阅读说明：本技术 灵芝免疫调节蛋白突变体及应用 (Ganoderma lucidum immunomodulatory protein mutant and application thereof ) 是由 周选围 黎刘定吉 毛培文 李奇嶂 于 2019-08-27 设计创作，主要内容包括：一种灵芝免疫调节蛋白突变体,包括：FIP-gluM<Sub>N31S</Sub>、FIP-gluM<Sub>T36N</Sub>和FIP-gluM<Sub>N31S/T36N</Sub>,其中：FIP-gluM<Sub>N31S</Sub>的氨基酸序列及核苷酸序列分别如SEQ ID NO.1和SEQ ID NO.4所示；FIP-gluM<Sub>T36N</Sub>的氨基酸序列及核苷酸序列分别如SEQ ID NO.2和SEQ ID NO.5所示；FIP-gluM<Sub>N31S/T36N</Sub>的氨基酸序列及核苷酸序列分别如SEQ ID NO.3和SEQ ID NO.6所示。本发明能够解决FIP-glu在应用对细胞毒性较大的问题,经过改造的突变体对细胞产生的毒性相较于灵芝免疫调节蛋白(FIP-glu)有显著性降低,可以有效提高其利用价值。(A mutant of ganoderma lucidum immunomodulatory protein, comprising: FIP-gluM N31S 、FIP‑gluM T36N And FIP-gluM N31S/T36N Wherein: FIP-gluM N31S The amino acid sequence and the nucleotide sequence of (A) are respectively shown as SEQ ID NO.1 and SEQ ID NO. 4; FIP-gluM T36N The amino acid sequence and the nucleotide sequence of (A) are respectively shown as SEQ ID NO.2 and SEQ ID NO. 5; FIP-gluM N31S/T36N The amino acid sequence and the nucleotide sequence of (A) are respectively shown as SEQ ID NO.3 and SEQ ID NO. 6. The invention can solve the problem that FIP-glu has high cytotoxicity in application, and the modified mutationCompared with ganoderma lucidum immunomodulatory protein (FIP-glu), the toxicity of the body to cells is remarkably reduced, and the utilization value of the ganoderma lucidum immunomodulatory protein can be effectively improved.)

1. A ganoderma lucidum immunomodulatory protein mutant is characterized by comprising: FIP-gluM_N31S、FIP-gluM_T36NAnd FIP-gluM_N31S/T36NWherein: FIP-gluM_N31Sthe amino acid sequence and the nucleotide sequence of (A) are respectively shown as SEQ ID NO.1 and SEQ ID NO. 4; FIP-gluM_T36NThe amino acid sequence and the nucleotide sequence of (A) are respectively shown as SEQ ID NO.2 and SEQ ID NO. 5; FIP-gluM_N31S/T36NThe amino acid sequence and the nucleotide sequence of (A) are respectively shown as SEQ ID NO.3 and SEQ ID NO. 6.

2. The method for preparing the mutant according to claim 1, wherein the mutant is obtained by using a nucleotide sequence of the ganoderma lucidum immunomodulatory protein FIP-glu shown in SEQ ID No.7 as a template, and a nucleotide sequence of the ganoderma lucidum immunomodulatory protein FIP-glu shown in SEQ ID No.7 and SEQ ID No.8 as templates, and performing PCR amplification by using corresponding primer sequences;

The corresponding primer sequences refer to:

Firstly, adopting primers shown in SEQ ID NO. 9-12 to carry out PCR amplification to obtain FIP-gluM_N31S；

Secondly, primers shown in SEQ ID NO.9, 12, 13 and 14 are adopted to obtain FIP-gluM by PCR amplification_T36N；

Thirdly, FIP-gluM is obtained by PCR amplification by adopting primers shown in SEQ ID NO.9, 10, 12 and 15_N31S/T36N。

3. The method of claim 2, wherein the PCR amplification is carried out using a vector including, but not limited to, a plasmid, a phage, or a viral vector.

4. The method of claim 3, wherein the vector is eukaryotic expression vector pPIC 9K.

5. the method as claimed in claim 2, wherein the nucleotide sequence obtained after PCR amplification is cloned into a vector, and is induced to express and purified after constructing a recombinant vector and transforming into an engineering bacterium.

6. The use of the mutant immunomodulatory protein of any preceding claim for the preparation of a medicament for the treatment of a tumor.

7. The use of claim 6, wherein said tumor comprises: tumors of the adrenal gland, bile duct, bladder, blood, bone and connective tissue, brain and central nervous system, breast, cervix, colorectal (colorectal), endometrium, oesophagus, gall bladder, head and neck, Hodgkin's (Hodgkin's) lymphoma, hypopharynx, kidney, larynx, leukaemia, liver, lung, lymphoma, mediastinal, melanoma (malignant melanoma), mesothelioma, multiple myeloma, nasal cavity, nasopharynx, neuroendocrine, non-Hodgkin's lymphoma, oral cavity, oesophagus, oropharynx, ovary, pancreas, sinuses, parathyroid, pituitary, prostate, salivary gland, sarcoma, skin, spine, adrenal, penis, larynx, pancreas, penis, prostate, salivary gland, pancreas, skin, spinal column, spinal cord, bone, cartilage, or cartilage, Gastric, testicular, thyroid, urethral, uterine, vaginal, and vulvar tumors.

8. The use of claim 6, wherein the medicament comprises a pharmaceutical carrier and dispersed therein a therapeutically effective amount of the mutant immunomodulatory proteins of Ganoderma lucidum of the invention.

9. Use according to claim 6, wherein the medicament is in the form of a tablet, coated tablet, hard or soft gelatin capsule, solution, emulsion, suspension, suppository or injection.

Technical Field

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

Background

Fungal Immunomodulatory Protein (FIP) was originally isolated from higher basidiomycetes and is a small molecule protein with immunomodulatory activity. After Kino et al isolated the first FIP (LZ-8 or FIP-glu) from Ganoderma lucidum (Ganodermaspp.) in 1989, researchers continued to find more FIP from various edible and medicinal fungi. They constitute a new family of proteins, FIP. The protein of FIP family has the functions of anti-tumor, anti-allergy, stimulating immune cells to produce various cytokines and other immunoregulation functions, and has good clinical application prospect and medicinal health care value. The fungal immunomodulatory protein consists of 110-140 amino acids, has a molecular weight of about 13kD, is deficient in histidine, cysteine and methionine, and is rich in aspartic acid and valine. The amino acids at the N-terminal are all acetylated amino acids. Most FIP is present as homodimers. In addition, FIP has great similarity to the immunoglobulin heavy chain variable region.

FIP has been found to exhibit significant growth inhibitory activity against a variety of cancer cells. For example, recombinant FIP-gts expressed in E.coli can inhibit the growth of human lung cancer cell A549 in endosomes and in vitro (Chien-Huang Liao, Yi-Min Hsiao, Chung-Ping Hsu, Meii-Yn Lin, James Chun-Huang Wang, & Yu-Lu Huang, et al (2006.) transcription mediated inhibition of transcriptional enzyme of transcriptional modulation protein from genomic tsugae in a549 human genomic cell. molecular Carcinogenesis, 45.) by down-regulating the transcription of the telomerase catalytic subunit (hTERT), inhibiting telomerase activity results in the inhibition of cell growth. Furthermore, recombinant FIP-glu obtained from yeast significantly inhibited human leukemia cells 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 genetic engineering protein from microbial leukemia, world Journal of Microbiology & Biotechnology, 25(3),

383-390.), human lung carcinoma cell A549 and Lewis lung carcinoma cell LLC1(Wu, C.T., Lin, T.Y., Hsu, H.Y., Sheu, F., Ho, C.M., & Chen, I.T. (2011.) Ling zhi-8 means p53-dependent growth of the lung cancer cells of lung cancer cells promotion a the ribosomal protein s7-mdm2-p53pathway. Carbonogenesis, 32(12), 1890-1896.). We have also found that Recombinant FIP-gat has the effect of inhibiting the growth of human breast cancer MDA-MB-231 (Xu, H., Kong, Y.Y., Chen, X., Guo, M.Y., & ZHou, X. (2016), Recombinant FIP-gat, a functional immunological regulatory protein from cancer arm, indecs growth inhibition and cell death in branched cell, journal of Agricultural and Food Chemistry, 64 (13)), and that by chip analysis, 669 differentially expressed genes, which have fold changes of at least 2-fold, are found in Recombinant FIP-gat treated MDA-MB-231, and apoptosis-related genes, such as TNFSF8, DRD1, PD1 and BCL-2, are also regulated. According to research, the recombinant FIP-glu produced by utilizing a yeast expression system has toxicity to the murine macrophage RAW264.7 in a certain range through a genetic engineering means, and the research result further enables the application of FIP in the field of medicine to be greatly limited.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a ganoderma lucidum immunomodulatory protein mutant, which can solve the problem of high cytotoxicity in application of FIP-glu and is a modified ganoderma lucidum immunomodulatory protein mutant FIP-gluM_N31S、FIP-gluM_T36NAnd FIP-gluM_N31S/T36NCompared with ganoderma lucidum immunomodulatory protein (FIP-glu), the toxicity generated to cells is remarkably reduced, and the utilization value of the ganoderma lucidum immunomodulatory protein can be effectively improved.

The invention is realized by the following technical scheme:

The invention relates to a ganoderma lucidum immunomodulatory protein mutant, which comprises the following components: FIP-gluM_N31S、FIP-gluM_T36Nand FIP-gluM_N31S/T36NWherein: FIP-gluM_N31SThe amino acid sequence and the nucleotide sequence of (A) are respectively shown as SEQ ID NO.1 and SEQ ID NO. 4; FIP-gluM_T36NThe amino acid sequence and the nucleotide sequence of (A) are respectively shown as SEQ ID NO.2 and SEQ ID NO. 5; FIP-gluM_N31S/T36NThe amino acid sequence and the nucleotide sequence of (A) are respectively shown as SEQ ID NO.3 and SEQ ID NO. 6.

the invention relates to a preparation method of the mutant, which is obtained by taking a nucleotide sequence of ganoderma lucidum immunomodulatory protein (FIP-glu) with nucleotide and amino acid sequences of SEQ ID No.7 and SEQ ID No.8 as templates and adopting corresponding primer sequences to perform PCR amplification.

The corresponding primer sequences refer to:

Firstly, adopting primers shown in SEQ ID NO. 9-12 to carry out PCR amplification to obtain FIP-gluM_N31S；

Secondly, primers shown in SEQ ID NO.9, 12, 13 and 14 are adopted to obtain FIP-gluM by PCR amplification_T36N；

Thirdly, FIP-gluM is obtained by PCR amplification by adopting primers shown in SEQ ID NO.9, 10, 12 and 15_N31S/T36N。

The PCR amplification is carried out by using a vector including but not limited to a plasmid, a phage or a viral vector, and preferably a eukaryotic expression vector pPIC 9K.

The preparation method preferably clones the nucleotide sequence obtained after amplification to a vector, constructs a recombinant vector, transforms the recombinant vector to engineering bacteria, and then carries out induced expression and purification.

The engineering bacteria are preferably yeasts.

The invention relates to application of the ganoderma lucidum immunomodulatory protein mutant in preparing a medicament for treating tumors.

The tumor comprises: tumors of the adrenal gland, bile duct, bladder, blood, bone and connective tissue, brain and central nervous system, breast, cervix, colorectal (colorectal), endometrium, oesophagus, gall bladder, head and neck, Hodgkin's (Hodgkin's) lymphoma, hypopharynx, kidney, larynx, leukaemia, liver, lung, lymphoma, mediastinal, melanoma (malignant melanoma), mesothelioma, multiple myeloma, nasal cavity, nasopharynx, neuroendocrine, non-Hodgkin's lymphoma, oral cavity, oesophagus, oropharynx, ovary, pancreas, sinuses, parathyroid, pituitary, prostate, salivary gland, sarcoma, skin, spine, adrenal, penis, larynx, pancreas, penis, prostate, salivary gland, pancreas, skin, spinal column, spinal cord, bone, cartilage, or cartilage, Gastric, testicular, thyroid, urethral, uterine, vaginal, and vulvar tumors.

The medicament comprises a medicament carrier and a therapeutically effective amount of the ganoderma lucidum immunomodulatory protein mutant dispersed in the medicament carrier. The composition may be a solid or a liquid. The pharmaceutical carrier is generally selected according to the type of administration used, and may be solid or liquid. The ganoderma lucidum immunomodulatory protein mutant can be in the same phase or different phases with a drug carrier.

The medicament is in the form of tablets, coated tablets, hard or soft gelatin capsules, solutions, emulsions, suspensions, suppositories or injections.

Technical effects

Compared with the prior art, the mutant of the invention has simple obtaining mode, expression method and purification process, and the prepared ganoderma lucidum immunomodulatory protein mutant has lower cytotoxicity and higher biological activity compared with the wild ganoderma lucidum immunomodulatory protein.

Drawings

FIG. 1 is a schematic diagram of detection of recombinant ganoderma lucidum immunomodulatory protein according to an embodiment;

In the figure: a: SDS-PAGE detection; b: western Blot detection;

FIG. 2 is a schematic illustration of an example cytotoxicity assay;

FIG. 3 is a schematic representation of the TNF-. alpha.transcript level assays of the examples.

Detailed Description