CN110563822A - Ganoderma lucidum immunomodulatory protein mutant and application thereof - Google Patents

Abstract

A Ganoderma lucidum immunomodulatory protein mutant, comprising: FIP-gluM _N31S , FIP-gluM _T36N and FIP-gluM _N31S/T36N , wherein: the amino acid sequence and nucleotide sequence of FIP-gluM _N31S are respectively as SEQ ID NO.1 and Shown in SEQ ID NO.4; the amino acid sequence and nucleotide sequence of FIP-gluM _T36N are shown in SEQ ID NO.2 and SEQ ID NO.5 respectively; the amino acid sequence and nucleotide sequence of FIP-gluM _N31S/T36N Shown as SEQ ID NO.3 and SEQ ID NO.6 respectively. The invention can solve the problem that FIP-glu is more toxic to cells in application, and the toxicity of the modified mutant to cells is significantly lower than that of Ganoderma lucidum immunoregulatory protein (FIP-glu), which can effectively improve its utilization value .

Description

Ganoderma lucidum immunoregulatory protein mutant and its application

technical field

The invention relates to a technique in the field of bioengineering, in particular to a mutant of ganoderma lucidum immunoregulatory protein.

Background technique

Fungal immunomodulatory protein (fugal immunomodulatory protein, FIP), which was originally isolated from higher basidiomycetes, is a small molecule protein with immune regulation function. After Kino et al. isolated the first FIP (LZ-8 or FIP-glu) from Ganoderma lucidum (Ganodermaspp.) in 1989, researchers successively discovered more FIPs from various edible and medicinal fungi. They constitute a new protein family - FIP. The proteins of the FIP family have various immunoregulatory effects such as anti-tumor, anti-allergic, and stimulating immune cells to produce various cytokines, and have good clinical application prospects and medicinal and health care values. Fungal immunomodulatory protein consists of 110-140 amino acids, with a molecular weight of about 13kD, which lacks histidine, cysteine and methionine, and is rich in aspartic acid and valine. The N-terminal amino acids are all acetylated amino acids. The vast majority of FIP exists in the form of homodimers. Furthermore, FIP shares great similarities with the variable domains of immunoglobulin heavy chains.

Studies have found that FIP has obvious growth inhibitory activity on a variety of cancer cells. For example, recombinant FIP-gts expressed in Escherichia coli can inhibit the growth of human lung cancer cell A549 in vivo and in vitro (Chien-Huang Liao, Yi-Min Hsiao, Chung-Ping Hsu, Meii-Yn Lin, James Chun-Huan Wang, & Yu-Lu Huang, et al. (2006). Transcriptionally mediated inhibition of telomerase of fungalimmunomodulatory protein from ganoderma tsugae in a549 human lungadenocarcinoma cell line. Molecular Carcinogenesis, 45.), is by down-regulating the catalytic subunit of telomerase (hTERT ) transcriptional regulation, inhibition of telomerase activity to cause inhibition of cell growth. In addition, recombinant FIP-glu obtained from yeast significantly inhibited human leukemia cell NB4 (Lin, J.W., Hao, L.X., Xu, G.X., Sun, F., Gao, F., & Zhang, R., et al. (2009 ). Molecular cloning and recombinant expression of a geneencoding a fungal immunomodulatory protein from ganoderma lucidumin piciapastoris. World Journal of Microbiology & Biotechnology, 25(3),

383-390.), human lung cancer cell A549 and Lewis lung cancer cell LLC1 (Wu, C.T., Lin, T.Y., Hsu, H.Y., Sheu, F., Ho, C.M., & Chen, I.T..(2011).Ling zhi-8mediates p53 -dependent growth arrest of lung cancer cells proliferation via the ribosomal protein s7-mdm2-p53 pathway. Carcinogenesis, 32(12), 1890-1896.) Growth and proliferation. We also found that recombinant FIP-gat can inhibit the growth of human breast cancer cell line MDA-MB-231 (Xu, H., Kong, Y.Y., Chen, X., Guo, M.Y., & Zhou, X..(2016). Recombinant fip-gat, a fungal immunomodulatory protein from ganoderma atrum, induces growth inhibition and cell death in breast cancer cells. Journal of Agricultural and Food Chemistry, 64(13).), through chip analysis, it was found that after recombinant FIP-gat treatment, MDA- There were 669 differentially expressed genes in MB-231, and their fold changes were at least 2-fold, and genes related to apoptosis, such as TNFSF8, DRD1, SMPD1 and BCL-2, were also up-regulated. According to the research, the recombinant FIP-glu produced by the yeast expression system is toxic to the mouse macrophage RAW264.7 in a certain range by means of genetic engineering. Big restrictions.

Contents of the invention

Aiming at the above-mentioned deficiencies in the prior art, the present invention proposes a Ganoderma lucidum immunoregulatory protein mutant, which can solve the problem that FIP-glu is more toxic to cells in the application. The modified Ganoderma lucidum immune regulatory protein mutant FIP-gluM _N31S The toxicity of FIP-gluM _T36N and FIP-gluM _N31S/T36N to cells is significantly lower than that of Ganoderma lucidum immunomodulatory protein (FIP-glu), which can effectively improve its utilization value.

The present invention is achieved through the following technical solutions:

The present invention relates to a class of Ganoderma lucidum immunoregulatory protein mutants, including: FIP-gluM _N31S , FIP-gluM _T36N and FIP-gluM _N31S/T36N , wherein: the amino acid sequence and nucleotide sequence of FIP-gluM _N31S are respectively as SEQ ID NO .1 and shown in SEQID NO.4; the amino acid sequence and nucleotide sequence of FIP-gluM _T36N are shown in SEQ ID NO.2 and SEQ ID NO.5 respectively; the amino acid sequence and nucleosides of FIP-gluM _N31S/T36N The acid sequences are shown in SEQ ID NO.3 and SEQ ID NO.6, respectively.

The present invention relates to the preparation method of the above-mentioned mutant, using the nucleotide sequence of Ganoderma lucidum immunoregulatory protein (FIP-glu) shown in SEQ ID NO.7 and SEQ ID NO.8 as a template, It is obtained by PCR amplification using corresponding primer sequences.

The corresponding primer sequence refers to:

①Use the primers shown in SEQ ID NO.9-12 to obtain FIP-gluM _{N31S through} PCR amplification;

② using primers shown in SEQ ID NO.9, 12, 13, 14, PCR amplification to obtain FIP-gluM _T36N ;

③ Using the primers shown in SEQ ID NO.9, 10, 12 and 15, FIP-gluM _N31S/T36N was obtained by PCR amplification.

Said PCR amplification uses vectors including but not limited to plasmids, phages or virus vectors, preferably eukaryotic expression vector pPIC9K.

In the preparation method, it is further preferred that the amplified nucleotide sequence is cloned into a vector, and after constructing a recombinant vector and transforming into an engineering bacterium, it is induced to express and purified.

The engineering bacteria is preferably yeast.

The present invention relates to the application of the above-mentioned Ganoderma lucidum immunoregulatory protein mutant for preparing a drug for treating tumors.

The tumors mentioned include: adrenal gland tumors, bile duct tumors, bladder tumors, blood tumors, bone and connective tissue tumors, brain and central nervous system tumors, breast tumors, cervical tumors, colorectal tumors (colorectal tumors), endometrial tumors , esophageal tumors, gallbladder tumors, head and neck tumors, Hodgkin's lymphoma, hypopharyngeal tumors, kidney tumors, laryngeal tumors, leukemia, liver tumors, lung tumors, lymphoma, mediastinal tumors, melanoma (malignant melanoma), mesothelioma, multiple myeloma, nasal cavity tumors, nasopharyngeal tumors, neuroendocrine tumors, non-Hodgkin's lymphoma, oral cavity tumors, esophageal tumors, oropharyngeal tumors, ovarian tumors, pancreatic tumors, sinuses Tumors, parathyroid tumors, penile tumors, pituitary tumors, prostate tumors, salivary gland tumors, sarcoma, skin tumors, spine tumors, stomach tumors, testicular tumors, thyroid tumors, urethral tumors, uterine tumors, vaginal tumors, and vulvar tumors.

The medicine comprises a medicine carrier and a therapeutically effective dose of the Ganoderma lucidum immunoregulatory protein mutant of the present invention dispersed therein. The composition may be solid or liquid. The choice of pharmaceutical carrier will generally depend on the type of administration being used and can be solid or liquid. The Ganoderma lucidum immunoregulatory protein mutant of the present invention may be in the same or different phases as the drug carrier.

The medicine is in the form of tablet, coated tablet, hard or soft gelatin capsule, solution, emulsion, suspension, suppository or injection.

technical effect

Compared with the prior art, the mutants of the present invention have simple acquisition methods, expression methods, and purification processes, and the prepared Ganoderma lucidum immunomodulatory protein mutants have lower cytotoxicity and higher biological active.

Description of drawings

Fig. 1 is the schematic diagram of the detection of recombinant Ganoderma lucidum immunomodulatory protein in the embodiment;

In the figure: A: SDS-PAGE detection; B: Western Blot detection;

Fig. 2 is the schematic diagram of embodiment cytotoxicity detection;

Fig. 3 is a schematic diagram of detection of TNF-α transcription level in the embodiment.

Detailed ways

Example 1

Obtaining Ganoderma lucidum immunoregulatory protein (FIP-glu)

1) Extract the genomic DNA of Chizhi. Ganoderma lucidium 50044 was preserved by the Plant Biotechnology Research Center of Shanghai Jiaotong University. Ganoderma lucidum RNA extraction method refers to Tiangen (TIANGEN) RNA extraction kit.

2) Amplify the Ganoderma lucidum immunoregulatory protein gene FIP-glu by PCR, and recover and purify the amplified target gene.

3) The pPIC9K vector and the amplified FIP-glu gene PCR product were digested with EcoR I and Apa I, and the digested plasmid and target gene were recovered. The recovered plasmid and the target gene were ligated with T ₄ DNA ligase, and then transformed into competent E.coli DH5α cells. The above pPIC9K vector and competent E.coli DH5α cells were preserved by the laboratory of Fudan-Jiaotong University-Nottingham Plant Biotechnology Research and Development Center; the above-mentioned EcoR I enzyme, Apa I enzyme and T ₄ DNA ligase were purchased from Bao Bioengineering (Dalian) Co., Ltd. .

4) After sequencing by Shanghai Sangon Bioengineering Co., Ltd., a recombinant expression vector with the correct sequence, namely pPIC9K-glu, was obtained. The pPIC9K-glu plasmid was extracted, and the plasmid was linearized with restriction endonuclease Sac I, and then transformed into Pichia pastoris GS115. After identification by PCR, positive yeast transformants were obtained. The aforementioned restriction enzyme Sac I was purchased from New England Bio-labs (NEB); Pichia pastoris GS115 was preserved by the laboratory of Fudan-Jiaotong University-Nottingham Plant Biotechnology R&D Center.

5) Using BMMY medium, induce yeast transformants to produce recombinant FIP-glu protein. The expression products were identified by SDS-PAGE and Western Blot, and the results are shown in lane 1 of A and B in Figure 1 .

Example 2

Obtaining the Immunomodulatory Protein Mutant FIP-gluM _N31S of Ganoderma lucidum

Using the nucleotide sequence shown in SEQ ID NO.7 as a template, and using P1 and P2, and P3 and P4 as primers (sequences shown in SEQ ID NO.9-12) respectively, the PCR amplification reaction was carried out. After the obtained products are mixed, a PCR reaction is carried out again, so as to obtain the FIP-gluM _N31S sequence of the Ganoderma lucidum immunoregulatory protein mutant gene. The recombinant Ganoderma lucidum immunoregulatory protein mutant FIP-gluM _N31S was obtained in the same manner as in Example 1, and its expression product was identified by SDS-PAGE and Western Blot, and the obtained protein was determined to be the Ganoderma lucidum immunomodulatory protein mutant FIP-gluM _N31S , and the identification results were as follows Shown in lane 2 of A and B in Figure 1.

Table 1 is used for the primer sequence of this embodiment

Example 3

Obtaining the Immunomodulatory Protein Mutant FIP-gluM _T36N of Ganoderma lucidum

Using the nucleotide sequence shown in SEQ ID NO.7 as a template, P1 and P5 and P6 and P4 as primers (sequences shown in SEQ ID NO.9, 12, 13, 14) respectively, carry out PCR amplification reaction . After the obtained products are mixed, a PCR reaction is carried out again, so as to obtain the FIP-gluM _T36N sequence of the Ganoderma lucidum immunoregulatory protein mutant gene. The recombinant Ganoderma lucidum immunoregulatory protein mutant FIP-gluM _T36N was obtained in the same manner as in Example 1, and its expression product was identified by SDS-PAGE and Western Blot to determine that the resulting protein was the Ganoderma lucidum immunomodulatory protein mutant FIP-gluM _T36N . The identification results were as follows Lanes 3 of A and B in Figure 1.

Table 2 is used for the primer sequence of this embodiment

Example 4

Obtaining the Immunomodulatory Protein Mutant FIP-gluM _N31S/T36N of Ganoderma lucidum

Using the nucleotide sequence shown in SEQ ID NO.7 as a template, and using P1 and P2 and P7 and P4 as primers (sequences shown in SEQ ID NO.9, 10, 12, and 15) respectively, carry out PCR amplification reaction . After the obtained products are mixed, a PCR reaction is carried out again to obtain the FIP-gluM _N31S/T36N sequence of the Ganoderma lucidum immunoregulatory protein mutant gene. The recombinant Ganoderma lucidum immunoregulatory protein mutant FIP-gluM _N31S/T36N was obtained in the same manner as in Example 1, and its expression product was identified by SDS-PAGE and Western Blot, and the resulting protein was determined to be the Ganoderma lucidum immunomodulatory protein mutant FIP-gluM _N31S/T36N , The identification results are shown in lane 4 of A and B in Fig. 1 .

Table 3 is used for the primer sequence of this embodiment

Example 5

activity assay

1. Cytotoxicity experiment

The effects of FIP-glu, FIP-gluM _N31S , FIP-gluM _T36N and FIP-gluM _N31S/T36N on the viability of mouse peritoneal macrophage RAW264.7 were determined by methylene blue absorption method. RAW264.7 cells were treated with 10 μg/mL of FIP-glu, FIP-gluM _N31S , FIP-gluM _T36N and FIP-gluM _N31S/T36N , respectively. After 24 hours, the supernatant was discarded, and 50 μL of 0.6% methylene blue was added to each well to stain the cells. After the cell plate was incubated at 37°C for 60 minutes, the supernatant was aspirated. Wash the cells with phosphate buffered saline (PBS), add 50 μL of elution buffer (volume ratio: ethanol: PBS: acetic acid = 50: 49: 1) after air-drying, and after incubation for 20 minutes, use a microplate reader (BIO-TEK, USA) to measure the absorbance at 570 nm. The results are shown in Figure 2, the cytotoxicity of recombinant Ganoderma lucidum immunoregulatory protein mutants FIP-gluM _N31S , FIP-gluM _T36N and FIP-gluM _N31S/T36N on macrophage RAW264.7 was significantly less than that of FIP-glu.

2. Cytokine detection

Mouse peritoneal macrophages RAW264.7 were co-cultured with 10 μg/mL FIP-glu, FIP-gluM _N31S , FIP-gluM _T36N and FIP-gluM _{N31S/T36N for} 6 hours, then the total RNA was extracted and detected by qRT-PCR The yield of TNF-α mRNA, wherein the forward primer of TNF-α is 5'-TTCTATGGCCCAGACCCTCA-3', the reverse primer is 5'-ACAAGGTACAACCCCATCGGC-3'; the forward primer of internal reference β-actin is 5'-ATCGTGCGGGACATCAAGG-3 ', the reverse primer is 5'-TCGTTGCCGATGGTGATGAC-3'. qRT-PCR was performed using a Roche Light Cycler 96. The reaction program was as follows: samples were heated to 95 °C for 1 min, denatured at 95 °C for 20 s, annealed at 55 °C for 20 s, extended at 72 °C for 20 s, and cycled 45 times. The mRNA detection results of cytokine TNF-α are shown in Figure 3. The mRNA levels produced by macrophage RAW264.7 treated with recombinant Ganoderma lucidum immunoregulatory protein mutants FIP-gluM _N31S , FIP-gluM _T36N and FIP-gluM _N31S/T36N The level of TNF-αmRNA was significantly higher than that produced by FIP-glu-treated macrophage RAW264.7.

The above specific implementation can be partially adjusted in different ways by those skilled in the art without departing from the principle and purpose of the present invention. The scope of protection of the present invention is subject to the claims and is not limited by the above specific implementation. Each implementation within the scope is bound by the invention.

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Claims (9)

1. A kind of Ganoderma lucidum immunomodulatory protein mutant, is characterized in that, comprises: FIP-gluM _N31S , FIP-gluM _T36N and FIP-gluM _N31S/T36N , wherein: the amino acid sequence and the nucleotide sequence of FIP-gluM _N31S are respectively as follows Shown in SEQ ID NO.1 and SEQ ID NO.4; the amino acid sequence and nucleotide sequence of FIP-gluM _T36N are shown in SEQ ID NO.2 and SEQ ID NO.5 respectively; the amino acid sequence of FIP-gluM _N31S/T36N And the nucleotide sequence is shown in SEQ ID NO.3 and SEQ ID NO.6 respectively. 2. according to the preparation method of the described mutant of claim 1, it is characterized in that, with the nucleotide sequence of the ganoderma immunomodulatory protein FIP-glu shown in SEQ ID NO.7 as template, with nucleotide and aminoacid sequence respectively The nucleotide sequence of Ganoderma lucidum immunoregulatory protein FIP-glu shown in SEQ ID NO.7 and SEQ ID NO.8 is used as a template, which is obtained by PCR amplification using corresponding primer sequences; The corresponding primer sequence refers to: ①Use the primers shown in SEQ ID NO.9-12 to obtain FIP-gluM _{N31S through} PCR amplification; ② using primers shown in SEQ ID NO.9, 12, 13, 14, PCR amplification to obtain FIP-gluM _T36N ; ③ Using the primers shown in SEQ ID NO.9, 10, 12 and 15, FIP-gluM _N31S/T36N was obtained by PCR amplification. 3. The method according to claim 2, characterized in that, the vectors used in the PCR amplification include but are not limited to plasmids, phages or viral vectors. 4. The method according to claim 3, wherein the vector is a eukaryotic expression vector pPIC9K. 5. The method according to claim 2, characterized in that the nucleotide sequence obtained after PCR amplification is cloned into a vector, and after constructing a recombinant vector and transforming into an engineering bacterium, it is induced to express and purified. 6. An application of the Ganoderma lucidum immunoregulatory protein mutant according to any one of the above claims, characterized in that it is used to prepare a drug for treating tumors. 7. The application according to claim 6, wherein said tumors include: adrenal gland tumors, bile duct tumors, bladder tumors, blood tumors, bone and connective tissue tumors, brain and central nervous system tumors, breast tumors, cervical tumors Tumors, Colorectal Tumors (Colorectal Tumors), Endometrial Tumors, Esophageal Tumors, Gallbladder Tumors, Head and Neck Tumors, Hodgkin's Lymphoma, Hypopharyngeal Tumors, Kidney Tumors, Laryngeal Tumors, Leukemia, Liver Tumors, Lung Tumors, Lymphomas, Mediastinal Tumors, Melanoma (Malignant Melanoma), Mesothelioma, Multiple Myeloma, Nasal Tumors, Nasopharyngeal Tumors, Neuroendocrine Tumors, Non-Hodgkin's Lymphoma, Oral Tumors , Esophageal tumors, Oropharyngeal tumors, Ovarian tumors, Pancreatic tumors, Nasal sinus tumors, Parathyroid tumors, Penile tumors, Pituitary tumors, Prostate tumors, Salivary gland tumors, Sarcoma, Skin tumors, Spinal tumors, Stomach tumors, Testicular tumors, Thyroid tumors, urethral tumors, uterine tumors, vaginal tumors and vulvar tumors. 8. The application according to claim 6, characterized in that the drug comprises a drug carrier and a therapeutically effective amount of the Ganoderma lucidum immunoregulatory protein mutant of the present invention dispersed therein. 9. The application according to claim 6, characterized in that, the medicament is in the form of tablets, coated tablets, hard or soft gelatin capsules, solutions, emulsions, suspensions, suppositories or injections.