Study On Secretory Expression Of Rh-Bikunin In Soybean Hairy Roots

Agrobacterium rhizogenes are Gram-negative bacterium which can infect almost all dicotyledon and minority of monocotyledon. The hairy roots are induced by Agrobacterium rhizogenes which contain a Ri plasmid (root inducing plasmid) from the plant wounds. The Ri plasmid has two important regions: T-DNA and Vir region. Vir region does not transfer itself, but it is very important to T-DNA transfer. The T-DNA region on the Ri plasmid can be transformed into the plant cells and inserted into the genome when Agrobacterium rhizogenes infect the plants, its expression can induce a large number of hairy roots. The target gene mediated into the Ri plasmid via either intermediate vector or binary vector system is transformed into Agrobacterium rhizogenes which infect the explants like the stems, leaves, cotyledons and hypocotyledonary axes of aseptic seedlings. After a series of inducing culturing and resistance screening, the aseptic transgenic hairy roots can be obtained, which can proliferate dozens, even hundreds of times in the ordinary (growth hormone free) medium.The process of extracting target protein from the transgenic hairy roots is very complicated. If the protein can express by secretion way,it not only increases protein yield but also greatly simplifies the downstream purification procedure. It is the basis for producing high purity target protein at large scale in the future.Bikunin is an acid glycoprotein which is extracted from human urine. It contains two Kunitz-type trypsin inhibitor domains, and exhibits an inhibitory activity against several proteases including trypsin, chymotrypsin, plasmin and neutrophil elastase as a protease inhibitor. Bikunin can inhibit the expression of inflammatory cytokines, stabilize lysosomal membranes and inhibit tumor cell invasion. Clinically it has applied mainly in the treatment of pancreatitis, septic shock, postoperative renal functional recovery and the protection of either injury of lungs or ischemical reperfusion injury.At present, the protein of Bikunin sold in the market is isolated from human urine with traditional chemical method with many disadvantages like the limited source, virus-inactivation problem and poor purity. However, genetically engineered drugs contain good therapeutic effect, fewer side effects, easy to large-scale production and other characters. Thus, the research on obtaining Bikunin with genetic engineering approach has significant meanings. In this study, we chose the hairy roots of soybean as the bioreactor to express rh-Bikunin, and started our research as follows:(1) Two oligonucleotides sequences of seapsp were synthesized with the restriction endonuclease cohesive ends of NcoI and BstBⅠ, and were ligated into vector pCAMBIA1301 which was digested with NcoI and BstBⅠ, the recombinant plasmid was named pCAMBIA1301/seapsp.(2) AvrⅡand BstBⅠtarget sites were added into the plasmid pMD18-T-bikunin (constructed in our lab) using specific primers by PCR. Digested the above plasmid and vector pCAMBIA1301/seapsp with AvrⅡand BstBⅠ, and ligated the DNA fragments. The plant binary expression vector named pCAMBIA1301/seapsp-bikunin was constructed, and then identified by endonuclease digestion assay and gene sequencing.(3) The Bikunin expression vector pCAMBIA1301/seapsp-bikunin was transferred into Agrobacterium rhizogenes C58C1 via Freeze-Thaw method. The result was confirmed by PCR and showed that the Bikunin gene had been transferred into the Agrobacterium rhizogenes.(4) Pre-cultured the soybean cotyledonary nodes onto B5/2 medium for 2 days, and infected them by activated Agrobacterium rhizogenes with Bikunin gene for 5~15min; then put them onto B5/2 co-culture medium, cultivating 27℃3 days without light; transferred them onto B5/2 selective medium with hygromycin (15mg/L) and carbenicillin (500mg/L). Cotyledonary nodes without Bikunin gene were the comparison. Cut the hairy roots when they grew to 2~5cm, transferred them onto subculture medium until they were surely aseptic, then moved them onto non-antibiotic and non-screening agent B5/2 medium.(5) Extracted the genomic DNA from the transformed hairy roots, and identified by PCR, the untransformed hairy roots group were the comparison. The electrophoresis results showed that majority of the transformed soybean groups obtained a specific band of about 600bp, which could not be observed in the control group. The results confirmed that the Bikunin gene was transformed into the soybean hairy roots.(6) Extracted the total protein from the positive hairy roots group and detected Bikunin TIA, the negative hairy roots of soybean group was the comparison. The result shows that the average value of Bikunin TIA in transgenic hairy roots group(50.88±11.10 IU/g) is 20 times higher than the normal group(2.56±0.80 IU/g)(P<0.001).(7) Suspension cultured the positive hairy roots, weighed the hairy roots and collected culture fluid for analysis every 4 days, until 28 days. The hairy roots increased 21 times in rocking incubator in 28 days. The TIA of rhBikunin reached its maximum in the 20th day, the maximum activity was nearly 22.5 IU/ml.In short, in this research the Bikunin plant binary expression vector pCAMBIA1301/seapsp-bikunin was successfully constructed the first time. The transgenic hairy roots were obtained and identified by PCR and Bikunin TIA detection. The result showed that the rhBikunin can secretory express in soybean hairy roots. This can be the basis of using soybean hariy roots as a bioreactor to produce rhBikunin.