Jatropha curcas ribosome inactivation protein JcRIP12 as well as coding gene and application thereof
A technology for inactivating ribosomes and encoding genes, applied in the fields of genetic engineering and biomedicine, can solve the problems of complex process, low production efficiency, complicated separation and purification process, etc., achieve good anti-tumor activity, efficient production, and solve the separation and purification process. cumbersome effect
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Embodiment 1
[0033] Example 1: Acquisition of Jatropha curcas ribosome inactivating protein JcRIP12 encoding gene (JcRIP12 gene)
[0034] Using the Jatropha curcas genome JatCur_1.0 data obtained from the NCBI platform, 12 JcRIP members were identified from the Jatropha curcas genome JatCur_1.0 through bioinformatics homology alignment technology, and JcRIP12 was one of the family members. And by designing the primers shown in Table 1 below, using the Jatropha curcas cDNA as a template, the existence of the JcRIP12 gene was verified by PCR technology. The PCR verification diagram of the JcRIP12 gene is shown in the figure. figure 1 As shown, the nucleotide sequence of the JcRIP12 gene is shown in SEQ ID NO. 1 of the sequence listing.
[0035] Table 1
[0036]
Embodiment 2
[0037] Example 2: Gene synthesis and vector construction
[0038] 1. Artificial modification of target gene fragments
[0039] (1) Prediction of mature protein sequences
[0040] Prokaryotic expression systems cannot modify proteins and require manual excision of the signal peptide. The signal peptides of Curcin and Curcin C were predicted by the signal peptide online prediction software Signal P, and the results showed that the signal peptides of the two were 1-28 amino acid sequences. However, our previous N-terminal sequencing results of the mature protein sequences of Curcin and Curcin C obtained by natural extraction and purification showed that there were 42 amino acid sequence deletions in the N-terminal of these two proteins. This is a certain deviation from the results predicted by Signal P. Studies have shown that there is a mature cleavage site in addition to the cleavage site of the signal peptide at the front end of the mature protein sequence (see Hajar, Owji,...
Embodiment 3
[0050] Example 3: Expression and purification of Jatropha curcas ribosome inactivating protein JcRIP12 (JcRIP12 protein) in Escherichia coli
[0051] 1. Inducible expression of JcRIP12 protein
[0052] (1) Pick a single colony containing the correct gene fragment after PCR verification, put it into 5 mL of fresh LB liquid medium, and shake it overnight at 180 rpm on a constant temperature shaker at 37 °C to obtain seed bacteria.
[0053] (2) The seed bacteria were transferred to 20 mL of fresh LB liquid medium containing 50 ug / mL kanamycin sulfate according to the volume ratio of 1:100, and the culture was shaken at 180 rpm on a constant temperature shaker at 37°C. OD of the bacterial solution 600 When it reaches 0.5 to 0.8 (this process generally takes 3 hours, it is accurately measured with a microplate reader), and 1 mL of uninduced bacterial liquid is taken as a control group for future use. IPTG was added to the test tube to a final concentration of 0.2 mM, and the expr...
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