Ssbbi and ssbbi3 genes and their encoding products and use

Abstract

The present application relates to genetic engineering and relates to a trichosanthin trypsin inhibitor gene SsBBI and SsBBI3 gene and its encoded product and application SsBBI3 The nucleotide sequence of the gene is shown as SEQ ID NO. 1, the amino acid sequence of the encoded product is shown as SEQ ID NO. 2, and the trichosanthin trypsin inhibitor protein SsBBI3 is obtained by cloning, amplification and expression in engineering bacteria of the trichosanthin trypsin inhibitor gene SsBBI3 The protein is modified from the trichosanthin trypsin inhibitor protein SsBBI, and the modification method is to cut off the intramembrane region and the transmembrane region of SsBBI and retain the extramembrane region. The nucleotide sequence of the SsBBI protein is shown as SEQ ID NO. 3, and the amino acid sequence of the encoded product is shown as SEQ ID NO. 4. This trichosanthin trypsin inhibitor has the effect of significantly inhibiting tumor cell proliferation at a low dose, and has good application prospect.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a gene based on the trisparganum trypsin inhibitor. SsBBI of SsBBI3 Genes and their encoded products and applications. Background Technology

[0002] The Chinese medicinal herb Sparganii Rhizoma is derived from the plant Sparganii Rhizoma, belonging to the family Sparganaceae. Sparganium stoloniferum The tuber of *Buch.-Ham.* is classified as a blood-activating and stasis-removing herb. The *Chinese Pharmacopoeia* (2020 edition, Part I) records that *Sparganium stoloniferum* has the effects of breaking up blood stasis, promoting qi circulation, eliminating stagnation, and relieving pain; it is commonly used in modern cancer treatment. *Sparganium stoloniferum* can significantly inhibit tumor cell proliferation and growth, prevent tumor invasion and metastasis, induce apoptosis, and regulate the cell cycle. Currently, the pharmacological basis of *Sparganium stoloniferum* is not fully understood. While there is considerable research on its small molecule compounds, no single characteristic component with significant pharmacological activity has been identified. Macromolecules, as natural products of plants, represent a natural resource waiting to be explored.

[0003] Trypsin inhibitors, derived from plants, possess diverse pharmacological activities and hold broad clinical application potential. Numerous studies have demonstrated that protease inhibitors, particularly plant-derived Bowman-Birk trypsin inhibitors (BBIs), exhibit significant antitumor effects in animals, indicating promising clinical applications. For example, soybean BBIs have shown potent inhibitory effects on the progression of gastric and colorectal adenocarcinomas. Similarly, the antiproliferative effects of pea protease inhibitor variant TI1B and pea-derived BBIs on HT-29 cells are also achieved through protease inhibition. Furthermore, mung bean trypsin inhibitor mBTI and buckwheat trypsin inhibitor BTI have been reported to have anti-colorectal and hepatocellular carcinoma effects. These studies suggest that plant-derived trypsin inhibitors can inhibit the occurrence and development of various cancers, demonstrating potential application value in antitumor drugs.

[0004] Currently, with the rapid development of molecular biology and pharmacology, a large number of plant BBI family proteins have been identified and proven to have anti-tumor activity, but research on the identification of protease inhibitors in medicinal plants is relatively lacking. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to address the lack of clarity regarding the material basis of the antitumor effect of *Sparganium stoloniferum* in existing research, and to develop a *Sparganium stoloniferum* trypsin inhibitor gene-based inhibitor through extensive experiments. SsBBI and SsBBI3 Applications of genes and their encoded products in anti-tumor therapy.

[0006] Technical solution: To achieve the above objectives, this invention discloses a trigonelline trypsin inhibitor gene. SsBBI3 The nucleotide sequence is shown in SEQ ID NO.1.

[0007] This invention also discloses the above-mentioned trigonelline trypsin inhibitor gene. SsBBI3 The encoded product has the amino acid sequence shown in SEQ ID NO.2. The product may be RNA, polypeptide, or protein.

[0008] This invention discloses a trigonelline trypsin inhibitor gene. SsBBI The nucleotide sequence is shown in SEQ ID NO.3.

[0009] This invention also discloses the above-mentioned trigonelline trypsin inhibitor gene. SsBBI The encoded product has the amino acid sequence shown in SEQ ID NO.4. The product may be RNA, polypeptide, or protein.

[0010] This invention also discloses the above-mentioned trigonelline trypsin inhibitor gene. SsBBI3 Specific primers, SsBBI3 Cloning was performed using the upstream primer: SsBBI3-6P-CEF.

[0011] 5'-ttccaggggcccctgggatccGCTCGCTTACAACCTGGTGC-3' and downstream primer: SsBBI3-6P-CER: 5'-ctcgagtcgacccgggaattcTCACGCGTTGAGATTCAACAGC-3'.

[0012] This invention also discloses the above-mentioned trigonelline trypsin inhibitor gene. SsBBI Specific primers, SsBBI Cloning was performed using the upstream primer SsBBI-6P-CEF:

[0013] 5'-ttccaggggcccctgggatccAACATCAAGAGATCTAGAGAAGAGAGAATAA-3'

[0014] Downstream primer SsBBI-6P-CER:

[0015] 5'-ctcgagtcgacccgggaattcTCACGCGTTGAGATTCAACAGC-3'.

[0016] This invention also discloses a gene containing the above-mentioned trigonelline trypsin inhibitor. SsBBI and SsBBI3The recombinant expression vector is pGEX-6P-1.

[0017] The application further discloses a trichosanthin trypsin inhibitor gene SsBBI3 and an application of a product coded by the trichosanthin trypsin inhibitor gene in preparing a Bowman-Birk type protease inhibitor anticancer substance, which comprises the following steps: SsBBI3 expressing the trichosanthin trypsin inhibitor SsBBI3 protein in the host cell, and the trichosanthin trypsin inhibitor SsBBI3 protein inhibits the proliferation of breast cancer cells MCF-7, breast cancer cells MDA-MB-231, cervical cancer cells HeLa and non-small cell lung cancer cells A549.

[0018] The trichosanthin trypsin inhibitor gene SsBBI or the trichosanthin trypsin inhibitor gene SsBBI The coded product also has the effect of the Bowman-Birk type protease inhibitor and also has the anticancer activity.

[0019] The application has the beneficial effects compared with the prior art in that:

[0020] The trichosanthin trypsin inhibitor gene SsBBI and SsBBI3 The gene is prepared by cloning from the trichosanthin plant for the first time. SsBBI and SsBBI3 The protein structures of the gene expression products SsBBI and SsBBI3 are similar, have the same conserved sequence structure, but since SsBBI has a transmembrane domain and is difficult to express, the SsBBI3 protein retaining the conserved domain is expressed to explore the functional effect, and the experimental results show that the trichosanthin trypsin inhibitor gene SsBBI3 is a key material basis for the trichosanthin to play the anti-tumor effect, can be used for inhibiting the proliferation of breast cancer cells MCF-7, breast cancer cells MDA-MB-231, cervical cancer cells HeLa and non-small cell lung cancer cells A549, and the protein can be applied to the preparation of an anticancer agent based on the trichosanthin trypsin inhibitor. The gene provided by the application can be used for mass production of high-efficiency anticancer agents through genetic engineering technology, and provides a research direction for the preparation of a new anticancer agent. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The trichosanthin trypsin inhibitor gene SsBBI and SsBBI3 is an agarose gel electrophoresis diagram of the trichosanthin trypsin inhibitor gene SsBBI and the trichosanthin trypsin inhibitor gene SsBBI3 ;

[0022] Figure 2 Sequence alignment was performed between the triangular trypsin inhibitor protein SsBBI and other Bowman-birk inhibitor family proteins from other species; Note: DN866_c0_g1_orf1 is the SsBBI protein sequence.

[0023] Figure 3 This is the secondary mass spectrum of the only peptide segment of the trigonelline trypsin inhibitor protein SsBBI.

[0024] Figure 4 Prediction of the transmembrane domain of the trigonelline trypsin inhibitor protein SsBBI;

[0025] Figure 5 Prediction of the secondary structure of the trigonelline trypsin inhibitor protein SsBBI;

[0026] Figure 6 Disulfide bond prediction for the trigonelline trypsin inhibitor protein SsBBI;

[0027] Figure 7 Phylogenetic tree of the triangular trypsin inhibitor protein SsBBI; Note: TRINITY DN866_c0_g1 is the gene sequence encoding the SsBBI protein;

[0028] Figure 8 Prediction of the tertiary structures of the trigonelline trypsin inhibitor proteins SsBBI and SsBBI3;

[0029] Figure 9 For the predicted structure of SsBBI and trypsin and Ss BBI binding site;

[0030] Figure 10 SDS-PAGE gel electrophoresis results of the SsBBI3-GST fusion protein, a trypsin inhibitor protein; Note: M is the protein marker, B↑ is the supernatant of the p-GEX6P-1 empty negative control lysed bacterial suspension, SB3-6P↑ is the supernatant of the lysed bacterial suspension of the large-scale SsBBI3-GST expression system, and E1~E8 are fractions 1 to 8 of the purified SsBBI3-GST protein.

[0031] Figure 11 The results of SDS-PAGE gel electrophoresis of the purified SsBBI3 trypsin inhibitor protein are shown below. Note: M is the protein marker, B↑ is the supernatant of the empty negative control lysed bacterial culture, SB3-6P↑ is the supernatant of the large-scale expression SsBBI3-GST lysed bacterial culture, and SB3 is the SsBBI3 protein obtained by enzyme digestion and purification.

[0032] Figure 12MALDI-TOF MS spectrum of purified SsBBI3;

[0033] Figure 13 Bar chart of SsBBI3 on breast cancer cell MCF-7, breast cancer cell MDA-MB-231, cervical cancer cell HeLa and non-small cell lung cancer cell A549. DETAILED DESCRIPTION

[0034] The above and other technical features and advantages of the present application will be more apparent from the following detailed description, taken in conjunction with the accompanying drawings.

[0035] The technical means used in the examples are conventional means known to those skilled in the art, and the raw materials used are commercially available.

[0036] Non-toxic Super Gel Blue TM Nucleic acid dyes were purchased from Shanghai Baysi Biological Technology Co., Ltd.; Gel recovery kit GeneJET Gel Extraction Kit was purchased from Thermo Fisher Scientific; 2x Rapid Taq MasterMix (P222); polysaccharide polyphenolic plant total RNA extraction kit FastPure Plant Total RNA Isolation Kit (Polysaccharides & Polyphenolics-rich) (RC401), reverse transcription kit HiScript III 1stStrand cDNA Synthesis Kit (+gDNA wiper) (R312-01 / 02), DH5α chemically competent cells (C502), BL21(DE3) chemically competent cells (C504) were purchased from Novizen Biotechnology Co., Ltd. Eco RI (1014A), Bam HI (1010A) and other restriction endonucleases, 10xK Buffer (ALG016A), 10xLoading Buffer (9157) were purchased from TaKaRa; Elution Buffer (pH7.9~8.1) for GST-Sefinose TM Resin (C600325-0500), Binding / Wash Buffer (pH7.3~7.5) for GST-Sefinose TMResin (C600326-0500), GST 4FF (Pre-Packed Gravity Column) (C600327-0001) were purchased from Shanghai Generay Biotech Co., Ltd. BCA Protein Assay Kit (KGP903) was purchased from Jiangsu Keygen Biotech Co., Ltd.; primers were synthesized by Shanghai Generay Biotech Co., Ltd.; other reagents were imported or domestic analytical pure reagents.

[0037] One, tridacna trypsin inhibitor gene SsBBI and SsBBI3 Cloning

[0038] SsBBI The cloning of tridacna trypsin inhibitor gene utilized upstream primer SsBBI-6P-CEF (as shown in SEQ ID NO. 5 in the sequence listing):

[0039] 5'-ttccaggggcccctgggatccAACATCAAGAGATCTAGAGAAGAGAGAATAA-3'

[0040] and downstream primer SsBBI-6P-CER (as shown in SEQ ID NO. 6 in the sequence listing):

[0041] 5'-ctcgagtcgacccgggaattcTCACGCGTTGAGATTCAACAGC-3'.

[0042] SsBBI3 The cloning of tridacna trypsin inhibitor gene utilized upstream primer SsBBI3-6P-CEF (as shown in SEQ ID NO. 7 in the sequence listing):

[0043] 5'-ttccaggggcccctgggatccGCTCGCTTACAACCTGGTGC-3' and downstream primer SsBBI3-6P-CER (as shown in SEQ ID NO. 8 in the sequence listing):

[0044] 5'-ctcgagtcgacccgggaattcTCACGCGTTGAGATTCAACAGC-3'. The PCR amplification was carried out with the total RNA of tridacna tuber cDNA library as a template. The amplification system was as follows: 2x Rapid Taq Master Mix enzyme 5 μL, primer primer-F and primer-R each 0.4 μL, template 1 μL, the rest was supplemented with sterile double distilled water. The reaction conditions were as follows: 95℃ pre-denaturation for 2 min, 95℃ denaturation for 20 s, 60℃ annealing for 20 s, 72℃ extension for 1 min, 35 cycles, then 72℃ extension for 5 min, 4℃ preservation. Thus, the tridacna trypsin inhibitorSsBBI and SsBBI3 Gene cloning, SsBBI3 gene sequence is As shown in SEQ ID NO.1 in the sequence list.

[0045] II. Bioinformatics Analysis of the Trigonella Trichopsaccharide Trypsin Inhibitor Protein SsBBI

[0046] The full-length cDNA of the trigonelline trypsin inhibitor protein SsBBI obtained in this invention... SsBBI The open reading frame (ORF) of the gene is 384 bp in length, and its detailed sequence is shown in SEQ ID NO.3 of the sequence listing. The SsBBI protein was sequence-aligned with other Bowman-birk inhibitor family proteins from other species using DNAMAN software, such as... Figure 2 As shown, all exhibit a highly conserved nine-amino acid binding ring motif CTXSXPPXC, which is located in the Cys region of SsBBI. 64 To Cys 72 Within the trigoninome, the unique peptide sequence of the SsBBI protein is SIPPICFCR, which overlaps with the conserved motif of the Bowman-birk inhibitor family of proteins. The secondary mass spectrometry of the peptide is shown below. Figure 3 As shown. The distribution of the transmembrane region and disulfide bonds of the SsBBI protein was predicted using the Predictprotein online website. This protein is a transmembrane protein; amino acids 1-17 at the N-terminus form the intramembrane region, amino acids 18-35 form the transmembrane region, and amino acids 36-127 form the extramembrane region, as shown. Figure 4 As shown.

[0047] I-TASSER analysis revealed that the secondary structure of the SsBBI protein consists of α-helices, β-sheets, and random coils, as shown in the figure. Figure 5 As shown; Ss BBI can form more disulfide bonds between cysteine ​​residues, such as Figure 6 As shown. A phylogenetic tree of the SsBBI protein sequence was constructed using the Neighbor-joining mode in MEGA10 software. The SsBBI protein is related to the pineapple, which is also a monocotyledonous plant. Ananas comosus The two Bowman-Birk protease inhibitors in *Var. bracteatus* are highly related and clustered together, with the highest degree of phylogenetic relationship. This suggests that the SsBBI protein likely possesses similar functions to Bowman-Birk protease inhibitors in monocotyledonous plants, such as... Figure 7 As shown. The Alphafold online website predicts tertiary structure models for the SsBBI and SsBBI3 proteins. The SsBBI3 protein, except for the absence of an intramembrane and transmembrane region, is highly similar in structure to the SsBBI protein, as shown below. Figure 8As shown. Performed using Discovery Studio. Ss Dating BBI protein to trypsin model, such as Figure 9 The binding sites of SsBBI to trypsin include the Ile group near the N-terminus. 63 Cys 64 Thr 65 Arg 66 and Ala, which is closer to the C end 112 、Gln 114 Lys 115 Pro 116 、Gln 118 Leu 119 Leu 123 Its spatial location is very close to Cys 64 to Cys 72 The highly conserved nine-amino acid binding loop shows that the three-dimensional structures of the SsBBI and SsBBI3 proteins are highly similar in this binding region. The docking results indicate that the SsBBI protein has trypsin inhibitory function of Bowman-birk inhibitor family proteins, and the SsBBI3 protein also has the functional characteristics of Bowman-birk inhibitor family proteins.

[0048] Trigoninase inhibitor SsBBI3 Induction and purification of genes and engineered bacteria

[0049] To better adapt to the E. coli expression system, SsBBI Gene sequence optimization was obtained SsBBI3 The gene was cloned and ligated into the expression vector pGEX-6P-1 to obtain... SsBBI3 -pGEX-6P-1 recombinant plasmid.

[0050] Recombinant plasmid SsBBI3 Transform BL21(DE3) competent cells with pGEX-6P-1 and culture and screen for cells containing [specific cells]. SsBBI3 Positive strains of the -pGEX-6P-1 recombinant plasmid SsBBI3 -pGEX-6P-1-BL21. Add the transformed expression bacterial culture to LB medium containing Amp resistance at a ratio of 1:100, and incubate at 37°C with shaking at 200 rpm until OD... 600=0.6, added to a final concentration of 1 mMIPTG and induced at 10℃ for 24 h. pGEX-6P-1 empty vector was treated under the same conditions as a blank control. 800 mL of bacterial culture was centrifuged at 5000 rpm for 15 min at 4℃, the supernatant was discarded to obtain bacterial cells, 20 mL of Lysis Buffer was added, and the cells were resuspended. The centrifuge tubes were placed in an ultrasonic homogenizer for ultrasonic homogenization for 10 min (5 s intervals) at 40% efficiency. The centrifuge tubes were inserted into a beaker containing an ice-water mixture and operated on ice. The ultrasonically homogenized lysate was centrifuged at 12000 rpm for 30 min at 4℃ to obtain... SsBBI3 The supernatant of the gene.

[0051] Purification of the recombinant protein SsBBI3-pGEX-6P-1 using a GST 4FF gravity column was performed as follows: The GST 4FF gravity column was opened, and after the 20% ethanol buffer was allowed to flow out naturally, 5 mL of binding buffer was added to equilibrate the column. Simultaneously, binding buffer was added to the sample supernatant at a 1:1 volume ratio and mixed thoroughly. After the binding buffer had completely flowed out, the sample was loaded onto the column, allowing it to flow down naturally under gravity. The effluent was collected. After the first loading of the sample, the effluent was loaded onto the column again, using a 20 mL syringe with a pore stopper for assistance. After two loadings, 2 mL of binding buffer was added each time to elute contaminating proteins, and A was measured. 280 Reduce to below 0.01 mg / mL. Add Wash Buffer to elute the target protein, 2 mL each time, for A. 280 Protein concentration was determined and detected by SDS-PAGE electrophoresis. A standard curve was plotted and protein concentration was determined using the KGI BCA protein assay kit. 20 μL of protein eluent was added to 4 μL of 5× Protein Loading Dye and incubated at 100℃ for 5 min. The mixture was then analyzed by 12.5% ​​SDS-PAGE gel electrophoresis (220 V, 60 min). Clear specific protein expression bands appeared at molecular weights of approximately 26 kDa and 36 kDa. Figure 10 As shown.

[0052] Recombinant plasmids SsBBI3-pGEX-6P-1 transformed BL21 (DE3) competent cells to the collection and broken step, using GST 4FF gravity column for three-prism recombinant protein SsBBI3-pGEX-6P-1 on-column enzyme cutting purification operation, the specific operation is as follows: open GST 4FF gravity column, natural flow out 20% ethanol buffer, add 5 mL Binding Buffer to balance the column, at the same time, add Binding Buffer to the sample supernatant at a volume ratio of 1:1, mix evenly, and flow out in Binding Buffer. After the column is filled, rely on gravity to flow down, and after the first column filling of the sample is completed, the flow-out liquid is filled into the column, and after three times of repetition, 20 mL of Binding Buffer is added to elute the impurities each time. A 280 to less than 0.01 mg / mL. Add 2 mL of enzyme cutting buffer containing 100 μg of PreScission Protease (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.0), 4°C, 240 rpm shaking for 36 h, then stand for 30 min, and then flow out. Use KJBCA protein content detection kit to draw standard curve and determine protein concentration. 20 μL protein eluent plus 4 μL 5× Protein Loading Dye, incubate at 100°C for 5 min, and detect by 15% SDS-PAGE gel electrophoresis (220 V, 60 min). At a molecular weight of about 15 kDa, there is an obvious specific protein expression band, as shown in Figure 11 . The purified protein SsBBI3 was detected by MALDI-TOF MS (Smicroflex LRF, Bruker), and the purity was high, and the molecular weight was as expected, as shown in Figure 12 .

[0053] Four, in vitro enzyme function verification

[0054] Three-prism trypsin inhibitor protein SsBBI3 function verification: after the purified SsBBI3 protein was dosed to breast cancer cells MCF-7, breast cancer cells MDA-MB-231, cervical cancer cells HeLa, and non-small cell lung cancer cells A549. The dosing concentration of breast cancer cells MCF-7 and MDA-MB-231 cells was set to 1×10 -5 µM, 1×10 -4 µM, 1×10 -3 µM, 0.01 µM, 0.1 µM, 1 µM, the drug administration concentration for cervical cancer cells (HeLa) and non-small cell lung cancer cells (A549) was set at 0.015625 µM. µM, 0.03125 µM, 0.0625 µM, 0.125 µM, 0.25 µM, 0.5 µM, 1 µM, incubation time was 0.5 h, 1 h, 2 h, 3 h, and 4 h. Negative control was enzyme digestion buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.0), positive control was 1 mM 5-FU. Cell CCK-8 assay results demonstrated that SsBBI3 significantly inhibited the proliferation of MCF-7 breast cancer cells, MDA-MB-231 breast cancer cells, HeLa cervical cancer cells, and A549 non-small cell lung cancer cells. Figure 13 As shown.

[0055] The above description is merely a preferred embodiment of the present invention and is illustrative rather than restrictive. Those skilled in the art will understand that many changes, modifications, and even equivalents can be made within the spirit and scope defined by the claims of the present invention, all of which will fall within the protection scope of the present invention.

Claims

1. A triosephosphate isomerase inhibitor gene SsBBI3 characterized in that, The nucleotide sequence of the gene is shown as SEQ ID NO.

1.

2. The trypsin inhibitor gene of claim 1 SsBBI3 the encoded product, characterized in that, The amino acid sequence of the product is shown as SEQ ID NO.

2.

3. The specific primer of the trypsin inhibitor gene SsBBI3 according to claim 1, characterized in that, The upstream primer comprises: 5'-ttccaggggcccctgggatccGCTCGCTTACAACCTGGTGC-3' and the downstream primer: 5'-ctcgagtcgacccgggaattcTCACGCGTTGAGATTCAACAGC-3'.

4. A recombinant expression vector comprising the triose phosphate isomerase inhibitor gene of claim 1. SsBBI3 4. A recombinant expression vector comprising the triose phosphate isomerase inhibitor gene of claim 1.​ 5. The recombinant expression vector comprising the triose phosphate isomerase inhibitor gene according to claim 4, characterized in that, SsBBI3 The expression vector is pGEX-6P-1. ​ 6. A gene for a trypsin inhibitor from Scorzonera SsBBI3 or a gene for a trypsin inhibitor from Scorzonera SsBBI3 Use of the encoded product in the manufacture of a Bowman-Birk type protease inhibitor anticancer drug; said anticancer drug being an anticancer drug against breast cancer, cervical cancer or non-small cell lung cancer.

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