| Human type?collagen is the most abundant collagen in articular cartilage, and it is also a kind of macromolecular proteins. Spiral of collagen, elastin and glycoprotein intertwined to form network structure of human type?collagen. So it has mechanical strength, and it is also the material basis for the healthy skin. Type?collagen is considered to be one of the most important rheumatoid arthritis autoantigen. In addition, type ?collagen is the main part of the transparent cartilage, so it is very useful for cartilage repair,and can be expected to be artificial cartilage material, artificial cornea, materials and other biomedical materials. Collagen production is mainly by means of acidolysis and alkaline hydrolysis or enzymatic extraction from animal connective tissue(pig skin, cattle hide,donkey skin, fish, etc.) at present. Its biological activity is somewhat lost in the process of extraction. Furthermore, the extracted animal-derived products, when applied to the human body, can often produce allograft rejection. This is even more complicated by the very real possibility and danger of hidden viruses. In addition, collagen extracted from animal tissue is water insoluble protein. It is impossible to dissolve without changing the molecular weight or structure of the protein, which has adverse effects for workability and plasticity,thus limiting the applicability of the production of collagen. Although the method of chemical synthesis of collagen can solve the above problem, collagen produced in such a manner usually does lacks the necessary hallmarks of biological activity structures(i.e.triple-helix configurations), and synthesis technology is relatively complex and high-cost.The protein expressed with the heterologous recombinant expression system produced protein that not only had good biocompatible, biodegradable, and cell adhesion characteristics, but also could promote epithelial cell formation and stop bleeding. In addition the workability of collagen was superior to collagen of animal origin and had no hidden viruses, low solubility, or rejection issues.Baculovirus Multigene Expression System is a kind of advanced eukaryotic expression system, which can use baculovirus to express exogenous gene in insect cells or insect body.Ac MNPV, as a kind of ds DNA virus can infect a variety of lepidoptera insects, is used as the carrier of gene expression system. And the Bm NPV system allows for an enhanced level of expression at a lower cost, yielding great potential for the system's future adoption.We use this system to express a full-length gene, human type ? collagen(4,257 bp),in cultured Spodoptera frugiperda 9 cells, Bombyx mori cells, and silkworm larvae. The recombinant plasmid p FBDM-polh-col?-p10-IM was introduced into asd deletion type of E.coli sw106-inv components contained Ac Multi Bac or Bm Multi Bac by Tn7 transposition.So we get recombinant bacterial E.coli r Sw106-Ac-p FBDM-polh-col?-p10-IM and E.coli r Sw106-Bm-p FBDM-polh-col?-p10-IM. The recombinant Bacmid was then prepared and transfected into cultured insect cells, Sf9 or Bm N. When E. coli Sw106 with invasin protein incubated with insect cells, Sw106 can infect the insect cells. Sw106 can not replicate/reproduce under the absence of DAP(a major component of prokaryotic cell walls)and die in the insect cells. Thereby Bacmid is released from Sw106 cells., and the recombinant baculovirus begins to transcript, express and assemblyin the insect cells. Four days post-infection, insect cells transfected with recombinant virus are observed under 543 nm wavelength light(m Cherry). These results indicate that the Ac MNPV-Col?-IM and Bm NPV-Col?-IM recombinants are successfully constructed and that human type ?collagen is indeed expressed in Sf9 cells and Bm N cells. SDS-PAGE followed by Coomassie staining showed the presence of a major band corresponding to the triple-helical structure of type ? collagen ?-chains in Sf9 cells and Bm N cells at 300 k Da. Sf9 cells were purified by Ni-NTA affinity columns. The results demonstrated that 150 m M imidazole elution buffer could elute the target protein, and the band corresponding to the triple-helical structure of type ? collagen ?-chains was measured to be approximately 300 k Da. Early fifth-instar silkworm larvae, infected with Bm NPV-IM-col ?. After 4 days post-infection, the skin of infected larvae was collected and ready for purification.SDS-PAGE followed by Coomassie staining and Western blotting showed the presence of both major bands corresponding to both single and triple-helical ?-chains. Type?collagen?-chains presented at approximately 130 k Da, while triple-helical ?-chains presented atapproximately 300 k Da. The results indicate that our newly-constructed Bm Multi Bac expression system is able to express the human type?collagen in silkworms larvae. In this study, we extracted about 50 mg of biologically active human type ? collagen from 50 infected larvae, indicating the expression level is quite high.An assay of human type ?collagen expressed by silkworm larvae demonstrated that the recombinant protein has considerable bioactive properties. At the same time, scanning electron microscopy of purified proteins clearly reveals randomly distributed and pitted structures. We conclude that the new, more efficient baculovirus-silkworm multigene expression system can coexpression human type ? collagen and m Cherry and it is possible to obtain macromolecular protein with bioactivity through this system. |
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