Constrction And Effect For Fusion Gene Expression Vector Of Myostatin Propeptide And Antiviral Peptide

Myostatin(MSTN) acts as a negative regulator of skeletal muscle growth and is almost expressed exclusively in skeletal muscle. The biological function of myostatin will be inhibited by the combination of myostatin propeptide (MSTN pro) and thus the muscle growth will be promoted. EB peptide and CV-N protein are antiviral peptides with broad spectrum antiviral activity. The experiments aim to construct the fusion gene expression vector of myostatin propeptide and antiviral peptide and develop the genetic engineering drugs which can not only promote growth but also resist virus. It has important scientific and practical significance.The sequence of pig myostatin propeptide was amplified from the pEASY-T3-MSTN vector, and it was further subcloned into pcDNA3.1(+) vector, which was named pcDNA3.1(+)-MSTN pro. In order to construct the fusion gene expression vector, the linker was added to the primers, and the target gene sequence containing linker was amplified from the pEASY-T3-MSTN vector, then it was further subcloned into pcDNA3.1(+) vector, which was named pcDNA3.1(+)-MSTN pro-L. According to the amino acid sequence of EB peptide, the gene sequence of EB was deduced by DNAman and synthesized by the company,then it was further subcloned into pcDNA3.1(+)-MSTN pro-L vector, which was named pcDNA3.1(+)-MSTN pro-L-EB. According to the NCBI GenBanK (the accession number:DQ668406.1), the cyanobacteria antiviral protein N gene sequence was synthesized by the company, and it was further connected with pcDNA3.1(+)-MSTN pro-L vector, which was named pcDNA3.1(+)-MSTN pro-L-CV-N. Electrophoresis and sequencing showed that the vectors were successfully constructed.The constructed vectors including pcDNA3.1(+)-MSTN pro, pcDNA3.1(+)-MSTN pro-L-EB, pcDNA3.1(+)-MSTN pro-L-EB and pcDNA3.1(+)were transfected into C2C12mouse myoblasts. Thus MSTN pro group, MSTN pro-L-EB group, MSTN pro-L-CV-N group and pcDNA3.1(+) group were established respectively. At the same time, a non-transfected vector group was designed. The following three tests were conducted in this way.(1) Test for the expression effect of vectors:After transfecting48hours, the mRNA expressions of MSTN pro, MSTN pro and EB Fusion Gene, MSTN pro and CV-N Fusion Gene were detected through QRT-PCR. The results showed that compared with non-transfected group and pcDNA3.1(+) group, the MSTN pro mRNA expression in MSTN pro group was increased very significantly. Compared with the non-transfected vector group, the MSTN pro-L-EB mRNA expression in pcDNA3.1(+)-MSTN pro-L-EB group was increased significantly, and the MSTN pro-L-CV-N gene mRNA expression in pcDNA3.1(+)-MSTN pro-L-CV-N group was increased very significantly. These results showed that the three vectors were successfully transfected and expressed.(2) Test for the cell proliferation assay:After transfecting48hours, the effect of each transfected group on C2C12cell proliferation was detected through cell count under the microscope. The results showed that compared with non-transfected vector group, the number of cells in other four groups were significantly reduced. But compared with pcDNA3.1(+) group, the number of cells in the groups of MSTN pro, MSTN pro-L-EB and MSTN pro-L-CV-N were increased, particularly the number of cells in the group of MSTN pro was increased significantly.(3) Test for the anti-virus detection:After transfecting48hours, CSFV and PRRSV were added into the corresponding groups respectively. After4hours, the situation of apoptosis in each group was tested by Annexin V/PI double staining method. The results showed that EB peptide had antiviral activity against two kinds of virus under the same conditions. However, Cyanobacteria antiviral protein N had antiviral activity only against CSFV and stronger than EB.