surrounding the nuclei. Conclusions The eukaryotic expression vector has been successfully constructed for human CRP labeled with HA sequence. It has been observed that the constructed vector may be expressed in mammalian cell NIH3T3 under quiescent condition. Thus, the present work has provided an important tool for further study on the CRP function inside cells.
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