Expression Of Human Fibroblast Growth Factor(hFGF18) In Oil Body System

Objective: Recombinant vector p OTBar-FGF18 was constructed and transformed into Agrobacterium tumefaciens EHA105, which contains Ti(Tumour inducing) plasmid that can help the target gene be inserted into the plant genome, and thus Genetic Inheritance stability can be inherited into offspring through meiosis, then express the target protein FGF18. Arabidopsis thaliana(A. thaliana) was a model plant. Its unopened flower buds can be infected by Agrobacterium, thus the target gene can be inserted into the Agrobacterium can also infect safflower and insert target gene into its Genome. The effect of the FGF18 expressed by oil body expression system on proliferation of NIH3T3 fibroblasts was investigated.Methods: Human FGF18 sequence was found on the NCBI and processed after the sequence was optimized. The plasmid containing p OTBar vector and FGF18 gene was extracted. After it was digested by Nco I and Hind III, p OTBar vector and FGF18 fragment was obtained. These two fragments could form a recombinant plasmid oleosin-FGF18 when they were ligated by T4 ligase. The recombinant plasmid was transformed into E. coli DH5 a, verify positive bacterias and save. Plasmid of positive E. coli was extracted and transformed into Agrobacterium tumefaciens EHA105, then verified positive bacteria and save. Arabidopsis and safflower were infected by positive Agrobacterium. After infected, A. Thaliana was normal cultured to harvest T1 generation seeds. When T1-generation seeds were sown, basta was sparyed and PCR experiments of blade were done to screen transgenic A. Thaliana lines. SDS-PAGE and Western blotting further verified the transgenic lines. Protein level and proliferation of NIH3T3 fibroblasts were determinated of the transgenic A. Thaliana lines. When investigated the proliferation of NIH3T3 fibroblasts of FGF18 expressed in oil body system, FGF18 expressed in E. coli expression system as the positive control, oil body of wild-type A. Thaliana as the negative control. After infected, aseptic tissue culture of the safflower then got the plantlet and grafting.Result: Oleosin-FGF18 expression vector was constructed and transformed into Agrobacterium successfully. The result of PCR of Arabidopsis showed that there were a band around 552 bp the same as the size of theory FGF18. The results of SDS-PAGE and Western blotting showed that the fusion protein oleosin-FGF18 had the band in39.7KDa consistent with the size of theoretical value. The content of fusion protein expressed in seeds reached at 455 ug per gram, accounting for 0.29% of total protein in oil body per gram seeds. MTT experiments showed that oil body containing oleosin-FGF18 and positive FGF18 protein had significant proliferative activity on NIH3T3 cells, and had a dose-dependent manner. whether added the heparin or not, there was no difference of the effect on NIH3T3 cells. The wild-type Arabidopsis oil bodies had no effect on NIH3T3 cells. The process of culture Safflower, substantially all of the seeds germination, the germination rate reached to 95%. After co-culture and antibacterial culture, buds grew about 50%, but only little buds can be transferred to elongation medium. There was a bad situation of rooting cultivation stage, so the use of grafting to make safflower buds which had basta resistance can grow further, and harvest seeds.Conclusion: Recombinant protein oleosin-FGF18 was obtained successfully in Arabidopsis seeds, and had proliferation activity on NIH3T3 cells. As using the oil-body system express FGF18 protein produced Magnifying great reference value. The fusion protein had its own unique properties, it can be directly applied on the skin and promote absorption, which can provide a reference for the development of a direct topical medication type. Agrobacterium-mediated transformation of safflower did not have the positive one.