| Spider silk as an extraordinary natural material with high molecular weight has intrigued researchers and scientists from various fields.Not only does it possess great application potential in the field of biomedical engineering,but it also plays an outstanding role in scientific fields related to physics.For instance,it's reported that spider silk had been used to manufacture super lenses.Realizing the fact that applications of the natural fiber are broad and ubiquitous,scientists are devoted to decoding its secret and unearthing effective approaches to produce it in artificial ways.Benefitting from advancement of biological technologies,recombinant spidroin could be produced through heterogeneous organisms.Up to now,there were several reports on pyriform silk due to its lower production in nature and its mixed form with other spidroins.Therefore,there are no available mechanical data on this kind silk.In this study,a fragment of DNA of pyriform spidroin repeat unit was exploited to produce recombinant spidroin by using molecular biology technology.Continuous and fine fibers were obtained with the wet-spinning techniques.The DNA fragment of repeat unit in pyriform spidroin screened in our lab composes of 213 amino acid residues composed of two specific repeat motifs,PXPXP and QQ.The fragment was exploited as a template.Four different PySp duplicates were amplified by PCR with different primers,and the PCR products were spliced together with the help of seamless cloning technology.Finally,the recombinant PySp genes were sub-cloned into the modified expression vector.Three plasmids were successfully obtained to express recombinant spider silk proteins containing 1 to 3 tandem repeat units.The previous spidroins,including R,2R and 3R could be expressed in E.coli BL21(DE3)after adjusting the condition for expressing proteins.SDS-PAGE showed that the recombinant spidrons existed in inclusion body.Spidroins with purity over 90% were obtained under denature condition.The three recombinant spidroins are insoluble in water but soluble in HFIP.To date,various spinning methods suitable for recombinant spidroins are feasible,including manual spinning,electrospinning and wet spinning.In this study,three recombinant fibers were gathered by continuous stretch using wet spinning apparatus.The results of nano tensile test showed that R,2R and 3R exhibited different mechanical properties.The R performance reached 100.21 MPa,exceeding the previously reported mechanical properties of recombinant spider silk.It,however,possesses low extensibility.2R exhibited good tensile strength and ductility which was 153.92 MPa and 68%,respectively.3R showed excellent extensibility,reaching 132% which was far more than that of reported synthetic silk fibers.However,its tenacity was at 67 MPa,which was relatively weak.Different spinning parameters affect the mechanical properties of the fiber.With optimized spinning conditions,it was expected to obtain better synthesis fibers of these spidroins in future.The results of FTIR indicated that there was no significant difference among secondary structures(?-helix,?-sheet,random coil and?-turn)of the three recombinant spidroins.The fraction of the secondary structures is more than 20%.Apparently the result differs from previous researches on recombinant spidroins.Further analysis uncovered that the fraction of ?-helix of each of the three spidroins was around 25%.To some extent,this results confirmed that these fibers were native-like ones at molecular scale.This research obtained data of mechanical properties of PySp for the first time,and paved the way for pragmatic applications of PySp in the future. |
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