Prokaryotic Expression And Antibody Preparation Of BGT Insecticidal Gene And Preliminary Study On Its Expression In Transgenic Birch

In recent years the plant genetic engineering research progressed very rapidly,utilizing plant genetic engineering technology,anti-virus,insect-resistant,herbicide-resistant and salt-resistant plants have been cultivated.But the problems that if transgenic plants could show a particular character in long term and the characteristic of foreign genes in transgenic plants should be detailed research.And environment ecological safety problem arise people's concerns,according to an exogenous protein,it is necessary to establish a simple and quick method for detection.According to those problems,this paper took expressed product of BGT gene consisting of the insecticidal toxin gene from the spider,Atrax robustus,and the C terminal of Cry IA(b) gene from Bacillus thuringiensis in transgenic birch as research target.Through IPTG induction and metal chelate affinity chromatography purification,fusion protein His-BGT with high purity and its specific antibody have been obtained.Preliminary establish quick enzyme linked immunosorbent assay method,and the expression characteristic of BGT gene in transgenic birch have been preliminary studied.This paper of main results summarized as follows:1 The prokaryotic expression vector pET28a-BGT was constructed successfully,and transferred into E.coli BL21(DE3) and expressed in efficiency.Fusion protein expressed was purified by immobilized metal ion affinity chromatography and the purity of the purified protein was above 90%.2 The fusion protein as antigen was used to immunize rabbit for the preparation of polyclonal antibodies with high titer.The titer of serum antibodies was above 1:10 000 as detected by ELISA and purified by immuno-affinity chromatography.The high purity of IgG was obtained.Western blot assay demonstrated that the IgG anti fusion protein after purification showed definite specificity.With the method of NaIO₄ anti-BGT antibody was labeled with HRP to make enzyme-labeled antibody.The labeling rate was above 85%.3 An ELISA bi-antibody sandwich for the detection of BGT protein in transgenic birch has been established.The method has been preliminary optimized.The optimal coating concentration of antibody,diluted ratio of serum and McAb with HRP was 2μg/ml,1:200. The standard curve was obtained.Regression equation was got by a regression analysis.It was y = 0.0015x - 0.0694,R²= 0.99.The lowest detectable limit of ELISA was 40.0 ng/ml.A rapid and sensitive method for detecting BGT insecticidal protein was preliminary established.4 The BGT expression level of 5 month in the plants of BGT transgenic birch ranged from 0.3%to 0.05%of total soluble protein by established detection method.And this method was reliable that was validated by western blot.At the same time,we had also tested the BGT content in different time and part,fining that the expression level of BGT increased with plant development ranged from 5 to 9 month in the leaves of the most of different transgenic birch.And in differ part of birch,there is no definite pattern,but the expression level of BGT was consistent with that of single transgenic birch.Transgenic birch had resistance to Lymantria dispar,Malacosoma neustria testacea Motschulsky and lostera anastomosis L.The resistance to Lymantria dispar was the best.