Construction Of Recombinant Adenovirus Containing Mechano-growth Factor,24E Gene And Expression In Neural Cells

Insulin-like growth factor (IGF-1) is a physiological endocrine regulator of the circulatory system, which plays its role through modifying the cell proliferation, differentiation and apoptosis in the organism development process. The mature IGF-1is a single basic protein chain composed of70amino acids and with the same structure in all variants. IGF-1gene contains6exons which can produce different mRNA by alternative splicing, thus forming different variants. Each variant is composed of a signal peptide, an IGF-1peptide and an E peptide.Mechano-growth factor (MGF) is a kind of splicing variants from IGF-1, what it is different from IGF-1is that it has a carboxy-terminal extension peptide. With the exon5of the49bP (Rodentia animal is52bP) sequence insertion, resulting in frameshift mutations of exon6, a special E peptide sequence of the C terminal24amino acid residues (MGF-24E) is yielded. Studies have found that MGF can activate muscle satellite cells, promote tissue regeneration, in addition, it showed high expression of MGF in local muscle, bone and nerve tissues under the stimulation of stress or injury. What’s more, the synthetic MGF-24E peptide can also promote a variety of cells proliferation and survival. MGF-24E is the extension peptide which makes MGF different from other IGF-1splicing isoforms. Thereby, the researchers suppose that MGF-24E may be the functional center of MGF. But there is still no strong argument, this issue has become the focus of attention.Several variants of IGF-1gene alternative splicing have the neuro-protective effect and the effect of MGF is more significant. In2004, Aperghis M found that MGF protected lacerated nervous tissue from neuron loss effectively for the first time which had opened a prelude to research the neural repair effect of MGF. In addition, injection of the MGF plasmid to the mice muscle cells suffering from amyotrophic lateral sclerosis could promote the survival of the spinal motor neuron. Recent research showed that, MGF could induce the increase of heme oxygenase expression, protect SH-SY5Y cells against the apoptosis induced by6-hydroxy dopamine. All the results of these studies provide a new idea for the treatment of some nerve injury diseases such as Parkinsonism. As a local factor, the action mode of MGF is independent of other IGF-1variants. Synthetical MGF can activate ERK1/2signal channel without affecting the phosphorylation of Akt, while IGF-1functions through the Akt pathway. Presumably, MGF may be independent of IGF-land play its proliferation role through the ERK signal pathway. As a stress response factors, the membrane receptor of MGF is still not clear and its molecular mechanism needs further exploration.It has made a great progress in nerve injury repair of mechano-growth factor. In view of the past, the means used to research MGF are as follows:inject the MGF recombinant plasmid into the damaged tissue directly or obtain the target protein of MGF by traditional prokaryotic expression and chemical synthesis method. These are simple, but there are still some problems such as low expression of protein, obscure effect, short action cycle and the incomplete function of a synthetical protein, which limit the application of MGF in clinic. In recent years, with the deep research of gene therapy and recombinant vaccine, the basis vector development has made a spurt of progress, adenoviral vector is the most widely used viral vector in gene therapy now. Using the Ad-Easy recombinant adenovirus packaging system, we constructed the recombinant adenovirus of the human MGF and its E terminal peptidc for the first time in this study. We chose the SH-SY5Y and N9as the typical representation of neuron and glial cells, MGF and MGF-24E were over expressed in target cells respectively, which had solved the limitation of the traditional methods. Then we compared the differences on the role of neural cell proliferation of MGF and MGF-24E to expound the neuroprotective effect of mechano-growth factor scientifically, and laid the theoretical foundation for determining the action site of MGF. The following contents are mainly included:1. Ad-Easy packaging system was used to construct the MGF and MGF-24E recombinant adenovirus. After the sequence alignment, we obtained human MGF and terminal24peptide gene through the synthetic method and connected them to the shuttle plasmid pAdTrack-cmv, the recombinant plasmid pAdTrack-MGF and pAdTrack-24E were constructed. After linearized with restriction endonuclease Pme I, the recombinant shuttle plasmid was transferred to the competent bacteria BJ5183with backbone plasmid pAdEasy-1through electroporation, linear DNA can generate stable transformants of BJ5183via homologous recombination between the transforming DNA and regions of homology within the genome of backbone plasmid. Adenovirus plasmids were transfected HEK293A cells by Lipofectamine, we obtained the recombinant adenovirus containing MGF and MGF-24E gene.2. Expression and identification of recombinant adenovirus in nerve cells. After repeated infection and multiplication, the titer of the constructed recombinant adenovirus increased. We used TCID50(50%tissue culture infection dose method) to determine the titer of virus and calculated it by Karber method. The result showed that TCID50(MGF)=10-6.2/100μL; TCID50(MGF-24E)=10-5.3/100μ L, which achieved the required titer for infection. With MGF and MGF-24E recombinant adenovirus infecting N9and SH-SY5Y cells, we observed the fluorescence under the microscope after24h and found that the two kinds of cells can be effectively infected by the packaged adenovirus with the expression of GFP in both cytoplasm and nucleus. After the collection of infected cells, we extracted the cell lysate through the ultrasonic method. SDS-PAGE analysis and Western-blot detection with specific antibody Anti-6*His-Tag, protein bands with molecular weight of20kD and2.94kD were detected in the N9, SH-SY5Y cells infected with MGF and MGF-24E recombinant adenovirus respectively. Besides, the size of which is consistent with the expected value. All the above showed that, both MGF and MGF-24E could be highly expressed in cells of N9, SH-SY5Y with the intermediary adenovirus.3. Effects of MGF and MGF-24E peptide on the proliferation of neural cells. N9and SH-SY5Y cells in logarithmic growth phase were inoculated in96well plates last night. While the cells fusion degree reached about30%, we added the recombinant adenovirus MGF and MGF-24E diluted by the serum-free culture medium to the wells. Each set of three replicates and set the blank control group. Finally, the time and concentration effects on the proliferation of N9, SH-SY5Y cells of MGF and MGF-24E were detected by MTT assay. The result showed that different concentrations of MGF and MGF-24had obviously promoted the proliferation of N9and SH-SY5Y cells. What’s more, with the increase of the adenovirus titer, the role in promoting the cell proliferation turned stronger. Both MGF and MGF-24recombinant adenovirus had the same promoting effect on N9and SH-SY5Y cell proliferation in some concentration. Compared with the control group, the proliferation effect had an extremely significant difference in36h and48h (P<0.01). However, the MGF treatment group and MGF-24E treatment group had no difference (P>0.05). Thus, MGF could promote the nerve cell proliferation and play an important role in repair of the damaged nervous system. While MGF-24E and MGF showed the same biological activity, it is testified that MGF-24E might be the active site of MGF, namely MGF took its effect through the end of the short peptide.