Construction Of Prokaryotic Expression Vector Of Antigen Determinant Fragment Of TaNRT2.1 Gene Of Wheat Roots, The Expression Of The Recombinant And The Purification Of Fusion Protein In The E.coli

In recent years, many high affinity nitrate transporter gene was cloned from plants. For example: BnNRT2 from Brassica napus, NRT2.1Np from Nicotiana plumbaginifolia, AtNRT2.1 and AtNRT2.2 from Arabidopsis thalaiana, GmNRT2 from Glycine max, BCH1 and BCH2 from Hordeum valgare, and LeNrtl-1 and LeNrt1-2 from Lycopersicon esculentum etc.The final purpose of our study was to sublocalize the product of the high affinity nitrate transporter gene-TaNRT2.1, which was cloned by our laboratory from Triticum aestivum. According to the amino acid sequence which was encoded by the TaNRT2.1, we found a fragment which was considred had the highest antigenicity. And this fragment "was named by Ag. The corresponding cDNA sequence of this fragment was designed again according to the codons which was preferred by the E. coli, and was synthesized by the SBS Genetech Co., Ltd. Then, this fragment was cloned to the fusion protein expression vector pGEX-2T.The early stage of my work went very well: I cloned Ag fragment into the fusion protein expression vector pGEX-2T, that was pGEX-2T-Ag for short, and then constructed the recombinant plasmid successfully. The vector pGEX-2T has a glutathione-S-transferase (GST) gene, which was in favor of the purification of the fusion protein subsequently. And there is a thrombin site at the end of the GST, which was in favor of the separation of the target protein from the mixture fusion protein in E.coli. These have been the foundation of the production of antibody, and Western blotting too. I had induced the expression of the fusion protein by the induction of IPTG. But now, I encountered a difficult problem: the fusion protein has become the insoluble inclusion body for the over-expression. This makes the purification of fusion protein was impossible. It was not resolved yet, although I had tried by many different ways.At the same time, 1 make use of the network resources sufficiently. I analysis and predict the characterization of the TaNRT2.1. And I make a conclusion that TaNRT2.1 is the membrane protein, and has 12 transmembrane helixes by many different methods. And I analysis the amino acid composition thoroughly, and predict the secondary structure and tertiary structure too.