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Expression of the Bacillus Thuringiensis Cry2a Insecticidal Protein in E. Coli and Development of Colloidal Gold Immunochromatographic Strip

Author: YangQi
Tutor: TangXueMing
School: Shanghai Ocean University,
Course: Crop Genetics and Breeding
Keywords: Biosafety GMO detection Cry2A insecticidal protein prokaryotic expression colloidal gold immunochromatography test strip
CLC: S188
Type: Master's thesis
Year: 2012
Downloads: 30
Quote: 0
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Abstract


With the rapid development of transgenic technology, genetically modified cropsand its products is growing and the safety of genetically modified organisms become thefocus of global concern. The major purpose of transgenic detection for agriculturalproducts is to identify whether they are GMO（sGenetically Modified Organism）. GMOdetection and identification has been an important means and basis for marketsupervision, safety evaluation, import and export inspection of GMO products, and alsohas great significance for transgenic biotechnology research.Polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay(ELISA) are commonly used as detection methods, however these two methods hassome shortcomings, including complicated procedures, equipment requirements, timeconsuming, high cost, and the need for professional personnel, therefore they can’t adaptto rapid detection of large number samples in market and field.The codon optimized cry2A gene from transgenic rice was cloned by PCR in thisstudy. Cry2A protein was highly expressed in E. coli expression system and itspolyclonal antibody was prepared. Based on the techniques of colloidal goldimmunochromatographic, test strip was developed for rapid detection of Bt-Cry2Agenetically modified products by using the antibody. This study mainly contains thefollowing aspects:1. Heterologous expression vector and host of insect-resistant genes cry2A wereconstructed. The cry2A gene from transgenic rice was cloned by polymerase chainreaction (PCR). Then the PCR products of cry2A gene were inserted into expressionvector pET-30a(+) using restriction endonucleases EcoR I and BamH I, resulting inthe recombinant expression plasmid pET-30a(+)/Cry2A. And the recombinantvector was introduced into E. coli BL21(DE3).2. Methods of expression and purification for Cry2A recombinant protein wereestablished. Cry2A protein was highly expressed in E. coli BL21as inclusion bodiesunder the optimized conditions of isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration0.4mmol/L for6h at30℃. The inclusion bodies can bedissolved and purified by Ni-NTA affinity chromatography under denaturingconditions. The purified Cry2A protein was dialyzed against the refolding buffer toobtain a high purity and biologically active protein.3. Specific antibody of recombinant protein Cry2A has been obtained, and thecondition to label with colloidal gold was optimized. The recombinant protein ofCry2A was purified and prepared to obtain antibody. Colloidal gold was preparedand labeled with affinity purified polyclonal antibody. After experiments, theoptimized conditions was determined, the optimum pH value of labeling is8.0, theoptimal proportion in colloidal gold and antibody is18μg/mL, the concentration ofantibodies used for test line is1.0mg/mL.4. Colloidal Gold Immunochromatographic Rapid Test strip was prepared. After thepreparation of gold labeled pad, the nitrocellulose (NC) membrane and othercomponents, Immunochromatographic rapid test strip was assembled, followed bycutting, packaging and performance testing. This is the first rapid test strip forCry2A protein in domestic. The test results showed that the detection limit of thetest strip for Cry2A insecticidal protein was l μg/mL; the test strip had weak crossreaction with Cry1C insecticidal protein; the test strip was able to detecte Cry2Aprotein after15days placed at37°C.In this study, the Cry2A recombinant protein obtained with insecticidal activity,can be used not only in the detection technology for the Cry2A transgenic crops, butalso in the safety evaluation of the Cry2A protein. In this study, the colloidal goldimmunochromatographic test strip developed can be used for rapid detection ofBt-Cry2A genetically modified products. This study laid a foundation for thedevelopment of rapid detection of the transgenic products in filed and large-scale, andhas great value of application in the field of testing for genetically modified products.

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CLC: > Agricultural Sciences > Agriculture as the foundation of science > Agriculture and Biology > Agricultural Biological Engineering
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