The Heterogenous Production,Fiber-formation Mechanism Of A New Type Chimeric Spidroins And The Spinning,Applications Of Artificial Spider Silks

It is well known that spider silks not only reveal excellent mechanical properties but also have excellent biocompatibility.Furthermore,the mechanical properties of these natural materials are even better than natural silk from bombyx mori.Therefore,it is considered that spider silks have extensive application prospect in aerospace,military,clothing,biomedical materials and some other fields.However,as predators,spiders are difficult to keep in captivity and spinning extremely low yields of fibers.So,it is necessary to seek other ways for producing mass amounts of spider silk proteins?spi-droins?and spinning high-performance spider fibers.Recent years,as spider silks have been more and more concerned,a lot of research production about the structure,formation,production and application of these natural materials has been carried out.So far,exogenous expression,particularly prokaryotic expression,is the most common way in producing recombinant spidroins and obtaining spider fibers.However,the large,complex structure and highly repetitive sequences of spidroin genes impose high metabolism burden to hosts,and result in the unpredictable expression.Additionally,due to the spinning mechanism of spider silk is still not un-raveled,the artificial spider fibers formed through conventional wet-spinning way re-veal much weaker mechanical properties than natural spider silk.Since uncertain pro-duction of recombinant spidroins and the immature artificial spider silk spinning tech-nique,industrial mass production of high-performance spider fibers has not been achieved yet.In this dissertation,a novel type of chimeric spidroin sequences has been designed.Systematically studying of the new type spidroins provided a new idea to produce mass amounts of spidroins in prokaryotic hosts and filled the blank in the domain of spider silk formation process.Meanwhile,the research also evaluated the potential application for tissue engineering and explored the technical route for large-scale production of spidroins and spider silk,laying a steady foundation for large-scale production of high-performance spider silk.Part 1.The production,structure and spinning mechanism of new type chimeric spidroins.Spidroins usually consist of three main parts;two conserved terminal do-mains?NT&CT?and a core repeat domain.The proportion and arrangement of amino acid sequence of the repeat region are characteristic for each spidroin type.Several studies have been undertaken to investigate the fiber formation process of spidroins.However,these researches could not rule out the potential effects from co-evolution of the three spidroin domains because all recombinant spidroins were fused with similar NT,CT and repeat domain.Therefore,fusing NT,CT and repeat modules from three different spider species and spidroins into chimeric spidroins can effectively avoid the interference of three domains co-evolution.Additionally,to overcome the difficulties in prokaryotic expression of spidroins,I chose NT from Euprothenops australis,CT from Araneus ventricosus,MiSp1 to 1⁴ repeat modules?W?from Argiope trifasciata,AcSp1 to construct triple chimeric spidroins NTW_1-4CT.As these novel spidroins carry two highly soluble terminal domains,the solubility and expression level of spidroins sequence in prokaryotic hosts?E.coli?was greatly improved.Besides,just like some other artificial spidroins,the solubility and yields of NTW_1-4CT were correlated nega-tively to the amounts of W modules.Analysis of spidroins showed that,although the NT,W,and CT modules are relatively distantly related,they still retain their respective characteristics and functions.NT and CT modules can also regulate conformational changes of repeat modules in a fiber formation process based mainly on shear forces,thereby improving mechanical properties of manual-drawing spidroin fibers.In this chapter,the results revealed that suitable terminal domains of spidroins could mediate high prokaryotic expression level and solubility of chimeric spidroins.It also showed that characteristics and functions of NT,W,and CT domains in spidroins were inde-pendent of any potential influences from co-evolution.This research provides new points to design suitable recombinant spidroins for prokaryotic expression,and lays a foundation for conventionalizing spidroin fibers with specific mechanical properties.Part 2.Mimicking the spider silk gland and duct to spin chimeric spidroins.Since manual-drawing chimeric spidroin fibers displayed low output and unstable properties,they cannot be produced on a large scale.Furthermore,conventional spidroins wet-spinning ways usually need highly volatile organic reagents,such as hexafluoroisopro-panol?HFIP?,formic acid and methanol.These spinning technologies were not only harmful to the environment,but also different from the forming process of natural spi-der silk.As a result,the mechanical strength of the recombinant spidroin fibers was much weaker than natural spider silk.Hence,in this chapter,by mimicking the structure and internal environment of the main ampulla silk gland and duct of Nephila clavipes,I built a corresponding wet-spinning platform and sought the biomimetic wet-spinning conditions.Through the biomimetic spidroins wet-spinning way,NTW₁CT spidroins were spun into biomimetic fibers successfully.Compared with the fibers formed by conventional wet-spinning way,the biomimetic fibers NTW₁CT-BNB?pH 5.3,pH 5.6?displayed smoother surface and denser cross-section structure.NTW₁CT-BNB?pH 5.6?fibers also showed higher mechanical strength and Young's modulus when compared with hand-drawing NTW₁CT fibers and conventional wet-spinning fibers NTW₁CT-DCB.Nevertheless,toughness and elongation of NTW₁CT-BNB?pH 5.6?fibers were decreased.Compared with the secondary structure of hand-drawing NTW₁CT fibers,the?-structure content of NTW₁CT-BNB?pH 5.6?fibers were increased,but the per-cent of?-helix were greatly decreased.It might cause the mechanical properties of NTW₁CT-BNB?pH 5.6?fibers differed from other fibers formed by the same spidroins.However,the structure of NTW₁CT-BNB?pH 5.6?fibers was still very different from natural aciniform silks.Therefore,although both mechanical strength and Young's mod-ulus of biomimetic fibers NTW₁CT-BNB?pH 5.6?is relatively higher than other fibers,it is still necessary to research the structure and inner-environment of aciniform glands and ducts in the future.It seems that mimic the natural aciniform silk formation condi-tion is an essential way to produce high-performance chimeric spidroin fibers.Part 3.Using bombyx mori silk-gland bioreactors to obtain chimeric spider silk in one step.From ancient time,sericulture is the principal way to obtain natural silks.The silk glands of Bombyx mori are ideal bioreactors to produce proteins as they have the advantages of low cost,high safety,and easily realizing mass production.Besides,the structure of spidroins is similar with silk fibroins.Hence,it is highly feasible to express spidroins in silk glands of Bombyx mori.In recent years,domestic and foreign research-ers have conducted a series of studies on the production of spider fibers by transgenic silkworms,but all of them were concentrated on MaSps and MiSps.In this chapter,CRISPER/Cas9 gene editing technology was used to obtain transgenic silkworms which could express chimeric spidroins with core repeat domain from AcSp1.Based on the previous donor plasmid framework,I designed and constructed donor plasmid pMD-18-S-NTW_1or4CT and prepared corresponding single-stranded donor templates?ssODN?.Then ssODNs were mixed with sgRNA1,sgRNA2 and Cas9 plasmid and were injected into silkworm eggs by a microinjector.sgRNA and sgRNA2 could rec-ognize the upstream and downstream sequences of silk fibroin heavy chain protein?Fib-H?gene respectively,and guide Cas9 to create double strand breaks in the specific lo-cations.After that,chimeric spidroin genes were integrated into the genome of silk-worms by homology-directed repair?HDR?mechanism and replaced the Fib-H gene.The transgenic silkworms finding through report gene could stably inherit NTW₁CT and NTW₄CT spidroin genes.Among them,NTW₄CT spidroins could be expressed by silk gland reactors of silkworms and formed hybrid silk in the silkworm cocoons.These results confirm that the spidroins containing repeat module from AcSp1 could also be expressed by transgenic silkworms,which makes it possible to prepare chimeric spi-droin fibers in a large scale.Part 4.The promising application of electrospun spidroins/PLCL scaffolds for vas-cular tissue engineering.Poly?L-lactic-co-?-caprolactone??PLCL?is one type of ma-terials that commonly used in electrospinning process.The electrospun PLCL nano-fibrous scaffolds performed great mechanical properties.However,due to the surface of PLCL scaffold is highly hydrophobic and lacking of cell binding sites,its cytocom-patibility is low.In this chapter,PLCL were blended with new chimeric spidroins NTW_1⁴CT to obtain a new type of electrospun nanofibrous scaffolds NTW_1⁴CT/PLCL?25/75,w/w?.The appearance,structure,and material properties of blended scaffolds were analyzed.Results showed that the blending with spidroins in-creased the conductivity and reduced the viscosity of spinning dopes,which caused the smaller average diameters and higher porosity of blended nanofibers scaffolds than pure PLCL ones.With the number of W modules increasing,mechanical properties of NTW_1⁴CT/PLCL?25/75?scaffolds were improved.Among them,NTW₄CT/PLCL?25/75?blend scaffolds,which contained the spidroins consisted of NT,CT and four W modules,showed much stronger strength than pure PLCL scaffolds.Additionally,in electrospinning process,the secondary structure of chimeric spidroins changed from a significant?-helix structure to an?-helix/?-sheet mixed conformation.The transfor-mation process was similar to the formation process of natural spider silks.Also,this process might make the chimeric spidroins containing three or more repeat modules can form hydrophilic structures close to natural spider silks,while those only with one or two cannot.As a result,the surface of NTW3⁴CT/PLCL?25/75?blend scaffolds changed from hydrophobic to hydrophilic.In vitro experiments,human umbilical vein endothelial cells?HUVEC?cultured on NTW_1or4CT/PLCL?25/75?blend scaffolds showed much better proliferation and adhesion than that on pure PLCL scaffolds.More-over,due to the moderate hydrophilicity surface of NTW₄CT/PLCL?25/75?blend scaf-folds,they exhibited the highest cytocompatibility.All results suggested that spi-droins/PLCL blend nanofibrous scaffolds,particularly NTW₄CT/PLCL?25/75?blend scaffolds have potential applications in vascular tissue engineering fields.