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| Cloning and Expression of PLCE1 Gene and Its Haplotypes of rs2274223 and rs3765524 |
| LI Da, DAI Peng, WANG Wei, ZHANG Wen-tao, WANG Qin, SHU Yi, ZHU Chun-lai, JI Qi-feng, LIANG Ping, YAN Zhen |
| Department of Pharmacogenomics, School of Pharmacy, Fourth Military Medical University, Xi'an 710032, China |
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Abstract Objective: The aimed is to address the question of how PLCE1 gene polymorphism or genotypes affect its expression by cloning human PLCE1 gene and constructing eukaryotic expression vector of PLCE1 rs2274223G-rs3765524T(PLCE1 Minor) and PLCE1 rs2274223A-rs3765524C(PLCE1 Major). Methods: Overlap extension PCR (SOE-PCR) and In-Fusion Technology were adopted to construct PLCE1 eukaryotic expression vector. The targeted gene was acquired by double point mutations based on homologous recombination. The expression of overexpressed PLCE1 by gene transfection was observed by real-time quantitative PCR and Western blotting. Results: The full-length human PLCE1 that contains 6 927 base pairs was acquired and confirmed by DNA sequencing. By comparing with the reference sequence (NM_016341) in NCBI database, it was found to exactly matched the reference sequence except a 18bp insertion in the region of 3 554~3 572bp. Additionally, the recombinant plasmids of two PLCE1 haplotypes, PLCE1 Minor and PLCE1 Major, were constructed respectively. By transfection, it was found that the expression levels of mRNA and protein of PLCE1 Minor were significantly higher than that of PLCE1 Major. Conclusion: A new PLCE1 transcript variant was discovered and the eukaryotic expression vectors of two haplotypes of human PLCE1 were successfully constructed. It was found that PLCE1 rs2274223G-rs3765524T promotes its mRNA and protein expression. These findings laid the foundation of revealing the correlation between PLCE1 single nucleotide polymorphisms (SNP) and cancer susceptibility.
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Received: 18 September 2016
Published: 25 December 2016
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Cite this article:
LI Da, DAI Peng, WANG Wei, ZHANG Wen-tao, WANG Qin, SHU Yi, ZHU Chun-lai, JI Qi-feng, LIANG Ping, YAN Zhen. Cloning and Expression of PLCE1 Gene and Its Haplotypes of rs2274223 and rs3765524. China Biotechnology, 2016, 36(12): 1-7.
URL:
https://manu60.magtech.com.cn/biotech/DOI:10.13523/j.cb.20161201 OR https://manu60.magtech.com.cn/biotech/Y2016/V36/I12/1
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