Study On Expression Of Recombinant HVEGF165 In The Middle Silk Gland Of Transgenic Silkworm

The silkworm?Bombyx mori.?,is an important silk-producing insect,has been raised and utilized by humans for more than 5 000 years.The silk gland of silkworm is a special place to synthesize and secrete silk protein?one commercial B.mori larvae can produce 0.5 g pure silk protein?,which is the core biological basis of silk industry.In recent years,with the vigorous development of biotechnology,especially with the promotion of completion of the silkworm genome project,the unique phenomenon of fast synthesis and secretion of silk proteins in silk gland of silkworm has been extensively elucidated,valued and re-realized that silk gland not only can be used for silk production,but also can be used as a bioreactor.Since the application of piggyBac transposon-mediated transgenic techniques in the silkworm and the first successful expression of recombinant human type III procollagen in cocoons of transgenic silkworms,dozens of recombinant proteins have been successfully expressed in the silk glands of transgenic silkworm.Indeed,a small number of recombinant protein materials have entered the stage of medium-term test or industrial trial production.The production of recombinant protein from silk gland of silkworm as bioreactor has great potential for development and application.The endothelial cell-specific vascular endothelial growth factor?VEGF?is a major mitogen that contributes to normal vasculogenesis and angiogenesis.hVEGF165 is not only the most frequently expressed isoform in tissues,but also the most physiologically relevant isoform,remarkable effect on therapeutic vascular regeneration in peripheral ischemic disease or chronic wound.At present,the recombinant hVEGF165 protein is mainly expressed and studied in vitro by prokaryotic expression system and eukaryotic expression systems:escherichia coli and yeast.It is not clear whether it is suitable for the expression of middle silk gland of transgenic silkworm and whether it has biological activity.Therefore,the aim of this paper is to explore the feasibility of expressing hVEGF165 protein in the middle silk gland of transgenic silkworm by using hVEGF165as a target and to provide theoretical reference and transgenic material basis for further development of recombinant hVEGF165 protein.The main findings obtained are as follows:1.Generation of hVEGF165 transgenic silkwormFirst of all,the sequence of hVEGF165 gene?np₀01020539.2?was downloaded from NCBI and the optimized hVEGF165 gene with a silkworm codon bias fusing a His6 tag in the C terminus.Secondly,the synthesized sequence was inserted into the subclone vector pSL1180[hSer1sp-DsRed-Ser1_PA]with the promoter of sericin-1 gene?sericin-1,ser1?as the regulatory element,and then was inserted into the piggybac skeleton vector by enzyme digestion method.The transgenic expression vector pB[hSer1sp-hVEGF165-Ser1PA,3×P3EGFP]?abbreviated as pBhSer1VEGF165?was constructed.Finally,the ultra-pure plasmid DNA of pBhser1VEGF165was injected into the early embryos of silkworm by microinjection,and five EGFP-positive brood was obtained and used to establish the transgenic line.The results of genomic PCR and sequencing showed that the hVEGF165 transgenic silkworm was successful and named S1-V165.2.Expression analysis of hVEGF165Western blot and qRT-PCR were used to detect the middle silk gland and cocoon shell of the fifth instar larvae of hVEGF165 transgenic silkworm.The results of qRT-PCR showed that the expression of hVEGF165 was significantly increased in the middle silk gland of the fifth instar larvae,and the expression level was increased gradually from the day 1 to day 6 of the 5th instar.The transcription level of hVEGF165 was about 56%of Ser1 mRNA at the 6th day of 5th instar.Subsequently,the recombinant proteins from silk gland and cocoon were extracted and analyzed by SDS-PAGE and Western Blot.The results showed that recombinant hVEGF165 protein was successfully synthesized and secreted into cocoon shell.3.Cell proliferation analysis of recombinant hVEGF165 proteinThe dimerization of hVEGF165 is the basis of its bioactivity.Cocoon powder was dissolved with PBS,8 M Urea and alkaline solution?AS,5g/L Na₂CO₃,4g/L NaHCO3?respectively,and then detected by western blot.in the presence of?ME,rhVEGF165 existed mainly in monomers in the samples.In the absence of?ME,rhVEGF165 mainly existed in dimeric form.In brief,these results indicated that rhVEGF165 is dimerized in the cocoon of S1-V165,and AS seems to be an efficient solvent for dissolving dimerized recombinant hVEGF165 from transgenic cocoons.Subsequently,the concentrated protein solution was sterilized and co-cultured with the starved cell.After 24 hours,the biological activity of recombinant hVEGF165 protein was analyzed by CCK-8kit and EdU staining respectively.The CCK-8 results suggested that cells treated with recombinant hVEGF165 or hVEGF165 standard exhibited increased absorbance at 450 nm compared with the control group and EdU incorporation results showed that HUVECs treated with recombinant hVEGF165 or hVEGF165 standard exhibited strong RFP fluorescence signals compared with the control group,Taken together,these results indicated that recombinant hVEGF165 significantly stimulated HUVECs proliferation,and its cell proliferation activity is even better than that of the hVEGF165 standard.Finally,the cells were treated with recombinant hVEGF165 proteins for 0,5,15,30 min respectively,and the results of western blot showed that ERK1 and ERK2phosphorylation levels induced by recombinant hVEGF165 at four time points were all increased compared with the control group,which further confirmed the bioactivity of recombinant hVEGF165.4.Release characteristics of recombinant hVEGF165To investigate the release characteristics of recombinant hVEGF165 protein from cocoon powder,we determined the release profiles of the AS supernatant by Western blot.The release behavior of recombinant hVEGF165 protein show that an initial burst of approximately 80%after one day.Thereafter,the release was sustained through the end of the 3-day experiment.5.Cell Proliferation activity of S1-V165 slik sliceDried and sterilized silk slices were used to culture Human umbilical vein endothelial cells by immersing directly in the medium containing 10%?v/v?FBS co-cultured with human umbilical vein endothelial cells for 24 hours.The morphology and growth state of the cells were good,and there was no obvious death and cytotoxicity compared with the control group.Afterwards,HUVECs starved in 100?L of DMEM containing 0.5%?w/v?FBS for 12 h.the WT and transgenic thin slices of cocoon shell were then each immersed in the DMEM on the surface of the HUVECs.After cultivation for 72h,the proliferation of HUVECs was measured by using CCK-8 kit.The result show that cells treated with the S1-V165 silk slice grew with the typical morphology of HUVECs and the absorbance at 450 nm was significantly higher than that of the WT.In this study,piggyBac transposon-mediated transgenic silkworm was successfully obtained by microinjection,fluorescence screening and molecular detection,and the recombinant hVEGF165 protein was specifically expressed in the middle silk gland of silkworm.The biological activity was determined at the cell level,which provided a theoretical basis for the follow-up clinical development and application.