Construction And Expression Of Eukaryotic Expression Vectors For Rat Annexin â…¡ And Tubulin Beta-5 In DRG Cell

[Background]Neuropathic pain is a chronic state, caused by the injured nerve system, and it has been a serious problem for human health for a long time. DRG neuron is the primary afferent neuron that carries the sensory signals to spinal cord, therefore it plays an important role in the peripheral mechanism of neuropathic pain. Now some studies have found the close relationships between cytoskeleton and related proteins with the DRG's nociceptive pain conduction. However the molecular mechanism is unknown. This study aims at obtaining the genes of annexin II (Anxa2) and tubulin beta-5 (Tubb5), constructing their eukaryotic expression vectors and observing their expression separately by genetic engineering technology.[Objective]To obtain annexinⅡ/tubulin beta-5 genes, construct their eukaryotic expression vectors and observe their expressions.[Methods]According to the Genbank, the coding sequence of rat annexinⅡand tubulin beta-5 genes were cloned by reverse transcription PCR method from rat DRG cells, and pGMT-Anxa2/pGMT-Tubb5 was constructed, according to which, Anxa2-flag/Tubb5-flag was amplified by inserting the flag-tagged sequences to the C-terminal. Then Anxa2-flag/Tubb5-flag was ligated into the eukaryotic expression vector pcDNA3.1(+). And the new recombinant plasmid pcDNA3.1(+)-Anxa2-flag and pcDNA3.1(+)-Tubb5-flag were constructed, which had to be evaluated by HindⅢand NotⅠdouble enzyme incision, PCR and sequence analysis.Then pcDNA3.1(+)-Anxa2-flag/pcDNA3.1(+)-Tubb5-flag was transfected into HEK293 cells by using lipofectamine 2000, which was the experimental group. There were also two control groups:pcDNA3.1(+) empty plasmid control group and negative control group with non-plasmid transfected. After 48-72 hours, western blot and immunofluorescence methods were used to detect the expression and localization of rat annexinⅡand tubulin beta-5 protein in the transfected cells in vitro.[Results]1. Anxa2/Tubb5 was amplified from mRNA: The size of PCR amplified product of Anxa2 is about 1020bp and Tubb5 is 1335bp, which are both consistent with the expected fragments.2. Identification of cloning vector pGMT-Anxa2/pGMT-Tubb5: pGMT-Anxa2/pGMT-Tubb5 was proved to be correct by PCR and sequence analysis.3. Anxa2-flag/Tubb5-flag was amplified: The size of PCR amplified product of Anxa2-flag is about 1044bp and Tubb5-flag is 1359bp, which are both consistent with the expected fragments.4. Idenfication of recombinant plasmids:pcDNA3.1(+)-Anxa2-flag was proved to be correct by HindⅢand NotⅠdouble enzyme incision, PCR and sequence analysis. And pcDNA3.1(+)-Tubb5-flag was also proved to be successfully constructed by the same way.5. Results of immunofluorescence: Immunofluorescence has detected the green-light widely distributed in, which indicates the expression and location of the goal protein. Both annexinⅡand tubulin beta-5 protein have been found expressed stably in HEK293 cell, and diffusely distributed throughout the plasma membrane and the cytoplasm separately. While no green-light has been detected in the pcDNA3.1(+)plasmid control group and negative control group.6. Results of Western blot: a 36-KD band has been found in the pcDNA3.1(+)-Anxa2-flag group, while a 50-KD band has been found in the pcDNA3.1(+)-Tubb5-flag group, which indicated that rat annexin II protein and tubulin beta-5 protein were both expressed stably in HEK293 cell. There were none specific bands in both of the two control groups. The internal controlβ-actin in different groups was expressed equally.[Conclusions]The eukaryotic expression vectors pcDNA3.1(+)-Anxa2-flag and pcDNA3.1(+)-Tubb5-flag were constructed successfully, which could effectively expressed in HEK293. This would facilitate the following study of the molecular mechanism of annexin II and tubulin beta-5 in the DRG's nociceptive pain conduction and may provide some clues for the prevention and treatment of the neuropathic pain.