Genetically Modified And Functional Study Of PDGF-BB Recombinant Silk

Silkworm(Bombyx mori.)is an important agricultual economic insect.Silk fiber was synthesized and secreted by silk gland and regarded as core ingredient in silk industry as well as the fundation of sericulture industry,which is epitomization of the economic value of the silkworm.Recently,the research on silk genetic manipulation technology has developed rapidly.With the completion of the Genome Atlas of Bombyx Mori and the gradual clarity of the mecahnism of the synthesis and secretion of silk protein,the research of producing functional exogenous proteins and improving functional silk materials based on silk gland and silk fiberby using genetic manipulation technology has been epidemic.It would not only to stabilize the current application market of silk,but also to open up a new direction in the application field for silk if it can be given a certain function.Based on the above research background,this study successfully expressed recombinant PDGF protein in silk gland and silk fiber,creating a higher functional silk which can promote cell proliferation.The specific findings are as follows:1.The acquisition and molecular detection of PDGF transgenic silkworm.Human platelet derived growth factor(PDGF)belongs to the fimaly of dimer glycoprotein exsisted in human platelet granules,it has strong chemotactic and mitosis activity which can play an important role in wound healing,vascular remolding and bone repairing.The results shows that: 1)After synthesized of PDGF gene artificially and constructed of silkworm transgenic expression vector,results of Asc? degestion showed that the expression vector piggy Bac[3xp3EGFP,h SPDGFSer1PA] has been successfully constructed;2)Progenies were screened by fluorescence after microinjection of transgenic vectors,5 positive germlines were obtained,and the positive rate was 38%;3)Genomic PCR results showed that the PDGF gene was successfully inserted into the silkworm genome,and the insert site was in chromosome 26 th Bm_scaff 25 determined by reverse PCR;4)The results of RT-PCR showed that PDGF gene was successfully transcribed while the transcription level of endogenous Ser1 gene decreased by 37.5%;5)The SDS-PAGE electrophoresis of the protein sample extracted from the silkworm cocoons of 26 positive individuals silkworm cocoons were carried out,and the specific bands appeared the similiar molecular weight as the PDGF gene.Western blot results showed that the protein can be specifically combined with PDGF antibody and the dimer formation can be detected at the same time.2.Purification and acticity evaluation of recombinant PDGF in transgenic silkworm cocoons.PDGF protein possesses the activity of promoting cell migration and proliferation.In order to verify the activity of recombinant PDGF protein,the cocoon shells of transgenic silkworm were purified and protein activity was detected.The results showed that: 1)Using urea to extract sericin from silkworm cocoons,and the Hi TrapTM Heparin HP column was used for heparin affinity chromatography according to the structure of PDGF protein with heparin binding domain.The yield of this process was approximatly 80%.In order to improve the purity of purified products,using the Hi TrapTM SP HP column combined with the PDGF protein,the yield rate is about 70%.Using Hi TrapTM Q HP column to combine with the impurity protein and collecting fluid flow,the final purity of PDGF protein solution was about 80%.The whole process can obtain 0.2mg recombinant protein from 1g cocoon,and the yield was 50.2%.2)The cells were treated by the medium of 0.5% serum concentration for 12 h and then treated by the purified recombinant proteins and the equivalent standard for 24 h.The results of Ed U and CCK-8 cell proliferation test showed that the purified PDGF protein could maintain the growth of NIH/3T3 cells and promote the proliferation of cells compared with the control group,and its activity was not significantly different from the equivalent standard.3)Using a medium with a serum concentration of 0.5% to starvate the cells for 12 h and scratch the cell surface.Then add the purified recombinant protein and the equivalent standard to treat for 24 h.Compared with the control group,purified PDGF protein can significantly reduce the area of scratches,while the number of cells migrated significantly more than the control group,which indicates that the purified PDGF protein can maintain the growth of NIH/3T3 cells and promote cell migration,and has no significant difference compared with the same amount of standard.3.Functional study of PDGF transgenic silk materials.When the body tissue has been damaged,platelet-releasing PDGF can be used as a mitogen to promote the proliferation of multiple cells and accelerate wound healing.In this study,the PDGF protein was specifically expressed in silk by the silk gland bioreactor,and the transgenic silk was given new biological activity to creating the functional silk material.The research results showed that: 1)Genetically modified silk has no significant difference compared with wild type silk in appearance,fiber diameter,sencondary structure and mechanical properties.2)The PBS buffer can extract recombinant protein from genetically modified silk.Continuous extraction for 20 days,13.7?g PDGF protein has been slowly and continuously released from 90 mg transgenic silk which accounts for about 45.65% of the total recombinant protein content.3)Using a medium with a 10% serum concentration to cultitivate cells for 12 h,the cell grew well after treated by transgenic silkworm cocoon slices for 24 h,and shows less death without cytotoxicity;4)Cells were starvated for 12 h by the medium of 0.5% serum concentration,and the wild type silk cocoon slices,transgenic cocoons slices and the equal amount standard were added for 24 h.The results of Ed U and CCK-8 cell proliferation test showed that compared with the control group,the transgenic silk cocoon slices could maintain the growth of NIH/3T3 cells and promote cell proliferation without any significant difference compared with the equivalent standard products.5)Using the medium of serum concentration of 0.5% to starvate of cells for 12 h,adding wild type silkworm cocoon slices,genetically modified silkworm cocoon slices and the equivalent standard treatment cells for 24 h,Western blot detection results showed that the transgenic silk cocoon slices can activate the ERK pathway without treatment and can promote NIH/3T3 proliferation without significant difference compared with that of the equal amount of standard products.All in all,this study utilizes the silk gland bioreactor and the high efficiency transgenic expression system to obtain the transgenic silkworm strains which can be used to express the active PDGF protein in the silk gland and silk,and genetically improve the functional silk material which can promote cell proliferation.Using the transgenic silk material,it is expected that the preparation of medical wound dressing by this kind of novel silk can promote wound healing and has great application prospect in biomedicine and tissue engineering fields.