Construction and expression of eucaryotic expression vector containing TNFAIP3 gene

Objectives: To construct a eukaryotic expression vector containing human TNFAIP3 gene and observe its expression, to lay a foundation for further study on TNFAIP3 gene. Methods: To obtain the totle RNA of the ACC-M cell line by Trizol method. The TNFAIP3 cDNA was amplified by reverse transcription polymerase chain reaction(RT-PCR) .The gene fragment of TNFAIP3 and eukaryotic expression vector PEGFP-N3 all were digested by restriction endonuclease EcoRI and kpnI. Then, the gene fragment of TNFAIP3 was linked into vector PEGFP-N3 by T4 DNA ligase.,and the recombined expression plasmid was identified by digestion analysis of endonuclease EcoRI and KpnI and sequence analysis.The recombined expression plasmid and blank plasmid were transduced into ACC-A2 cell line respectively,and the expression of TNFAIP3 was measured by RealTime-PCR.Also,the expression of recombined protein was observed by fluorescence microscope. Results:The molecular size of RT-PCR products was 2373bp by gel electrophoresis. The recombined expression plasmid PEGFP-N3-TNFAIP3 was digested into two ftagments of about 2373bp and 4.7kb by endonuclease EcorI and KpnI, and sequence analysis of RT-PCR products demonstrated that it contained the complete code region of human TNFAIP3 gene. The result of RealTime-PCR indicated the expression of TNFAIP3 gene in recombined expression plasmid group was higher than other groups.Conclusion:A eukaryotic expression vector containing human TNFAIP3 gene is successfully constructed, and it can express out the recombined protein , which has laid a concrete foundation for further study on TNFAIP3 gene.