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Tat-Cry1Ac domain gene expression vector

Author: YangJing
Tutor: YangFengShan
School: Heilongjiang University
Course: Microbiology
Keywords: Bacillus thuringiensis Domain Protein Transduction Domain
CLC: Q78
Type: Master's thesis
Year: 2009
Downloads: 54
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Abstract


Bacillus thuringiensis is the widest use and largest production microorganism insecticide in the world now. Insecticidal crystal proteins (ICPs) of Bacillus thuringiensis were successfully used in transgene as insecticidal proteins. Domains of ICPs were very important to insecticidal activity. The function of ICP Domains and activity is not amplified exactly now. How to explain the relationship between the function of different Domains of pesticidal crystal protein and pesticidal activity in molecular biological methods is very usefulin gene modification and genetic recombination. A new and efficient delivery technology termed protein transduction has been proved to cargo exogenous macromolecules or charged compounds into living cells. Small sequences in some proteins, full of arginine, have been reported as protein transduction Domain (PTD),which can take biomacromolecules into cellular membranes. PTD is widely used in medicine.The role of each ICP Domains and the effect of Tat to Domains were studied. 9 primers were synthsised which can amplify cry1Ac Domain sequences by PCR, and 12 fusion expression vectors containing different Domains and combinations with Tat were constructed. Fusion vectors of pET1AcI、pET1AcI-II、pET1AcI-II-Ⅲ、pET1AcII、pET1AcII-Ⅲ、pET1AcIII、pETT1AcI、pETT1AcI-II、pETT1AcI-II-III、pETT1AcII、pETT1AcII-III、pETT1AcIII were successfully identified by PCR, single enzyme digestion and double digestion of plasmid and sequence method.Effects of various conditions on the expression of 12 fusion expression vectors including IPTG leve,time and temperature were investigated. The induced products were analysised by SDS-PAGE. Results showed that the suitable expression conditions were as follows: 0.1mM IPTG, 15℃and 24h induction time. 12 target protein were obtained on the suitable expression conditions. expression form identificition show that pET1AcI,pET1AcI-II,pET1AcI,pETT1AcI-II and pETT1AcII-III are soluble.pET1AcI-II-III, pET1AcII-III, pET1AcIII, pETT1AcI, pETT1AcI-II-II, pETT1AcII and pETT1AcIII were roduced as inclusions The protein purifying condition of pETT1AcI-II-III was studying. Target protein can be fully eluted with 40mM imidazole stock solution . pET1AcI protein was purified in the same condition.Bioassay result against Plutella xylotella showed that the insecticidal activity of the original pETT1AcI-II-III containing Tat was raised, and the mortality rate is 92.31%.The active fragment of 6 fusion expression vectors containing Tat was raised too. The insecticidal activity of combinations with different Domains did not raise ,but decreased.

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CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)
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