NKAIN2真核表达载体的构建与表达鉴定

Construction and expression identification of eukaryotic expression vector of NKAIN2

目的 NKAIN2基因位于6号染色体长臂,6号染色体长臂的缺失现象经常发生于各种肿瘤。现构建候选抑癌基因NKAIN2真核表达载体,使其在前列腺癌细胞22RV1中过表达并观察其细胞定位,为进一步功能研究奠定实验基础。方法采用PCR方法扩增NKAIN2基因序列,将其重组于pc DNA4载体中,利用PCR和DNA测序鉴定克隆正确性;并转染到22RV1细胞,Western blot检测其蛋白表达,免疫荧光检测其在细胞中的定位。结果重组质粒中插入正确目的片段,片段大小为627 bp,与预计位置相符。Western blot检测表明,在25 000处检测到目的蛋白条带,与预计大小相符,而阴性对照未检测到相应条带,说明真核表达载体成功转染细胞,并且能够瞬时表达。转染了pc DNA4-FLAG-NKAIN2质粒的22RV1细胞在Petri皿培养24 h后可测到转染的带FLAG-NKAIN2片段的蛋白表达,且过表达的蛋白主要定位于细胞膜和细胞质中。转染未带FLAG标签的pc DNA4质粒未检测到蛋白荧光信号。结论成功构建了pc DNA4-FLAG-NKAIN2真核表达载体,转染22RV1细胞后高表达,为进一步研究NKAIN2在前列腺癌中的生物行为和其发生发展的相互作用机制提供基础。

Objective NKAIN2 gene is located in the long arm of chromosome 6,and the lack of the long arm of chromosome6 occurs frequently in various tumor. We constructed the eukaryotic expression vector of NKAIN2 gene,a candidate tumor suppressor gene,which was overexpressed in prostate cancer cell 22RV1 and cell location was observed,building the foundation for further functional study. Methods The coding sequence of NKAIN2 was amplified by PCR,and then inserted into pc DNA4 vector. After being verified by PCR and DNA sequencing,the construct was transfected into 22RV1 cells. Western blotting and immunofluorescence were performed to analyze the expression and distribution of NKAIN2 gene. Results Sequence analysis confirmed that the correct target gene( 627 bp) was inserted into the vector and corresponded with predicted position. The target protein bands were detected at 25000 by western blotting and corresponded with predicted size. The corresponding bands of negative control group,however,did not exist. It indicated eukaryotic expression vector successfully transfected into cells and could transient expression. The protein expression with FLAG-NKAIN2 sequencing was detected in 22RV1 cell transfected with pc DNA4-FLAG-NKAIN2 vector 24 hours after culturing in Petri dish. It was highly expressed and displayed a cell membrane and cytoplasmic distribution in 22RV1 cells. Protein fluorescence signal was not detected in pc DNA4 vector transfected without FLAG.Conclusion The eukaryotic expression vector pc DNA4-FLAGNKAIN2 was constructed successfully,with a highly expression in the transfected 22RV1 cells,providing the foundation for further research of the interaction mechanism between behavior of NKAIN2 in prostate cancer and it's development.