小鼠丝氨酸/精氨酸蛋白特异激酶2基因真核重组表达质粒的构建及其对微管蛋白聚合的影响

Construction of eukaryotic expression vector for mouse serine/arginine-rich protein specific kinase 2 gene and its effect on microtubule polymerization

目的构建小鼠丝氨酸/精氨酸蛋白特异激酶2(serine/arginine-rich protein specific kinase 2,SRPK2)基因(srpk2)的真核重组表达质粒,并分析其对微管蛋白α-Tubulin聚合的影响。方法利用RT-PCR法扩增srpk2的全长cDNA序列,通过分子克隆技术构建真核重组表达质粒pLV-EGFP(2A)Puro-srpk2;在脂质体Lipofectamine 2000的介导下,将真核重组表达质粒及空载体pLV-EGFP2(A)Puro分别转染人胚肾HEK293T细胞,同时设空白对照组(未转染)。采用RT-PCR及Western blot法分别检测HEK293T细胞中srpk2基因mRNA转录及SRPK2蛋白的表达情况;Western blot法分析HEK293T细胞中聚合态和游离态α-Tubulin的含量。结果双酶切及DNA序列鉴定证明真核重组表达质粒pLV-EGFP2(A)Puro-srpk2构建正确,且在HEK293T细胞中实现了srpk2基因过表达。真核重组表达质粒转染组HEK293T细胞中游离态α-Tubulin含量显著低于空载体转染组及空白对照组(P<0.05),而聚合态α-Tubulin水平明显高于空载体转染组及空白对照组(P<0.05)。结论成功构建了srpk2基因的真核重组表达质粒,SRPK2可促进α-Tubulin微管蛋白聚合。

Objective To construct a recombinant eukaryotic expression plasmid for mouse serine/arginine-rich protein specific kinase 2（SRPK2） gene（srpk2）, and analyze its effect on polymerization of α-Tubulin. Methods The full-length cDNA fragment of srpk2 gene was amplified by RT-PCR, based on which recombinant eukaryotic expression plasmid pLVEGFP（2A） Puro-srpk2 was constructed by molecular cloning techniques and transfected into HEK 393 T cells in medication of Lipofectamine 2000, using the cells transfected with empty vector pLV-EGFP2（A） Puro and those untransfected as controls. The transcription level of srpk2 mRNA was determined by RT-PCR, while the expression level of SRPK2 by Western blot. The polymeric and monomeric α-Tubulin contents in HEK293 T cells were analyzed by Western blot. Results Restriction analysis and DNA sequencing indicated that the recombinant eukaryotic expression plasmid pLV-EGFP（2A） Puro-srpk2 was constructed correctly. RT-PCR and Western blotting results showed that srpk2 were over-expressed in HEK293 T cells. The expression level of monomeric α-Tubulin in HEK293 T cells transfected with recombinant plasmid pLV-EGFP（2A） Puro-srpk2 was significantly lower, while that of polymeric α-Tubulin was significantly higher, than those in the cells transfected with empty vector and the untransfected cells（each P〈0. 05）. Conclusion Recombinant eukaryotic expression plasmid pLV-EGFP（2A） Puro-srpk2 was successfully constructed, and SRPK2 promoted the polymerization of α-Tubulin.