CN114774432A

CN114774432A - Jatropha curcas ribosome inactivating protein JcRIP12, and coding gene and application thereof

Info

Publication number: CN114774432A
Application number: CN202210519204.0A
Authority: CN
Inventors: 徐莺; 韩攀; 覃思颖; 邓聿杉; 马芮
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2022-05-12
Filing date: 2022-05-12
Publication date: 2022-07-22
Anticipated expiration: 2042-05-12
Also published as: CN114774432B

Abstract

The invention provides a coding gene of Jatropha curcas ribosome inactivating protein JcRIP12, a coding gene of codon optimized Jatropha curcas ribosome inactivating protein JcRIP12, and Jatropha curcas ribosome inactivating protein JcRIP12 coded by the two coding genes, and in-vitro anti-tumor experiments prove 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 stomach cancer cells, and accordingly the invention also provides the application of the Jatropha curcas ribosome inactivating protein JcRIP12 in preparing medicines for treating tumors. The invention not only enriches the types of barbadosnut ribosome inactivating proteins with anti-tumor activity, can provide support for research and development of anti-tumor drugs, but also solves the problems of complicated separation and purification process, complex process, low production efficiency and higher production cost in the prior art for separating and purifying the barbadosnut ribosome inactivating proteins from the barbadosnut.

Description

Jatropha curcas ribosome inactivating protein JcRIP12, and coding gene and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering and biological medicine, and relates to jatropha curcas ribosome inactivating protein JcRIP12, and a coding gene and application thereof.

Background

At present, malignant tumors are one of the main factors affecting the average life span of human beings, and the research and development of antitumor drugs are particularly important. Some active ingredients in the plant can selectively inhibit the proliferation of tumor cells, and the method has already achieved initial achievements in the aspect of developing new anti-cancer drugs, and provides a new way for the research of the anti-tumor activity of the plant protein and the development and utilization of resources thereof.

The research on the anti-tumor activity of Jatropha curcas ribosome-inactivating proteins (RIPs) is a major breakthrough in the development of the medicinal value of Jatropha curcas. In vitro experiments prove that the jatropha curcas seed kernel toxin protein Curcin purified from jatropha curcas has obvious inhibition effect on the proliferation of gastric cancer cells (SGC-7901), mouse myeloma cells (SP2/0) and human liver cancer cells (HepG2), but has no inhibition effect on human cervical cancer cells (hela) and human normal embryonic lung diploid cells (MRC). The Jatropha curcas bark ribosome inactivating protein Jc-SCRIP also shows the proliferation inhibiting effect on MCF-7 cells, SW620 and HepG2, and the IC of the protein Jc-SCRIP on MCF-7 cells, SW620 and HepG2₅₀0.15mM, 0.25mM and 0.40mM, respectively). Zhang et al found that Jatropha curcas ribosome inactivating protein Curcin C obtained by purification from Jatropha curcas can significantly inhibit the in vitro proliferation of non-small cell lung cancer (A549), colon cancer (HCT116), leukemia (MV411) and osteosarcoma (U2OS), but the proliferation inhibition effect on human embryonic normal kidney cells (HEK-293) is small, and the antitumor activity of Curcin C is stronger than that of Curcin. The Jatropha curcas ribosome inactivating protein gene 26SK is cloned from the kernel of Jatropha curcas by Danula et al and ligated to the vector pET28a⁽⁺⁾In the above, the protein was successfully expressed in Escherichia coli Rosetta (DE3), and the 26SK protein was confirmed to be significantly inhibited by cell experimentsThe growth of triple negative human breast cancer (MDA-MB-231) cells was made, but the growth of African green monkey kidney epithelial cells (Vero cells) was not affected. The research results show that the ribosome inactivating protein of the barbadosnut has selective toxicity in the aspect of anti-tumor and has huge application potential in the research field of anti-tumor drugs. Therefore, it is one of the research focuses in the field to find out whether there are other members with higher anti-tumor activity in the jatropha curcas ribosome inactivating protein family, and if the jatropha curcas ribosome inactivating protein with better anti-tumor activity can be found out, the research and development of drugs for resisting malignant tumor will have positive significance.

At present, the separation and purification process of natural ribosome inactivating protein is complicated, and the purification yield is very low. For example, CN101891798B discloses a separation and purification method of Curcin, and CN107058261B discloses a separation and purification method of Curcin ribosome inactivating protein, Curcin C. However, the two processes are complicated, not only have long process time and low production efficiency, but also have high production cost, are not beneficial to realizing large-scale production, and thus hinder the practical application of the barbadosnut ribosome inactivating protein. Therefore, if a simpler method can be provided for industrial production of the barbadosnut ribosome inactivating protein, a powerful promoting effect can be generated on the practical application of the barbadosnut ribosome inactivating protein in the anti-tumor field.

Disclosure of Invention

The invention provides a barbadosnut ribosome inactivating protein JcRIP12, a coding gene, a vector and a host, and application of the barbadosnut ribosome inactivating protein JcRIP12 in preparation of a medicine for treating tumors, so that the types of barbadosnut ribosome inactivating proteins with anti-tumor activity are enriched, support is provided for research and development of anti-tumor medicines, and the problems of complicated separation and purification processes, complex process, low production efficiency and high production cost in the process of separating and purifying the barbadosnut ribosome inactivating proteins from barbadosnut in the prior art are solved.

In order to realize the purpose of the invention, the technical scheme adopted by the invention is as follows:

the coding gene of jatropha curcas ribosome inactivating protein JcRIP12 provided by the invention has a coding amino acid sequence shown as a protein in a sequence table SEQ ID NO.3, and a nucleotide sequence shown as a sequence table SEQ ID NO. 1.

The invention also provides a codon-optimized jatropha curcas ribosome inactivating protein JcRIP12 encoding gene, wherein the encoding gene encodes a protein with an amino acid sequence shown as a sequence table SEQ ID NO.3, and a nucleotide sequence of the encoding gene is shown as a sequence table SEQ ID NO. 2. The coding gene with the nucleotide sequence shown as SEQ ID No.2 in the sequence table is obtained by optimizing codons of the coding gene with the nucleotide sequence shown as SEQ ID No.1 in the sequence table according to the codon preference of escherichia coli on the premise of not changing the amino acid sequence of the coding gene and in order to obtain the Jatropha curcas ribosome inactivating protein JcRIP12 in a prokaryotic expression mode.

On the basis of the technical scheme of the coding gene, the invention also provides a jatropha curcas ribosome inactivating protein JcRIP12, and the amino acid sequence of the jatropha curcas ribosome inactivating protein JcRIP12 is shown as a sequence table SEQ ID NO. 3. The jatropha curcas ribosome inactivating protein JcRIP12 is encoded by a coding gene with a nucleotide sequence shown as a sequence table SEQ ID NO.1 or SEQ ID NO. 2.

The invention also provides a recombinant vector containing the coding gene with the nucleotide sequence shown as the sequence table SEQ ID NO.2 and a host containing the coding gene with the nucleotide sequence shown as the sequence table SEQ ID NO. 2.

Furthermore, the recombinant vector is formed by inserting a coding gene with a nucleotide sequence shown in a sequence table SEQ ID NO.2 into a prokaryotic expression vector, for example, the prokaryotic expression vector can be a pET-30a prokaryotic expression vector and can also be other prokaryotic expression vectors. The host is an escherichia coli expression strain.

Certainly, on the basis of the coding gene with the nucleotide sequence shown as SEQ ID No.1 of the sequence table, in order to obtain the jatropha curcas ribosome inactivating protein JcRIP12 through other expression systems, the codons of the coding gene with the nucleotide sequence shown as SEQ ID No.1 of the sequence table can be optimized according to the codon preference of other expression systems on the premise of not changing the coding amino acid sequence of the gene, so as to construct the coding gene suitable for the corresponding expression system, and then a recombinant vector and a host are constructed by using the coding gene suitable for the corresponding expression system, so that the jatropha curcas ribosome inactivating protein JcRIP12 can be expressed.

The invention also provides a method for preparing the Jatropha curcas ribosome inactivating protein JcRIP12, which comprises the steps of culturing the host under the condition that the host is suitable for producing more Jatropha curcas ribosome inactivating protein JcRIP12, and then separating and purifying the Jatropha curcas ribosome inactivating protein JcRIP12 produced by the host.

The invention proves that the Jatropha curcas ribosome inactivating protein JcRIP12 has higher-level inhibition effect on the proliferation of human non-small cell lung cancer cells (A549), human osteosarcoma cells (U2OS), human hepatoma cells (HepG2) and human gastric cancer cells (SGC-7901) through in-vitro anti-tumor activity experiments, and has weaker inhibition effect on the proliferation of normal cell line Human Umbilical Vein Endothelial Cells (HUVEC). Particularly, the proliferation inhibition effect of the Jatropha curcas ribosome inactivating protein JcRIP12 on human non-small cell lung cancer cells (A549) is obviously superior to that of the existing Curcin and Curcin C; meanwhile, the proliferation inhibition effect of the Jatropha curcas ribosome inactivating protein JcRIP12 on human liver cancer cells (HepG2) is obviously superior to that of Curcin. The toxicity of the Jatropha curcas ribosome inactivating protein JcRIP12 to normal cells is smaller than that of Curcin and Curcin C. Therefore, the Jatropha curcas ribosome inactivating protein JcRIP12 provided by the invention can be used as a potential active substance for developing antitumor drugs and applied to preparing drugs for treating tumors.

Further, when the Jatropha curcas ribosome inactivating protein JcRIP12 is applied to preparation of a medicament for treating tumors, the tumors are human non-small cell lung cancer, human osteosarcoma, human hepatoma or human gastric cancer, and the tumors are preferably non-small cell lung cancer or human hepatoma.

Compared with the prior art, the technical scheme provided by the invention has the following beneficial technical effects:

1. the invention provides jatropha curcas ribosome inactivating protein JcRIP12 and a coding gene thereof, and application of the jatropha curcas ribosome inactivating protein JcRIP12 in preparing a medicament for treating tumors, enriches the types of the jatropha curcas ribosome inactivating protein with anti-tumor activity, and can provide support for research and development of anti-tumor medicaments.

2. On the basis of encoding genes of Jatropha curcas ribosome inactivating protein JcRIP12, codons of the encoding genes are optimized, optimized gene segments are inserted into prokaryotic expression vectors, then the prokaryotic expression vectors are transferred into an Escherichia coli expression strain for prokaryotic expression, and the preparation of the Jatropha curcas ribosome inactivating protein JcRIP12 is realized through purification. The invention realizes the high-efficiency production of the jatropha curcas ribosome inactivating protein JcRIP12 through prokaryotic expression, provides a way for the industrial production of the jatropha curcas ribosome inactivating protein JcRIP12, and simultaneously, the invention proves that the jatropha curcas ribosome inactivating protein JcRIP12 has the equivalent or better anti-tumor activity compared with the existing Curcin and Curcin C through an anti-tumor activity experiment. The invention solves the problems of complicated separation and purification process, complex process, low production efficiency and higher production cost in the prior art for separating and purifying the jatropha curcas ribosome inactivating protein from the jatropha curcas.

3. The invention is proved by in vitro anti-tumor activity experiments that: the Jatropha curcas ribosome inactivating protein JcRIP12 has high-level inhibition effect on the proliferation of human non-small cell lung cancer cells (A549), human osteosarcoma cells (U2OS), human liver cancer cells (HepG2) and human gastric cancer cells (SGC-7901). Particularly, the proliferation inhibition effect of the Jatropha curcas ribosome inactivating protein JcRIP12 on human non-small cell lung cancer cells (A549) is obviously superior to that of Curcin and Curcin C which are separated and purified from Jatropha curcas; meanwhile, the proliferation inhibition effect of the Jatropha curcas ribosome inactivating protein JcRIP12 on human liver cancer cells (HepG2) is also obviously superior to that of Curcin. The toxicity of the Jatropha curcas ribosome inactivating protein JcRIP12 on normal cells is less than that of Curcin and Curcin C. Therefore, the Jatropha curcas ribosome inactivating protein JcRIP12 provided by the invention can be applied to preparing medicines for treating tumors.

Drawings

FIG. 1 shows the PCR-verified image of JcRIP12 gene, and the arrow indicates the location of JcRIP12 gene.

FIG. 2 shows the JcRIP12 gene sequence before and after codon optimization.

FIG. 3 shows the results of the plasmid duplex enzyme assay.

FIG. 4 shows small expression of JcRIP12 protein, with the position of the JcRIP12 protein indicated by the arrow.

FIG. 5 shows the results of JcRIP12 protein purification by SDS-PAGE analysis.

FIG. 6 shows SDS-PAGE of JcRIP12 protein.

FIG. 7 shows the Western Blot detection of JcRIP12 protein.

FIG. 8 is the UV absorption spectra of JcRIP12 protein, Curcin, and Curcin C.

Detailed Description

The jatropha curcas ribosome inactivating protein jcprip 12, its coding gene and application are further described by the following examples. The following described examples are only a part of the embodiments of the present invention, and not all of them. Other embodiments, which can be derived by those skilled in the art from the summary and examples of the invention without creative efforts, are within the protection scope of the present invention.

The molecular biological experiments, which are not specifically described in the following examples, were carried out by referring to the specific methods listed in molecular cloning instructions (third edition) of J. SammBruk et al, or according to kits and product instructions.

In the following examples, Curcin refers to Jatropha curcas ribosome inactivating protein disclosed in Lin Juan et al (see, antibodies of fungi from seeds of Jatropha curcas, Acta Pharmacologica Sinica,2003,24(3), 241-. Curcin C refers to Jatropha curcas ribosome inactivating protein prepared according to the method disclosed in CN 107058261B.

The following examples mainly take the case of obtaining jatropha curcas ribosome inactivating protein jcprip 12 by means of prokaryotic expression of escherichia coli, and give a preparation method of jatropha curcas ribosome inactivating protein jcprip 12. Of course, the jatropha curcas ribosome inactivating protein jcrrip 12 can also be expressed by other expression systems. When the jatropha curcas ribosome inactivating protein JcRIP12 is obtained by means of prokaryotic expression of escherichia coli, firstly, a JcRIP12 gene is identified from jatCur _1.0 of a jatropha curcas genome through technologies such as bioinformatics homology comparison and the like, the nucleotide sequence of the gene is shown as a sequence table SEQ ID NO.1, and the existence of the JcRIP12 gene is verified through cloning. Then, the codon of the JcRIP12 gene is optimized according to the codon preference of escherichia coli, the optimized gene is inserted into a prokaryotic expression vector and is transformed into an escherichia coli expression strain, then induction expression is carried out, the expression of the jatropha curcas ribosome inactivating protein JcRIP12 is successfully realized, and the jatropha curcas ribosome inactivating protein JcRIP12 is obtained in the whole bacteria through purification and renaturation.

Certainly, on the basis of the jcrrip 12 gene, in order to obtain the jatropha curcas ribosome inactivating protein jcrrip 12 through other expression systems, the codon of the jcrrip 12 gene can also be optimized according to the codon preference of other expression systems on the premise of not changing the gene coding amino acid sequence, so as to construct a coding gene suitable for the corresponding expression system, and then a recombinant vector and a host are constructed by using the coding gene suitable for the corresponding expression system, namely the jatropha curcas ribosome inactivating protein jcrrip 12 can be expressed. Such embodiments are also within the scope of the present invention, based on the protection of the present invention from the jcirip 12 gene.

Example 1: acquisition of encoding gene (JcRIP12 gene) of Jatropha curcas ribosome inactivating protein JcRIP12

12 JcRIP members are identified from the jatCur _1.0 of the jatropha curcas genome by bioinformatics homology comparison and other technologies by utilizing jatCur _1.0 data of the jatropha curcas genome acquired by an NCBI platform, wherein the JcRIP12 is one family member. The existence of the JcRIP12 gene is verified by PCR technology by designing primers shown in the following table 1 and taking jatropha curcas cDNA as a template, the PCR verification picture of the JcRIP12 gene is shown in figure 1, and the nucleotide sequence of the JcRIP12 gene is shown in SEQ ID NO.1 of the sequence table.

TABLE 1

Example 2: synthesis of Gene and construction of vector

1. Artificial modification of target gene fragment

(1) Prediction of mature protein sequence

Prokaryotic expression systems do not allow for protein modification and require manual cleavage of the signal peptide. And predicting the Signal peptides of the Curcin and the Curcin C by using Signal peptide online prediction software Signal P, wherein the results show that the Signal peptides of the Curcin and the Curcin C are 1-28 amino acid sequences. However, the sequencing result of the N-terminal of the mature protein sequences of Curcin and Curcin C obtained by natural extraction and purification in the previous period shows that 42 amino acid sequence deletions exist at the N-terminal of the two proteins. This is a deviation from the Signal P prediction result. It has been shown that the protein has a mature cleavage site in addition to the cleavage site for the signal peptide at the front of the mature protein sequence (see Hajar, Owji, Navid, et al. Acomprehensive review of signal peptides: structures, roles, and applications [ J ]. European Journal of cell Biology,2018,97(6): 422-. Therefore, we speculate that secondary cleavage may occur during translation of both Curcin and Curcin C. As the protein, the Curcin and the Curcin C are all belonging to the same gene family, when the mature protein sequence is predicted, the mature protein sequence is determined by taking the mature protein sequence of the Curcin and the protein sequence of the Curcin C as a template, adopting the first-level structure sequence comparison and using the third-level structure to verify the comparison result. The final prediction of the mature protein sequence has 38 amino acid deletions. The method can retain the characteristics of the natural protein, eliminate excessive interference factors, reduce the structure of the natural protein and provide a theoretical basis for the subsequent research of antitumor activity.

(2) Codon bias analysis and optimization thereof

The codon usage frequency of the JcRIP12 gene was analyzed using an online website (http:// www.detaibio.com/tools/codon-usage-computer. html). After determining that a large number of rare codons influencing expression do exist, codon optimization is carried out by using an online website (https:// www.genscript.com) on the premise of not changing a gene coding amino acid sequence according to the codon preference of escherichia coli. Finally, the Codon Adaptation Index (CAI) was calculated using the online tool EMBoss (http:// www.bioinformatics.nl/EMboss-explorer /). The Escherichia coli codon usage frequency list is from the Kazusa database. After codon optimization, the codon adaptation index was increased from 0.649 before optimization to 0.79, and the control before and after codon optimization is shown in FIG. 2.

In order to ensure the prokaryotic expression efficiency and avoid gene mutation in the gene cloning process, the invention adopts a gene synthesis mode to obtain a gene segment after codon optimization. Enzyme cutting sites NdeI and HindIII are additionally arranged at the N end and the C end of a mature sequence obtained after analysis, and the recombinant plasmid pET30a-JcRIP12a with Amp resistance is obtained by being committed to the Shanghai biology for synthesis after the confirmation of no errors. The nucleotide sequence of the finally obtained codon-optimized jatropha curcas ribosome inactivating protein JcRIP12 encoding gene is shown as a sequence table SEQ ID NO. 2.

3.(1) extraction of recombinant plasmid

Taking 2-5 mL of a bacterial liquid containing recombinant plasmid pET30a-JcRIP12a, extracting plasmids, and specifically referring to the instruction of a TIANGEN plasmid miniextract kit for operation. The extracted plasmid solution is stored at-20 ℃.

(2) Restriction enzyme digestion verification of plasmid

And determining the accuracy of the extracted plasmid by double enzyme digestion verification and sequencing verification. The enzyme should be cleaved to avoid the presence of the selected cleavage site in the desired gene fragment. The result of the plasmid double-enzyme digestion test is shown in figure 3, the obtained plasmids all obtain target bands under enzyme digestion, and the success of the construction of the recombinant vector is shown by combining with a sequencing result.

(3) Transformation of BL21(DE3) competence

The recombinant plasmid pET30a-JcRIP12a which is confirmed to be correct by enzyme digestion and sequencing verification is transformed into the competent cells of Escherichia coli BL21(DE3) by a heat shock method (see Zhang, Lizhi, Zhang, etc.) one-step method for preparing competent cells of Escherichia coli BL21(DE3) strain and optimizing transformation conditions [ J ]. Jiangsu agricultural science, 2016,44(12): 529-.

Example 3: expression and purification of jatropha curcas ribosome inactivating protein JcRIP12(JcRIP12 protein) in escherichia coli

Inducible expression of JcRIP12 protein

(1) And (3) selecting a single colony containing the correct gene fragment after PCR verification to 5mL of fresh LB liquid culture medium, and shaking the single colony on a constant temperature shaker at 37 ℃ at a rotating speed of 180rpm overnight to obtain the seed bacteria.

(2) The seed bacteria were inoculated into 20mL of fresh LB liquid medium containing 50ug/mL kanamycin sulfate at a volume ratio of 1:100, and cultured on a constant temperature shaker at 37 ℃ with shaking at 180 rpm. OD of bacterial liquid₆₀₀When the concentration reaches 0.5-0.8 (the process generally needs 3 hours, and the enzyme-linked immunosorbent assay is used for accurate determination), 1mL of non-induced bacteria liquid is taken as a control group for later use. IPTG was added to the tube to a final concentration of 0.2mM, and the tubes were each allowed to induce expression at 15 ℃ and 37 ℃. After 16h of induction expression, SDS-PAGE is used for analyzing and identifying induction expression results, specifically, the bacterial liquid is collected, a sample of the induced bacterial liquid is centrifuged for 5min at 4 ℃ and 12000rpm, supernatant is discarded, thalli are collected, and expression quantity of the thalli is detected by SDS-PAGE, and the results are shown in figure 4.

Small expression results showed that jcrrip 12 protein was expressed higher at 15 ℃, so we set induction conditions to: the final concentration of IPTG was 0.2mM, induction was carried out at 15 ℃ for 16h, and since the expression level of JcRIP12 protein was too low in a small amount of expression, the protein was obtained from the whole strain in this example.

Purification of JcRIP12 protein

The whole strain obtained in the previous step was sonicated in 50mM Tris (pH8.0), 300mM NaCl, 8M Urea, 20mM Imidazole buffer containing 1mM DTT while the Ni-IDA affinity column was equilibrated with 50mM Tris (pH8.0), 300mM NaCl, 8M Urea, 20mM Imidazole buffer, after which the target protein was eluted with different concentrations of Imidazole equilibration buffer and each eluted fraction was collected for SDS-PAGE analysis. The results of JcRIP12 protein purification analyzed by SDS-PAGE are shown in FIG. 5.

Purifying and analyzing by Ni-IDA affinity chromatography column, collecting Lane 5 protein with relatively high purity (Lane 3-7 are all eluted by 500mM Imidazole equilibrium buffer), adding into treated dialysis bag, dialyzing into buffer solution [1 XPBS (pH7.4), 4mM GSH, 0.4mM GSSG, 0.4M L-Arginine, 1M Urea ] at 4 deg.C, renaturing JcRIP12 protein, and finally dialyzing into storage solution [1 XPBS (pH7.4), 5% Glycerol solution ] for about 6-8 h. After the completion of the renaturation by dialysis, the obtained supernatant was filtered through a filter having a pore size of 0.22 μm, aliquoted, and frozen at-80 ℃.

Example 4: performance testing of JcRIP12 protein

1. Protein stability test (Freeze-thaw experiment)

Taking a piece of JcRIP12 protein frozen at-80 ℃, placing in an ice-water bath for 5-10 min until the protein is slowly melted, and placing in a refrigerator at 4 ℃ for 0.5h after the protein is melted. No abnormal phenomenon exists, which indicates that the freeze-thaw experiment of the JcRIP12 protein is normal.

2. Protein concentration detection

The concentration of JcRIP12 protein was determined using the Bradford protein concentration assay kit, and the final concentration of JcRIP12 protein was found to be 0.3 mg/mL.

3. SDS-PAGE detection of proteins

A plurality of JcRIP12 protein samples were taken, SDS-PAGE loading buffer (Biyun day, SDS-PAGE protein loading buffer (5X, odorless) product number: P0286-15 ml) was added, and the samples were heated at 100 ℃ for 7min, and then centrifuged to take the supernatant for electrophoresis. And (3) performing voltage-stabilized electrophoresis at 120V until a bromophenol blue band moves to a position 1cm away from the bottom of the gel, taking out the gel, and dyeing and decoloring by using a protein gel rapid treatment system. The measurement was carried out by using a Bio-Rad gel imager, and the results are shown in FIG. 6.

4. Western Blot detection of proteins

A plurality of JcRIP12 protein samples are taken, the samples are processed, then the proteins are separated after gel electrophoresis, then the samples are transferred to a PVDF membrane, the stable voltage of 100V is 1h, the PVDF membrane after the strip transfer is sealed by skimmed milk powder for 2h, primary antibody incubation liquid of 0.1% antibody is added for incubation for 2h, then secondary antibody diluted by the volume ratio of 1:5000 is used for incubation for 1h, and ECL chemiluminescence method is adopted for development, and the result is shown in figure 7.

5. Ultraviolet absorption spectrum of protein

And (3) putting 200 mu L of JcRIP12 protein solution into a quartz 96-well plate, and measuring the ultraviolet light absorption value of the JcRIP12 protein solution in a continuous wavelength microplate reader, wherein the scanning range is 200-500 nm, and the step length is 1 nm. The same test was also performed with Curcin, Curcin C as controls. The results are shown in FIG. 8.

UV absorption spectroscopy is a routine method for studying the conformation of protein molecules and allows some information to be obtained about the protein molecule (see: Zhang Tree. enzyme research techniques [ M ]. science publishers, 1987: 45-61.). As shown in fig. 8, there is a maximum absorption peak at b for each of the Curcin, Curcin C and jcrrip 12 proteins, which is mainly contributed by peptide bonds. There is a minimum absorption peak at c and an absorption peak at d, which is mainly contributed by the electron transition of phenylalanine and tryptophan residues. There is a fairly large but non-peaked segment of the curve at a near 200 nm. Compared with Curcin and Curcin C which are separated and purified from Jatropha curcas, the JcRIP2 protein has the same overall fluctuation trend of the ultraviolet absorption curve, but the absorption peak moves downwards.

6. Determination of relative molecular weight and isoelectric Point of proteins

The JcRIP12 protein is sent to Shanghai biological engineering Co., Ltd for molecular weight identification and sequencing, and isoelectric point determination is carried out by adopting a capillary method, and the result shows that the relative molecular mass of the JcRIP12 protein is 30588.4Da, the isoelectric point is 9.89, the protein consists of 274 amino acid sequences, and the amino acid sequence is shown as SEQ ID NO.3 of a sequence table.

TABLE 2

Example 5: exploration of antitumor activity

In this example, the anti-tumor activity of jcrrip 12 protein was tested, and jcrrip 12 protein, Curcin and Curcin C were applied to 4 tumor cell lines, i.e., human non-small cell lung cancer cell (a549), human osteosarcoma cell (U2OS), human hepatoma cell (HepG2) and human gastric cancer cell (SGC-7901), and 1 normal cell line, i.e., Human Umbilical Vein Endothelial Cell (HUVEC), respectively. The antitumor activity was evaluated by using Curcin and Curcin C as control groups. The method comprises the following specific steps:

a549, U2OS, HepG2, SGC-7901 and HUVEC in logarithmic growth phase are selected, and monolayers of A549, U2OS, HepG2, SGC-7901 and HUVEC are digested with 0.25% pancreatin and prepared into cell suspensions with corresponding media. Specifically, the medium of HepG2 cell line was: dmem (high glucose); the culture medium of the U2OS cell line was: McCoy's5A media (Modifiedwith TRicine); a549, SGC-7901 and HUVEC cell lines are cultured in the following culture media: RPMI-1640; all media were supplemented with 10% FBS and 1% Penicillin-Streptomyces-Gentamicin.

According to 6X 10⁴Cell density per ml cells were plated evenly in disposable sterile 96-well plates, 100uL cell suspension per well, placed at 37 ℃ in 5% CO₂Culturing for 24 hours in the cell culture box, removing supernatant after cells adhere normally, adding corresponding amounts of protein and culture medium according to the final concentrations of JcRIP12, Curcin and Curcin C of 1.25,2.5,5,10,20 and 40 mu g/ml respectively, repeating 5 holes for each concentration, continuing culturing for 48 hours under the same condition, removing the culture medium, adding 10% of CCK-8, incubating for 30-60 min in the culture box, and measuring the light absorption value at 450nm by using a microplate reader.

Medium without added protein was used as a blank.

The proliferation inhibition rate of the cells was calculated according to the following formula, statistically analyzed by Graphpad software and calculated the median inhibitory concentration IC₅₀The results are shown in Table 3 below.

Wherein X represents the absorbance at 450nm of the administered group, the blank group and the control group.

TABLE 3

As shown in Table 3, the JcRIP12 protein has good proliferation inhibition effect on tumor cells A549, U2OS, HepG2 and SGC-7901 in vitro, and has weak proliferation inhibition effect on normal cells HUVEC. The toxicity of the JcRIP12 protein to tumor cells A549 and HepG2 is obviously stronger than that of Curcin, the toxicity of the JcRIP12 protein to the tumor cells A549 is obviously stronger than that of the JcRIP12 protein to the Curcin C, and the anti-tumor activity of the JcRIP12 protein to the tumor cells U2OS and SGC-7901 is also at a higher level. The toxicity to normal cells is less than that of Curcin and Curcin C, the compound has wide antitumor spectrum, is a potential active substance for developing antitumor drugs, and can be applied to preparing drugs for treating tumors.

Sequence listing

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ggagataaat cttatttctt taacgatccg actgaattga aggatgcaca aactcatctt 420

ttcaaagaca caaagcaaac cgccataaaa attacaggta gctacgactc tctcaaagct 480

caaggaggag atcgagaatc agtagattta ggaatcgggc aattagactc tcacatctat 540

acactccata aatcgacagc gttaaaagac gttgctaagt ctcttgtttg tattattcag 600

atggtttcag aagcagcccg atttaagtct attgagaata agatagtaga taagattgat 660

ggaagtttta aaccgaagct tgatataatc actcgtgaga acaactgggg tgacctttct 720

gaaggaattc aaaacgctga taagaaggga aattttaaaa caaaagttag attgcagaag 780

gaagatggta aggaggatat tatatctaac gttaatcaaa taataggaga gatgggaatt 840

ttgctgtata agaagaagaa gatctacaat attccaagtt ttggacaaac aaattttgga 900

aaccttatcc agaactaa 918

<210> 2

<211> 837

<212> DNA

<213> Artificial Synthesis ()

<400> 2

catatgcatc atcatcatca tcacgcgatt ccgagcgtta gctttaccat tacccgtatt 60

ccgggcgacg ataaaaccga ctacaagcag ctgatggtcg atctgcgtaa aaaactgagc 120

agcggtacca ccagtaacgg cgttccggtt ctgcgtagta ccgcgagcaa agaagcgaaa 180

tacctgctgg tcaacatcat caacagcggc aagaaagaaa ttaccctggg tctgaacgtc 240

atcaacgcgt acgttctggc gtataaagtt ggcgataaga gctacttctt caacgacccg 300

accgaactga aagacgcaca gacccacctg tttaaggaca ccaaacagac cgcgatcaaa 360

atcaccggta gctacgatag cctgaaagca caaggtggcg atcgcgaaag cgtggatctg 420

ggtattggcc aactggatag ccatatctat accctgcata aaagcaccgc gctgaaagac 480

gttgcaaaga gcctggtctg catcattcag atggtctctg aagcggcgcg ctttaaaagc 540

atcgaaaaca agatcgtcga caagattgac ggcagcttca agccgaaact ggatatcatc 600

acccgcgaaa acaactgggg cgatctgtct gaaggtattc agaacgcgga caagaagggc 660

aacttcaaga ccaaagtccg cctgcagaaa gaagacggca aagaggacat catcagcaac 720

gtcaaccaga tcatcggcga aatgggtatc ctgctgtaca agaagaagaa gatctacaac 780

atcccgagct ttggccagac caactttggt aacctgattc agaactaatg aaagctt 837

<210> 3

<211> 274

<212> PRT

<213> Artificial Synthesis ()

<400> 3

Met His His His His His His Ala Ile Pro Ser Val Ser Phe Thr Ile

1 5 10 15

Thr Arg Ile Pro Gly Asp Asp Lys Thr Asp Tyr Lys Gln Leu Met Val

20 25 30

Asp Leu Arg Lys Lys Leu Ser Ser Gly Thr Thr Ser Asn Gly Val Pro

35 40 45

Val Leu Arg Ser Thr Ala Ser Lys Glu Ala Lys Tyr Leu Leu Val Asn

50 55 60

Ile Ile Asn Ser Gly Lys Lys Glu Ile Thr Leu Gly Leu Asn Val Ile

65 70 75 80

Asn Ala Tyr Val Leu Ala Tyr Lys Val Gly Asp Lys Ser Tyr Phe Phe

85 90 95

Asn Asp Pro Thr Glu Leu Lys Asp Ala Gln Thr His Leu Phe Lys Asp

100 105 110

Thr Lys Gln Thr Ala Ile Lys Ile Thr Gly Ser Tyr Asp Ser Leu Lys

115 120 125

Ala Gln Gly Gly Asp Arg Glu Ser Val Asp Leu Gly Ile Gly Gln Leu

130 135 140

Asp Ser His Ile Tyr Thr Leu His Lys Ser Thr Ala Leu Lys Asp Val

145 150 155 160

Ala Lys Ser Leu Val Cys Ile Ile Gln Met Val Ser Glu Ala Ala Arg

165 170 175

Phe Lys Ser Ile Glu Asn Lys Ile Val Asp Lys Ile Asp Gly Ser Phe

180 185 190

Lys Pro Lys Leu Asp Ile Ile Thr Arg Glu Asn Asn Trp Gly Asp Leu

195 200 205

Ser Glu Gly Ile Gln Asn Ala Asp Lys Lys Gly Asn Phe Lys Thr Lys

210 215 220

Val Arg Leu Gln Lys Glu Asp Gly Lys Glu Asp Ile Ile Ser Asn Val

225 230 235 240

Asn Gln Ile Ile Gly Glu Met Gly Ile Leu Leu Tyr Lys Lys Lys Lys

245 250 255

Ile Tyr Asn Ile Pro Ser Phe Gly Gln Thr Asn Phe Gly Asn Leu Ile

260 265 270

Gln Asn

Claims

1. The jatropha curcas ribosome inactivating protein JcRIP12 encoding gene is characterized in that the encoding gene encodes a protein with an amino acid sequence shown as a sequence table SEQ ID NO.3, and the nucleotide sequence of the encoding gene is shown as the sequence table SEQ ID NO. 1.

2. A codon-optimized jatropha curcas ribosome inactivating protein JcRIP12 encoding gene is characterized in that the encoding gene encodes a protein with an amino acid sequence shown as a sequence table SEQ ID NO.3, and the nucleotide sequence of the encoding gene is shown as a sequence table SEQ ID NO. 2.

3. The jatropha curcas ribosome inactivating protein JcRIP12 is characterized in that the amino acid sequence of the jatropha curcas ribosome inactivating protein JcRIP12 is shown as a sequence table SEQ ID NO. 3.

4. A recombinant vector comprising the coding gene of claim 2.

5. A host comprising the gene of claim 2.

6. A method for preparing the Jatropha curcas ribosome inactivating protein JcRIP12 as claimed in claim 3, which is characterized in that the host is cultured under the condition that the host produces more Jatropha curcas ribosome inactivating protein JcRIP12, and then the Jatropha curcas ribosome inactivating protein JcRIP12 produced by the host is separated and purified.

7. The use of the jatropha curcas ribosome inactivating protein jcrrip 12 in the manufacture of a medicament for treating tumors as claimed in claim 3.

8. The use according to claim 6, wherein the tumor is human non-small cell lung cancer, human osteosarcoma, human hepatoma or human gastric cancer.