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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

Pending Publication Date: 2022-07-22
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The present invention provides Jatropha curcas ribosome-inactivating protein JcRIP12, encoding gene, carrier and host, and the application of said Jatropha curcas ribosome-inactivating protein JcRIP12 in the preparation of medicines for treating tumors, so as to enrich the anti-tumor activity The types of Jatropha curcas ribosome-inactivating protein provide support for the research and development of anti-tumor drugs, and solve the cumbersome separation and purification process, complex process, and Problems of low production efficiency and high production cost

Method used

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  • Jatropha curcas ribosome inactivation protein JcRIP12 as well as coding gene and application thereof
  • Jatropha curcas ribosome inactivation protein JcRIP12 as well as coding gene and application thereof
  • Jatropha curcas ribosome inactivation protein JcRIP12 as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention provides a jatropha curcas ribosome inactivation protein JcRIP12 coding gene, a codon-optimized jatropha curcas ribosome inactivation protein JcRIP12 coding gene, and a jatropha curcas ribosome inactivation protein JcRIP12 coded by the two coding genes. The jatropha curcas ribosome-inactivating protein JcRIP12 is used for preparing the jatropha curcas ribosome-inactivating protein JcRIP12, and an in-vitro anti-tumor experiment proves that the jatropha curcas ribosome-inactivating protein JcRIP12 has good anti-tumor activity on human non-small cell lung cancer cells, human osteosarcoma cells, human liver cancer cells or human gastric cancer cells. Therefore, the invention also provides application of the jatropha curcas ribosome-inactivating protein JcRIP12 in preparation of medicines for treating tumors. According to the invention, the variety of jatropha curcas ribosome inactivating proteins with anti-tumor activity is enriched, and support can be provided for research and development of anti-tumor drugs; and the problems of tedious separation and purification process, complex process, low production efficiency and relatively high production cost of separation and purification of jatropha curcas ribosome inactivated protein from jatropha curcas in the prior art are solved.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and biomedicine, and relates to a Jatropha curcas ribosome inactivating protein JcRIP12 and its encoding gene and application. Background technique [0002] At present, malignant tumors are one of the main factors affecting the average life expectancy of human beings, and the research and development of anti-tumor drugs is particularly important. Some active ingredients in plants can selectively inhibit the proliferation of tumor cells, and preliminary results have been achieved in the development of new anti-cancer drugs, providing a new way for the research on the anti-tumor activity of plant proteins and the development and utilization of resources. [0003] The research on the antitumor activity of Jatropha curcas ribosome-inactivating proteins (RIPs) is a major breakthrough in the development of the medicinal value of Jatropha curcas. Lin Juan has proved through in vitro experime...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/70C12N1/21A61K38/16A61P35/00C12R1/19
CPCC07K14/415C12N15/70A61P35/00A61K38/00
Inventor 徐莺韩攀覃思颖邓聿杉马芮
Owner SICHUAN UNIV
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