Cloning And Expression Of 5’- Nucleotidase Partial Sequence Of Cordycepin Metabolism Related Gene And Related Research

Cordycepin(3’-deoxyadenosine) is a kind of nucleoside analogue, one of the important compounds in Cordyceps militaris. It has many pharmacological function such as antitumor, anti-tumor, anti-aging, enhancing immune and so on. But for cordycepin biosynthetic pathway is not clear, but also need for further studies confirm.In this study, firstly used HPLC method to determine the yield of cordycepin in fermentation liquid of Cordyceps militaris, then draw the curves of cordycepin. According to the known putative cordycepin metabolism pathway selected the related genes. Actin and 18 S rRNA as the reference gene. Have used the q RT-PCR for quantitative analysis of the relevant gene. Depending to the relative value of detection showed that 5’-nucleotidase and adenylate kinase genes up-regulated trend same as cordycepin generate curves in mycelium. According to cordycepin generate curves, known putative cordycepin metabolism pathway, qRT-PCR were initially identified that cordycepin synthesis via AMP→ADP→dADP→dAMP→ cordycepin deoxyadenosine pathway. Preliminarily determined 5’-nucleotidase was the important related gene to the synthesis of cordycepin.Have used the 3’ RACE kit amplificated 5’-nucleotidase gene sequence of 3’ end, the sequence is 1270 bp, and build the 3’ end of prokaryotic expression plasmid. It turn to induce expression in E.coli BL21 and obtained the expressed proteins. Was identified as inclusion bodies. The inclusion body protein was washed and dissolved, then used the gradient dialysis method to refolding. Purified by His Trap TM FF affinity chromatography and obtained high purity recombinant proteins. Recombinant proteins relative molecular mass is 42 kDa. After bioinformatic analysis, the structure contains 13 α-helices and 26 β-strands.The purified protein was emulsified with freund’s complete adjuvant and incomplete adjuvant, to immunize rabbit. In order to prepare the polyclonal antibody of rabbit. With positive serum as the primary antibody, HRP-labeled goat anti-rabbit IgG as secondary antibody, tested the recombinant protein by Western blotting. The purified protein showed a strong reaction with the positive sera. Specific bands appeared at 42 kDa place. Utilized TCA-acetone precipitation method to extract cordyceps militaris mycelium of total protein.The results showed that the positive serum can react with the total protein and the purpose of the strip in 135-180 kDa, but would like further identification.In summary, this experiment through relevant research, screening to the cordycepin synthesis related genes, and laid the foundation for the study of cordycepin metabolic pathway, the expression of related genes at the same time, in order to further study the role of enzymes in the metabolic pathway and regulation mechanism to provide the experimental basis.