| The plant genetic engineering has been greatly developed since the first transgenic plant came out in 1983, and the outstanding achievement has been made in the plant antivirus, insect-resistance, herbicide -resistance, stress resistant varieties improvement and heterogeneous gene expression. The transgenic plant has showed great potential as a cheap bioreactor to produce various medicine material needed by mankind. The transgenic technology has an important breakthrough of gramineae with rice as a typical grass, and it is possible for rice to carry heterogeneity protein. The rice is taken as the expression system to produce safe and cheap transgenic vaccine, antibody and cytokines by the transgenic technology, which is not only the improvement of the research of medical genetic engineering, but also a important means for rice from a simple food plant to a high attached-value functional food.Helicobacter pylori(H.pylori,Hp) is the main causing factor of chronic gastritis and peptic ulcer diseases, and has been listed by WHO as the pathogenic bacteria closely related with gastric adenocarcinoma, and has also been categorized as the Gourp I carcinogen. The use of vaccine will be the best effective way to prevent Hp infection.This research will transfer the Hp ureB gene and adjuvants Cholera toxin B subunit gene(CTB) into rice chromosome DNA by the plant transgenic technology and get regenerated shoots with steady inheritance, which laid the foundation for the research of establishing the rice bioreactor to carry microbe antigen and Hp transgenic plant vaccine.Methods1.Construct rice expression vector of CTB gene p13W4-CTB: CTB gene was amplified by polymerase chain reaction with pGEM-CTB as model, and vector p13W4 was digested with BamHI ,then was dephosphorylated, digested product was ligated with the same digested CTB. The recombinant plasmid was identified with PCR and enzyme digestion, and was confirmed by sequence analyze.2.Construct rice expression vector of CTB-ureB fusion gene pCWUN:(1) Fusion and clone of CTB gene and ureB gene: CTB gene and Hp ureB gene were amplified by high-fidelity PCR respectively from plasmid pGEM-CTB and Hp genome, and then they were fused by PCR. Then insert the fusion gene CTB-ureB into the vector pUC19. The recombinant plasmid was identified with PCR and enzyme digestion, and was confirmed by sequence analyze.(2) Reconstruction of Wx promoter of rice: Wx promoter of rice was amplified by high-fidelity PCR respectively from plasmid p13W4. sequence and Kozak sequence(K sequence) was amplified by primer extension with primer K5'pro and K3' pro, and then fuse the two sequences by PCR to get the fused Wx-K sequence.(3) Construct rice expression vector of CTB-ureB pCWUN: NOS terminator was amplified by PCR, and cut CTB-ureB fusion gene from pUCU, and insert the NOS terminator CTB-ureB fusion gene and Wx-K fusion sequence into vector pCAMBIAl 300 sequently. The recombinant plasmid was identified with PCR and enzymatic digestion.3. The competent Agrobacterium tumefacien was transformed by rice expression vector p13W4-CTB and pCWUN respectively.and obtain Agrobacterium tumefacien transformant after identified with PCR and enzyme digestion.4. Embryogenic calli was induced from rice immature embryos. Rice embryogenic calli was transformed with p!3W4-CTB by Agrobaclerium tumefacien . Finally we obtained transgenic plant after resistence screen, differentiation and root culture,and the transgenic plant was identified with PCR.results1 .PCR analysis, enzyme digestion identify and DNA sequence analysis verify that CTB gene has been inserted into the carrier p13W4 in the correct direction, and sequence analysis showed that nucleotides of cloned CTB was 100% homologous with that of Genbank; NOS terminator CTB-ureB fusion gene and x-K fusion sequence has been inserted into the vector pCAMBIAl300 in the correct direction and in the right order, and sequence analysis showed that nucleotides of cloned CTB was 100% homologous with that of G...
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