| Dragline silk produced from spider’s major ampullary gland has excellentmechanical properties, is one of the best natural biological material in nature from aoverall performance, which has high strength, high elasticity and high work offracture characteristics, intensity5times stronger than steel, tough3times of Kelvarfibers. Under extreme conditions, such as low and high temperatures, it still hasexcellent performance. Based on these unique physical and biologicalcharacteristicsspider dragline silk presents great ranges of promising applications inmedicine, materials, and military. However, the nature of the spider decided that it isnot practical to get large amount of spider silk proteins by mass rearing. Using geneticengineering methods, heterologous expression of recombinant spider silk protein hasbecome an effective way to obtain large-scale spider silk protein.With examination and study of the composition, structure, properties andforming principle of natural spider silk through the application of modernbiotechnology and polymer chemistry, it has been proposed a novel proteinengineering approach, which combines the structure domain of synthetic repetitivesilk protein sequences and reliable spider silk. Protein obtained by means of geneticengineering is extremely similar to the spider silk and has a high yield, which couldbe applied in the production of bionic spider silk protein. This has provided the basisfor cost-effective mass production of spider silk analogs.In genetically engineered spider silk protein core sequence studies, the currentstudy has clearly understood the categories of repeated motifs of spider silk proteins,basically can be summarized as alanine-rich motif (An/(GA) n), GPGXXpentapeptide motif, GGX motif, and a spacer (Spacer) four motifs, these fourcharacteristic motifs form a certain structure to perform specific functions. Alanine-rich motif which is formed β-pleated sheet, to provide strength to the wire; GPGXXpentapeptide motif angle β-helix is formed to provide high flexibility for spidersilk.Structural basis based on the excellent characteristics of natural spider silkprotein is highly conserved sequence that is consist of four simple repetitive aminoacid motifs, you can take the key functional units to get repetitive splicing genetic engineering of spider silk protein coding gene sequences. Currently, the world iswidely used these highly conserved functional unit combination splicing, to achievefull-length construct genetically engineered spider silk sequences and get the syntheticspider silk whose performance close to natural spider silk. Thus, by way of generecombination, the construction of splicing of the core units in series is effectivegenetically engineered spider silk generic coding gene cloning strategies, which havebeen proven through research.In the use of prokaryotic expression of genetically engineered spider silk proteins,microbial metabolic engineering technology can transform Escherichia coli so that itcan be close to the natural size of the high-level expression of high molecular weightproteins genetically engineered spider silk, thereby expressing genetically engineeredspider silk spider silk fibers which is closer to the natural spider silk.Combine the progress and problems domestically and internationally in syntheticspider silk gene engineering research progress, this thesis research work carried out inthe following five areas:Part One: Design studies on genetic engineering spider silk proteinmoleculesThe part of the study firstly retrieved core structural unit of geneticallyengineered spider silk proteins which have been reported internationally, from whichto select genetically engineered spider silk core sequence of natural spider draglinesilk protein core sequence of a representative by computer-aided molecular modeling,simulation the possible conformation, thus pick out a structure in the space closest tonatural spider silk protein sequences; then according to E. coli codon triplets, selectspider silk core sequence corresponding to E. coli codon bias to design a geneticallyengineered spider silk core gene coding sequence applies to the E. coli expressionsystem; built in the last restriction enzyme sites required core sequence of the seriesbased on splicing and prokaryotic expression vector, respectively, at both ends of thecore sequence plus cloning restriction sites, and in the sequence ends with a protectivebase.Through the analysis and design, to obtain a135bp gene engineering lengthspider silk core gene sequence to the sequence as a monomer, a continuous series by asubsequent splicing, spider fibroin can get close to the natural sequence due to thelength of the target gene, so that the genetic engineering excellent performance spidersilk has a natural spider silk possible. Part Two: Study research on splicing and vector construction of geneticengineering of spider silk carrier core sequence.Genetically engineered spider silk to obtain recombinant vector is an importantbasic work carried out in this project, the key properties of genetically engineeredspider silk is the final decision to build close to the size of the series of natural spidersilk gene splicing by molecular biology techniques.This part of the study we used the same method to connect the tail enzymegenetically engineered spider fibroin tandem splice sequence due to pBSK1syntheticsequence based company, respectively Xma I/Sca I and BspE I/Sca I digested,digested recovery fragment, T4ligase fast connection, transformed into DH5αcompetent cells after Amp resistance screening recombinant cloning vectors andrecombinant enzyme digestion, get2times series recombinant clones, repeat theprocess on the basis of such doubly core sequence followed, ultimately,96recombinant clones series; for all recombinant clones were digested identificationpurposes to confirm the inserted gene fragment size is correct.This part of the study using genetic engineering techniques and molecularbiology, genetic engineering establishment of spider silk protein coding sequence ofthe gene-splicing techniques and cloning core vector technology system. Theobtained genetic engineered spider silk cloning vector has been proved that theinserted sequence is correct by the identification of restriction endonuclease reaction.The acquirement of above cloning vector provides basis for the following prokaryoticexpressiongenetically engineered spider silk.Part Three: Expression and identification of genetically engineered spidersilk proteinThis part of the experiment transformed prokaryotic expression vector into BL21(DE3) competent, and after obtaining engineering bacteria, culturedOD600to about0.6under30℃, added IPTG to a final concentration of1mmol/L, induced after6hours, prepared whole cell lysis supernatant, then identified the expression of thetarget protein with10%SDS-PAGE electrophoresis and protein immunoblottingexperiment.The results showed that: Despite related protein expression was not detectedinempty vector control pET28a (+), the construction of genetically engineered spidersilk protein expression vectors were detected with purposeful expression ofrecombinant protein, and the product sizes were relatively consistent with the expected molecular mass. In addition to these specific target proteins, other positivetruncated protein bands existence was not found, which explained that thephenomenon of early translation terminated expression and gene deletions did notoccur. This part of the study to achieve a breakthrough in genetic engineeringprokaryotic expression of spider silk the key technologies for the next biomimeticspinning genetically engineered spider silk protein provides an important rawmaterial.In summary, this study employed a haploid core sequence that is a geneticallyengineered spider silk protein sequence that contains varieties of good traits such aselasticity module and strength module. Mastered the natural spider silk protein-codinggene length genetically engineered spider silk expression vector construction methods;explored the conditions of high density fermentation engineering bacteria to expressthe target protein; established the separation and purification of genetically engineeredspider silk protein key technology system, to carry out these key technologies for thedownstream of genetically engineered spider silk spinning Physical and chemicalproperties of silk and analysis technology to provide the necessary prerequisite andguarantee, the next job will focus on the construction of genetically engineered spidersilk protein biomimetic spinning platform, allowing genetically engineered spider silkprotein biomimetic spinning key technology to break through. Establish these keytechnology breakthroughs and technological system and the development ofapplications for traction spider silk protein series of new genetically engineeredbiomaterials in the field of biological material foundation. |
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