| Recombinant spider silk,as a potential excellent material,has a great potential application value in the field of biomedical engineering.Recent research has demonstrated that the material could be used for biomaterials such as veins,nerve conduit and drug carrier.At present,the research of recombinant spider silk protein is mainly focused on major ampullate spidroin(MaSp),minor ampullate spidroin(MiSp)and flagelliform spidroin(Flag).The lack of PySp,especially the PySp of A.ventricosus has not been reported,the biological function remains to be studied.In order to analyze the function of PySp-Rp region,study the effect of C-terminal non-repeating region(CT)on the mechanism of filament formation,test the mechanical properties of the silk fibroin.And finally obtain a kind of recombinant spider silk protein with excellent material properties,which provides the theoretical basis for the bionic silk protein bionic.The following research work is carried out in this project and all the results are satisfactory.(1)The research designs specific primers and Araneus ventricosus genome as a template which has been created by our lab,cloned obtain a spider silk protein coding sequence.By Blast analysis,it was confirmed that the sequence contained a complete repeating region(Rp)of piriform spidroin(PySp).The sequence is A.ventricosus' s first complete sequence of PySp repeats,which filled the gaps in the domestic A.ventricosus silk protein-coding gene clone,adding a new member of silk protein gene to its research.(2)The PySp-Rp domain was recombined with the MiSp-NT/CT domain,established two types of recombinant spider silk protein expression vectors,which are MiSpNT-PySpRp without CT domain and MiSpNT-PySpRp-MiSpC with a complete function domain,so as to process proteins expression and purification.At the same time optimize the expression of the two proteins.Two kinds of recombinant spider silk protein were successfully expressed in E.coli,after SDS-PAGE glue and Western-blot identification.Since the expression of protein were high yield,inclusion bodies were formed.So after purified unde denaturant,obtained high purity of recombinant spider silk protein.(3)Using Circular dichroism(CD)analysis two kinds of recombinant spider silk proteins' secondary structure under different pHs and calculate the percentage change of them secondary structures.Using Fourier transform infrared spectroscopy(ATR-FTIR)infrared spectroscopy for the determination of two kinds of recombinant spider silk protein after lyophilization,the percentage of two grade structure calculation of the amide I band.Comparisoned of two kinds of silk protein secondary structure found that with the lower pH,CT domain will stimulate PySp polymerization in order to promote its silking;It spectra showed that CT domain could increases the percentage of ?-sheet in PySp silk,The results reveal the CT domain could promote assemble into silk fibroin protein that is good for improving PySp silk fiber mechanical properties.Reveals that CT domain can promote the assembly silk fibroin protein and improve the mechanical properties of silk fibers.(4)Using scanning electron microscope(SEM)observe the surface morphology of MiSpNT-PySpRp-MiSpCT recombinant spider silk protein fiber which obtaind by wet spinning.The result shows that the surface of the recombinant spider silk fiber is smooth and uniform in diameter.It is similar to the natural spider silk fiber.But its diameter is slightly thicker than natural spider silk,about 20 um.In conclusion,the study not only filled in the gap of the coding region of the repeat gene of PySp in the garden,but also further studied the mechanism of CT on silk filament mechanism.It is necessary to further explore the mechanical properties of the recombinant spider silk fiber by increasing the shear force and other conditions.The study provides a new theoretical basis for the future bionics of recombinant spider silk protein. |
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