Stems From Japanese Lamprey Gene Recombinant Mutant Protein RLj-112 Of Antimicrobial And Antitumor Function Research

Lj-RGD3 is three RGD functional protein come form of salivary gland japanese lamprey japonica which is named RGD toxic protein.Molecular weigh of Lj-RGD3 is 13.0 kD.Lj-RGD3 have the function of antiangiogenic,inhibit platelet aggregation,induce the apoptosis of tumor cells.The main mechanism of Lj-RGD3 is three RGD sequence combined with cell surface of integrin,and closed signal regulation between the extracellular and cells to inhibit the process of cell growth,proliferation,migration,transport.Lj-112 is a mutant of Lj-RGD3 which is deleted all of RGD motifs.Lj-112 contains rich-histidine which is rich-histidine protein.Reference data were consulted rich-histidine protein have have the function of antimicrobial activity,inhibiting tumor blood vessels,immune regulation,antithrombotic.Because of RGD toxic protein have nothing to antimicrobial activity,so taking rLj-RGD3 and rLj-26(lost three RGD and all His)as the control protein,the antibacterial function of Lj-112 is being studied.At the same time,this article also added to study the antitumor function of Lj-112 the further enrich the research contents of this subject.Through gene synthesis and prokaryotic expression system,we are obtain form histidine affinity chromatography purification of rLj-RGD3 protein,rLj-112 protein and rLj-26 protein.SDS-PAGE identification results show that molecular weigh of rLj-RGD3,rLj-112,rLj-26 is respectively 10.2kD.Sequence analysis show that rLj-112 with histidine rich glycoprotein and antimicrobial peptides have high homology.Using enzyme standard instrument results show that rLj-112 have strong killing effect to staphylococcus aureus with rLj-RGD3?rLj-26 as control.Using dilution coating method is used to prove that rLj-112 could significantly inhibit the growth of candida albicans with dose and time dependent,the inhibition rate was 100%.Using tablet confrontation method is used to prove that rLj-112 have antagonism effect on the different pathogenic fungi.Using optical microscope and electron microscope is used to observe that fungal hyphae became weak and the abnormal state after rLj-112 treatment.Oxford cup method is used to prove that temperature have no effect on antibacterial activity of rLj-112,metal cation reduce antibacterial activity of rLj-112.Using MTT assay is used to prove that rLj-112 could inhibit the proliferation of hela cells.Using transwell assay is used to prove that rLj-112 could inhibit the migration of hela cells.Giemsa's and FITC-phalloidine dye method observe show that after compared with the control group,the rLj-112 make bright blue fluorescence of Hela nuclei,cytoskeleton framework.Using the Swiss-Jim dye test show that after the treatment of rLj-112,Hela morphological changes,cell nucleus density increases,the corpuscle in apoptosis,cell death.Furthermore,the DNA ladder and flow cytometry instrument test show that rLj-112 could induce apoptosis of hela cells and DNA fragmentation.Conclusion: rLj-112 has the characteristics of broad spectrum antibacterial and antitumor activity,for subsequently functional protein of drug development laid a solid foundation.