Expression system of bacillus thur ingiens (Bt) insecticidal protein Cry1Ac-a

A technology of cry1ac-a and insecticidal protein, applied in the field of molecular biology and biology, can solve the problems of differences in insecticidal proteins, cumbersome and complicated operations, and difficulty in obtaining efficient expression of Cry1Ac protein, so as to reduce the secondary structure and overcome the code The effect of subpreferences

Inactive Publication Date: 2012-07-18
FUDAN UNIV
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Problems solved by technology

The imported high-purity Cry1Ac protein is very expensive, and it is also different from the insecticidal protein produced by the coding gene transferred into rice
However, the protein extracted from Bacillus thuringiensis is structurally different from the Cry1Ac protein transformed into rice, and as a protoxin, it cannot show biological activity outside the host body.
Miscellaneous proteins are introduced after activation by enzymatic digestion, so it cannot be directly used for scientific evaluation experiments
Due to the existence of im

Method used

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  • Expression system of bacillus thur ingiens (Bt) insecticidal protein Cry1Ac-a
  • Expression system of bacillus thur ingiens (Bt) insecticidal protein Cry1Ac-a
  • Expression system of bacillus thur ingiens (Bt) insecticidal protein Cry1Ac-a

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Embodiment Construction

[0022] 1. Cry1Ac-a nucleotide sequence optimization, synthesis and expression recombinant plasmid construction.

[0023] On the premise of ensuring insecticidal activity, in order to promote the expression of Cry1Ac-a protein in Escherichia coli, the coding frame and codon of insecticidal gene Cry1Ac were modified and optimized. The base sequence in the Cry1Ac-a protein gene was optimized according to the codon preference of Escherichia coli. The optimized sequence is shown in the sequence table SEQ ID NO.1, and its sequence length is 1857bp, in addition to including the start codon ATG and the stop codon TAA , and 5' NdeI and 3' EcoRI restriction sites.

[0024] Pass the gene fragment through Nde I and EcoR Ⅰ was connected to the pET43.1a vector that was also digested with the same restriction enzymes (see attached figure 1 ).

[0025] Restriction endonucleases were added to both ends of the target fragment Nde I 、EcoR The enzyme cutting site of I is connected to t...

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Abstract

The invention belongs to the field of molecular biology and biotechnology, and relates to an expression system of a bacillus thur ingiens (Bt) insecticidal protein Cry1Ac-a. The expression system of the Bt insecticidal protein Cry1Ac-a is synthetic. The expression system of the Bt insecticidal protein Cry1Ac-a comprises expression vectors and protein Cry1Ac-a gene fragments. The protein Cry1Ac-a gene fragments are obtained by optimization according to an escherichia coli codon preference. An optimized protein Cry1Ac-a comprises 615 amino acids, and has molecular weight of 68.7KD and an isoelectric point of 5.42. The invention also relates to a method for high-efficiency expression of a protein by recombinant strain IPTG induction. The expression system of the Bt insecticidal protein Cry1Ac-a overcomes the defect of an escherichia coli expression codon preference, obviously reduces a secondary structure of a mRNA, realizes high expression of the protein Cry1Ac-a in escherichia coli, and provides a feasible solution scheme for high-efficiency and low-cost expression of the protein Cry1Ac-a.

Description

technical field [0001] The invention belongs to the field of molecular biology and biotechnology, and specifically relates to an expression system of artificially synthesized Bt insecticidal protein Cry1Ac-a, including artificially synthesized Bt The gene of the insecticidal protein contains the expression vector of the gene and the product expressed by the gene. Background technique [0002] Bacillus thuringiensis ( Bacillus thur iniens , Bt ) Insecticidal crystal protein has high specific insecticidal activity to many important agricultural pests, but it is completely harmless to humans, vertebrates, and plants, and can be completely degraded in nature, so it is widely used in pest control. change Bt The application of crops has opened up new avenues for pest control and reduced the use of chemical pesticides. With the development of rice transgenic technology research and development and the cultivation of transgenic varieties, my country has cultivated a number of...

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Application Information

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IPC IPC(8): C12N15/32C12N15/70C12N1/21C07K14/325C12R1/19
Inventor 宋志平覃晓萍肖满秋冯延叶杨忠
Owner FUDAN UNIV
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