| Basic fibroblast growth factor (bFGF) was first obtainded from bovine pituitary and nerve tissue by Gospodarowicz in 1974. Because of its stimulus of 3T3 cells proliferation it was denominated fibroblast growth factor. It is sensitive to acid and heat, and its isoelectric point is alkalinous, so it is also called basic fibroblast growth factor. hbFGF with more than 38kb length locates on 4q25 of human being chromosome and it is made up of two introns and three exons.The distribution of basic fibroblast growth factor (bFGF) in vivo is very broad, they can be found in brain, heart, bone, eyes, adrenal gland and placenta etc. Cells cultivated in vitro, such as vascular smooth muscle cells, vascular endothelial cells, oligodendrocytes can also generate bFGF, so can some tumor cells including chondrosarcoma cells and neurospongiom cells etc. It has been proved that bFGF is a cytokine with multi-biological activities both in vivo and in vitro.bFCF which is a broad-spectrum mitogen and inducing factor of morphogenesia and differentiation, has evident chemotaxis to cells derived from mesoblastic and neuroectoderm. Studies have proved that bFCF is an angiogenesis factor, with the fuction of promoting wound healing, tissue repair, and certain cell regeneration. Its biological functions mainly are: encouraging neovascularization, helping damage repairing of soft tissue, cartilage, bone tissue and nerve tissue, and also promoting extremity regeneration.hbFGF is found widely in human tissues, its function as neurotrophic factor and cytokinin in clinical therapy has been proved, and it also shows a new therapeutic way of many incurable diseases such as alzheimer's disease, cerebral ischemia, nerve injury, nerve deafness, degeneration of optic nerve etc. hbFGF has a significant effect on clinical therapy of soft tissue injuries like trauma and empyrosis, especially facilitate to the healing of bedsore, ulcer and surgical trauma. It may become the drug to cure myocardial infarction, osteoporosis, alzheimer's disease and other diseases. Now, bFGF has been applied in surgery, cardiovasology, neurology, dentology, geratology etc, and we can see in the near future, it would be widely applied in spinal marrow transplant, vascular prosthesis construction, autoallergic epidermis cultivation and microsurgery.The praeparatums used in the market are proteins expressed from Escherichia coli. Pichia Pastoris express system which is widely used in genetic engineering with properties of simple operation, distinct genetic background, and lower cost price. The system has eukaryotes' folding and secreting mechanism. It can accomplish the post-translational modification process including proteolysis, folding, glycosylation etc which is the properties of eukaryotes. Also, it has rapid growth, requires simple nutritional ingredient, and can accommodate the needs of large-scale industrialization. So we build an expression system of recombinant bfgf and seek to produce recombinant proteins with genetic engineering technology. But it is very complex in technics and high in cost price. However, recombine hbFGF gene and express it in the plants is an effective way to solve above mentioned problems. What is more, it is a new important method to produce crude drugs via the transformed hairy root cultivation. Soybean as a common field crop has a high propagation coefficient and extensive application. It possesses the whole eukaryotic expression system in charge of genetic transcription, translation and regulation, also can do posttranslational processing such as glycosylation, amidation and phosphorylation to expression products which are stored in some organelle or secreted outside the cell. It is productive and quality stable, less effective to the plant growthThe advantages of hairy root culture technology make people pay more and more attention on the study of hairy root culture to produce natural drugs. There are many prominent advantages of hairy-root culture, as follows: (1) Quick growth. HuangJuhui etc had demonstrated mustard transformed hairy root grew 100 times quicker than non-transferred root. (2) High synthesis ability and strong stability. Hormones are not needed during cultivation. (3) Biotransformation function. Using hairy root's biotransformation ability can produce many new compounds, for example KAWAGUCHI KIICHIRO succeeded in getting cardiac glycoside from ginseng hairy-root. (4) Metabolites secretion to culture medium, profit for extraction of secondary metabolites. (5) Quantity of secondary metabolites can be increased via genetic engineering methods. Ri plasmid of Agrobacterium rhizogenes is a very good transgene vector, it can mediate aim gene to plant cells. (6) Strong breeding potential. Hairy-root has very strong breeding potential, and it can be made as artificial seeds for long term preservation. (7) Easy extraction of hairy-root and easy acquisition of its reproductive plant, this has significant meanings to transgenic plants and root reproductive plant exogenous gene transfer.In this study, we choose the hairy root of soybean as the bioreactor to express recombinant hbFGF, and started research as follows:1. Cloning of hbFGF gene and construction of the binary expression vector. (1) Chose the linearization plasmid pMD18-T-bFGF (constructed in this lab) as the template, and set up PCR reaction system. NcoI, BstBI cleavage sites were cloned into pCAMBIA1301 vector via PCR using the specific primers. Constructed recombinant hbFGF-pCAMBIA1301 plastid to transform Ecoli, hbFGF gene with NcoI and BstBI cleavage sites was subcloned into the vector. The correct recombinant plasmid identified by endonuclease digestion assay and gene sequencing was then transformed into Agrobacterium rhizogenes C58C1.(2) Competent cells preparation of Agrobacterium rhizogenes C58C1. bFGF plant expression vector pCAMBIA1301-bFGF was transferred into Agrobacterium rhizogenes C58C1 via Freeze-Thaw method. The result was confirmed by PCR.2. hbFGF gene was mediated into soybean cotyledonary node by Ri plasmid.hbFGF gene was mediated via agrobacterium rhizogenes and Hygresistant-hairy roots of soybean were obtained. The soybean cotyledonary nodes transformation: Put the soybean cotyledonary nodes into B5/2 medium pre-culture for 2 days, and infected them by activated agrobacterium rhizogenes with hbfgf gene for 5-15min. Then put them into B5/2 co-culture medium, cultivating 27℃3 days without light. Transferred them into B5/2 selective medium with hygromycin (15mg·L-1) and carbenicillin (500mg·L-1). Cotyledonary nodes without hbfgf gene were also transferred into B5/2 selective medium as the comparison. Cut the hairy roots when they grew to more than 2cm, transferred them into subculture medium until they were surely aseptic, then moved them into non-antibiotic and non-screening agent B5/2 medium. Sub-cultured the hairy roots every week, and detected their biomass and expression level after 20 days. 19 Hyg-resistant transgenic hairy roots of soybean were obtained successfully.3. Molecule and expression detection of the transgenic hairy roots of soybean (1) Identification of hbFGF gene by PCR.Extracted the genomic DNA with modified CTAB method as the templates, performed PCR with specific primers (as mentioned before), the untransformed hairy roots of soybean were the comparison. The electrophoresis results showed that the majority of the transformed soybean group (19 individuals) obtained a specific band of about 500bp, which could not be observed in the control group. The results showed that the hbFGF gene was transformed into the soybean.(2) Analysis of the expression of the hbFGF gene in the hairy roots.Extracted the mRNA from the positive hairy roots, and then performed RT-PCRwith specific primers. The electrophoresis results showed that the positive hairy roots obtained a specific band of about 500bp, which indicated that the hbFGF gene was expressed at the transcriptional level. Purified of the above positive hairy roots and analyzed by SDS-PAGE. The results of 15% SDS-PAGE showed that the molecular weight of the recombinant hbFGF is 18kDa which is exactly identical with the native one. It indicated that the expressed protein was accurate. At last, the Western Blot analysis confirmed that the hbFGF could be expressed in soybean steadily.4. Research on genetic stability of hbFGF expression in the hairy root of soybean.We analyzed the transverse genetic stability to the hbFGF highly expressed hairy roots of soybean. 20mg hairy roots which had been sub-cultured 5 times were purified and performed SDS-PAGE.The results showed that the hbFGF gene expressed as many as their parental generation which indicated a steady transverse inheritance. But it is necessary to further investigate the genetic stability of sexual multiplication.5. Optimization of the transgenic hairy roots scale cultivation system.Transgenic hairy roots with testified germ free, quick growth, and plenty branches were cut into 2-3cm length. Put 3-5 of these hairy roots into each 150ml germfree triangle flasks within liquid culture medium, suspension shaking culture, 120rpm.Set up 3 repeats for every physico-chemical factor, weighed the hairy roots every 4d, and cultivated until 20d. Detested different factors' effects on the differentiation and proliferation of the hairy roots, found the optimal conditions, and experimented with multi-factors culture.The optimal liquid culture conditions without light were: 1/3 volume liquid, 120 rpm rotation speed, 30 g/L sucrose, 1/2 B5 culture medium, 1.5g/L casein hydrolysate, 1mg/L gibberellin, 1mg/L indoleacetic acid, pH 6, temperature 25℃, and more than 70% dissolved oxygen. The hairy roots increased 35 times in rocking incubator in 20 days, 31 times in fermentor in 8 days.In short, in this research the hbFGF gene high expressed transgenic soybeans were obtained for the first time, and the optimal multi-factors culture conditions for scale culture were studied. The optimal liquid culture condition was determined, which showed great possibility to use hairy roots of soybean as the bioreactor to produce the medicative protein hbFGF on a large scale.
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