WO2002040675A1

WO2002040675A1 - Novel human tumor necrosis factor mutant proteins and their preparing methods and uses

Info

Publication number: WO2002040675A1
Application number: PCT/CN2000/000424
Authority: WO
Inventors: Changqing Chen; Hui Liu; Fang Lu
Original assignee: Shanghai Research Center Of Biotechnology, Chinese Academy Of Sciences
Priority date: 2000-11-15
Filing date: 2000-11-15
Publication date: 2002-05-23
Also published as: AU2001213790A1

Abstract

The present invention dislcoses a type of novel low toxic human tumor necrosis factor mutant proteins and their preparing methods, the coding sequences encoding the mutant proteins, the expression vectors and the transformed host cells containing the coding sequences, as well as pharmaceutical composition containing the novel human tumor necrosis factor mutant proteins. The novel human tumor necrosis factors have Arg32Trp/Leu157Phe mutants or Arg2Lys/Asn30Ser/Arg32Trp/Leu157Phe mutants. The type of mutant proteins have significantly lower toxicity to mice, but have equal or superior killing effect to human tumor cells compared with type protein, the four mutant proteins have some lower toxicity to macaque and have high inhibiting tumor effect to mice, and have better therapeutic effect to human carcinoma of bladder CP-3 transplanted into naked mice.

Description

Novel human tumor necrosis factor mutant protein, preparation method and application thereof

The invention relates to the fields of genetic engineering, protein engineering and disease treatment, and more particularly, to a new low-toxic human tumor necrosis factor mutant protein and a preparation method thereof, a coding sequence encoding the mutant protein, an expression vector containing the coding sequence, and transformation. Host cells, and pharmaceutical compositions containing the novel human tumor necrosis factor mutant protein.

Background of the invention

Human tumor necrosis factor TNF α is mainly produced by macrophages and monocytes. It can be induced by BCG, endotoxin, etc .; some tumor cells, such as human premyeloid leukemia cell HL-60, are induced by phorbol ester. Macrophages are differentiated in the direction and can also produce TNF. In 1984, Pennica et al. (Pennica D, et al, Nature 1984; 312: 724) first cloned the human TNF α gene _C DNA, and deduced that the human T NF α molecule consists of 157 amino acids, of which the 69 and 101 positions Two cysteines form a disulfide bond in the active state of TNF α, and their molecular weight is about 17KD. At the same time, M. Ikehara et al. (Ikehara M. et al, Proc Natl Ac ad Sci USA, 1984; 81: 5956; Ikehara M. et al, Chem Pharm Bull, 1988; 36 (1): 291) also cloned Human TNF alpha gene cDNA.

The results of clinical trials using recombinant human TNF a at home and abroad show that this factor has a significant killing effect on a variety of malignant tumor cells. Many patients have reduced or even disappeared tumors after treatment. Patients are relieved, their quality of life is improved, and their survival time is extended. Recombinant human TNF a has a good antitumor effect. However, in the actual application process, it has also been found that recombinant human TNF a has very obvious toxic and side effects such as fever, chills, hypotension, and weight loss, which limits the clinical popularization and application of recombinant human TNF a.

Studies on human tumor necrosis factor (hTNF-a) show that its most significant feature is that it can specifically kill a variety of tumor cells in vitro or in vivo without significant toxic and side effects on normal tissue cells. CWiedenmann B., Reichardt P., ath U.et al "" Phase-I trial of intravenous continuous infusion of tumor necrosis factor in advanced metastatic carcinomas ", J. Cancer Res. Clin. Oncol., 1989, 115 (2): 189-192; Hersh EM, Metch BS , Muggia FMet al., "Phase II studies of recombinant human tumor necrosis factor alpha in patients with malignant disease: A summary of southwest oncology group experience", J. Immunother., 1991, 10 (6): 426-431.) 'But in practical applications, hTNF-α has large toxic and side effects, which limits its clinical use (Old LJ., Tumor Necrosis Factor: Structure, Mechanism of Action, Role in Disease and Therapy, 1990, l- 30, Karger, Pasel.).

For this reason, many researchers are trying to genetically modify protein engineering methods in order to obtain a new type of high-efficiency and low-toxicity human tumor necrosis factor.

Yamagishi et al. Proposed four regions necessary for maintaining hTNF-α activity: I, 30-32 chloro acids; II, 82-89 amino acids; III, 115-117 amino acids; IV, 141: 146 amino acids (Yamagishi Confirmation ^ J., Kawashima H., Matsuo N., et al. 'Mutational analysis of structure-activity relationships in human tumor necrosis factor-alpha, Protein Engng, 1990, 3 (8): 713-719.).

In addition, Nanjing University Bai Chen et al. Found that deletion of this region 68-73 can reduce the biological activity and solubility of hTNF-α (Cen B., Jm W., Wu H. et al., Glycine 68 to histidine 73 has an important role in the function of human tumor necrosis factor alpha, Biochem Mol Biol int., 1997, 43 (l): 47- 52.)

It was reported that 30 asparagines had severely lost their viability after they were converted to isoleucine (Yamagishi J. et al., 1990, supra). After the arginine at position 32 is changed to valine, only half of its viability remains, and the viability after conversion to tryptophan is two orders of magnitude lower than that of wild type (Van Ostade X., Taveniiev J., Prange T. et al., Localization of the active site of human tumor necrosis factor (hTNF) by mutational analysis, EMBO J., 1991, 10 (4): 827-836.). Van Ostade et al.'S study also found that the activity of the alanine at position 84 was severely reduced after replacement with other amino acids, and the activity was completely lost when it was changed to valine; and the amino acid change at position 86 also had a great impact on the activity. In addition, the substitution of the C-terminal amino acid of hTNF-a has a greater effect on the viability, especially when the leucine at position 157 is changed to phenylalanine with a 5-fold higher activity than the wild type (Van Ostade X., 1991, supra).

In addition, the activity of arginine at position 2 can be doubled after being converted to lysine (high longevity, Mao Shenlan, Yu Yue, etc., a new high-efficiency, low-toxicity human tumor necrosis factor mutant in E. coli High-efficiency expression in Chinese, Journal of Biochemistry and Biophysics, 1996, 28 (1): 49-55) ₀

The two receptors R55 and R75 of hTNF- mainly mediate different biological functions. R55 mediates cytotoxic activity, while R75 mediates endotoxicity, (Louis A., Tartaglia LA, David V. et al ., Two TNF Receptors, Immun. Today, 1992, 13 (5): 151-153; Kamijo R., Takeda K., Nagumo lines by TNF mutants, Biochem. Biophys. Res. Commun., 1989, 160 (2) : 820-827.). In 1993, Van Ostade et al. Reported for the first time in "Nature" that after changing leucine 29 to serine and 32 arginine to tryptophan, the killing activity on mouse fibroblast cells decreased significantly, and Similar affinity to R55 receptor and wild type, and decreased affinity to R75 receptor (Loetscher H. ₅ Steinmetz M., Lesslauer W., Tumor necrosis' factor: receptors and inhibitors, Cancer Cells, 1991, 3 ( 2): 221 "226). Since then, there have been more and more reports on the study of hTNF-α muteins from the perspective of interaction with receptors.

Van Ostade et al. Reported that the 29th and 32th positions were replaced. After binding to the other 8 amino acids, the binding to R55 and R75 decreased (Van Ostade X., Vandenabeele P., Everaerdt B.et al "Human TNF mutants with selective activity on the R55 receptor, Nature, 1993, 361 (2): 166-169. When the serine at position 86 becomes threonine, its binding to the R55 receptor is the same as that of the wild type, but its binding to R75 decreases. Many. Aspartic acid at position 143 was changed to tyrosine, phenylalanine, and asparagine, respectively, and the affinity with R55 decreased more than the affinity with R75 (Van Ostade X., Vandenabeele P., Tavernier J. et al. Human tumor necrosis factor mutants with preferential binding to and activity on either the R55 or R75 rceptor, Eur J Biochem, 1994, 220 (3): 771 -779 .; Loetscher H., Stueber D., Banner D. et al "Human tumor necrosis factor alpha (TNF alpha) mutants with exclusive specificity for the 55-Kda or 75-Kda receptors, J Biol. C em., 1993, 268: 2635046357.).

After glutamic acid at position 146 changed to arginine, it hardly bound to the R75 receptor and its binding capacity to the R55 receptor was reduced by half compared to the wild type.

In summary, although the structure of human tumor necrosis factor TNF α and its various mutations have been extensively studied, there is still a need in the art to develop new types of human tumor necrosis factor TNF α with low toxicity and / or high efficiency. For clinical use.

The purpose of the present invention is to overcome the above disadvantages in the prior art, and provide a human tumor necrosis factor TNFa with low toxicity and / or high efficiency, thereby promoting its clinical application.

Summary of the invention ''

In the first aspect of the present invention, a low-toxicity and / or high-efficiency human tumor necrosis factor mutant protein is provided, which protein contains the Arg32Trp / Leul57Plie mutation. Preferably, the mutant protein may further contain the Arg2Lys / Asn30Ser mutation; Most preferably, the mutein has the amino acid sequence shown in SEQ ID NO. 1 or 2.

In a second aspect of the invention, a DNA sequence encoding the novel human tumor necrosis factor mutant protein is provided. .

In a third aspect of the present invention, a vector containing the above-mentioned DNA sequence and a corresponding transformed host cell are provided. ,

In a fourth aspect of the present invention, a method for producing the aforementioned human tumor necrosis factor mutant protein is provided, and the method includes the steps of:

(a) a DNA sequence encoding a human tumor necrosis factor TNFct mutant protein is operably linked to an expression control sequence to form a human tumor necrosis factor mutant egg. a white expression vector, wherein the human tumor necrosis factor mutant protein contains an Arg32Trp / Leul57Phe mutation Preferably, the mutant protein may further contain an Arg2Lys / Asn30Ser mutation; most preferably, the mutant protein has an amino acid sequence shown in SEQ ID N0.1 or 2;

() Transforming the expression vector in step (a) into a human host cell;

(c) culturing under conditions suitable for expressing the human tumor necrosis factor mutant protein, thereby expressing the human tumor necrosis factor mutant protein;

(d) isolating and purifying the human tumor necrosis factor mutant protein.

In a fifth aspect of the present invention, use of the novel human tumor necrosis factor mutant protein and a pharmaceutical composition containing the novel human tumor necrosis factor mutant protein are provided. The composition contains an effective amount of the human tumor necrosis factor mutation of the present invention. Protein and pharmaceutically acceptable carriers and / or excipients.

Detailed description of the invention

In the present invention, "tumor necrosis factor TNFcc" generally means human tumor necrosis factor TNFoc, but can also refer to tumor necrosis factor TNFoc that is then derived from mouse, pig, horse, or bovine. Preferably, it is human tumor necrosis Factor TNFa. In addition, the tumor necrosis factor TNFa may be a TNFa with a natural wild-type sequence, or a derivative or recombinant TNFa with a mutated sequence (as opposed to a wild-type sequence), as long as the mutated sequence does not include the Arg32Trp / Leul57Phe mutation. The amino acid sequence of natural wild-type human tumor necrosis factor TNFa is shown in SEQ ID No: 3.

In this context, "Arg32Trp / Leul57Phe" means that, relative to the natural wild-type sequence, the

Arginine (Arg) at position 32 is mutated to tryptophan (Trp), and leucine (Leu) at position 157 is mutated to (Phe). However, it should be understood that "Arg32Trp / Leul57Plie" is equivalent to "32Trp / 157Phe", that is, whether or not arginine at position 32 and / or leucine at position 157 is provided, as long as the mutation is tryptophan and benzene Alanine can be expressed as "Arg32Trp / Leul57Plie" or "32Trp / 157Phe". The description of mutations 2 and 30 is analogous. '

The novel mutein of the present invention can be prepared by synthesizing primers based on the published sequence of human tumor necrosis factor and amplifying the coding sequence of human tumor necrosis factor by the PCR method. For example, according to the method described in Li Changben et al., Science in China (Series B), 1991, 6: 497, a natural hTNFa gene was constructed. Human TNFa coding sequences can also be artificially combined. In addition, base sequences can be substituted for coding sequences to facilitate high expression (e.g., for expression in E. coli, natural codons can be replaced with E. coli preferred codons encoding the same amino acid). Then, the DNA sequence of human tumor necrosis factor is used to genetically modify the mutant form indicated in the present invention. The technique of genetic modification is known in the art, for example, see "Mutagenesis: a Practical Approach", MJ McPerson , Ed., (IRL Press, Oxford, UK. (1991), which include, for example, site-directed mutagenesis, cassette mutagenesis, and polymerase chain reaction (PCR) mutagenesis.

After obtaining the DNA sequence encoding the novel mutant protein of the present invention after site-directed mutation, it was ligated into a suitable expression vector and then transferred into a suitable host cell. Finally, the transformed main cells are cultured, and the new mutant protein of the present invention is obtained through isolation and purification.

In the present invention, various vectors known in the art such as commercially available vectors can be selected. For example, a commercially available vector is selected, and then a nucleotide sequence encoding the novel mutant protein of the present invention is operably linked to an expression control sequence to form a protein expression vector.

As used herein, "operably linked" refers to a condition where certain parts of a linear DNA sequence can affect the activity of other parts of the same linear DNA sequence. For example, if the signal peptide DNA is expressed as a precursor and is involved in the secretion of the polypeptide, then the signal peptide (secret leader) DNA is operably linked to the polypeptide DNA; if the promoter controls the transcription of the sequence, it is operably linked to the ¾k Coding sequence; if the ribosome binding site is placed at a position that enables it to translate, it is operably linked to the coding sequence. In general, "operably linked to" means adjacent, while for secreted leader sequences it means adjacent in reading frame.

In the present invention, the term "host cell" includes prokaryotic cells and eukaryotic cells. Examples of commonly used prokaryotic host cells include E. coli, Bacillus subtilis, and the like. Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells. Preferably, the host cell is a prokaryotic cell, more preferably E. coli. After years of research, the inventors have introduced various mutant forms such as 2Lys, 30Ser, 32Trp, 157Plie and their combinations into human tumor necrosis factor, and based on the spatial model of the interaction between hTNF-β and its receptor. The SGI protein imaging workstation has established a model for the binding of hTNF-α to its receptors (R55 and R75 receptors) and conducted extensive and in-depth research. Prior studies of the present invention have shown that mutation of arginine at position 32 to tryptophan results in a significant decrease in the killing activity of human tumor necrosis factor TNF α (Van Ostade, X., 1991, supra). However, the present inventors have unexpectedly discovered that, 'After mutating arginine at position 32 and leucine at position 157 of human tumor necrosis factor hTNF-α to tryptophan at position 32 and phenylalanine at position 157 (in other words, that is, The introduction of the Arg32Trp / Leul57Phe mutation) can significantly reduce the toxicity of human tumor necrosis factor TNF c [, and at the same time the killing activity of human tumor necrosis factor TNF α on human tumor cells does not decrease.

The exact mechanism leading to the decrease in toxicity is not fully understood. According to the detailed analysis of the relationship between the structure and function of TNF by the inventors of the present invention, it is likely that the result of the interaction between hTNP-ci's 32-arginine → tryptophan and the two types of receptors was compared with wild-type In contrast, binding to R55 was retained and binding to R75 was decreased. Specifically, hTNF-α mainly binds to receptors in a trisomy, and each receptor binds in a groove between two TNF molecules. The C-terminal 157-position becomes Phe, which stabilizes the TNF trisomy form due to its increased hydrophobicity, thereby increasing its biological activity.

In addition, the inventors of the present invention found that asparagine at position 30 does not participate in binding to two receptors based on the spatial structure of the hTNF-α trisomy. However, asparagine at position 30 can change to hTNF-α The trisomics are tighter, which increases the stability of the trisomics. 'Therefore, in order to further improve the human tumor necrosis factor TNF α, Asn30Ser mutation can be introduced again. In addition, research has shown that increasing the activity of hTNF-α at the N-terminal basic amino acid can increase its activity (the specific mechanism cannot be explained), for example, the activity can be doubled by changing arginine at position 2 to lysine.

In the present invention, the present inventors constructed a novel mutant protein of human tumor necrosis factor TNFa using the method of Over-Lap PCR. According to the measurement of the properties of the mutant proteins, they were found to be highly effective in killing human tumor cells and the LD50 of the acute toxicity test was much lower than that of the wild type.

The novel mutein of the present invention is helpful for people to further understand the mechanism of action of TNF, and to modify and modify the TNF molecule by using genetic engineering methods to locate functional regions with unique activity. In addition, since the novel mutant protein of the present invention improves the anti-tumor activity of TNF and reduces the toxic and side effects, it will undoubtedly provide a broad prospect for the anti-tumor clinical treatment of TNF.

In addition, based on the novel human TNFa mutein of the present invention, through further mutagenesis at other sites, a human tumor necrosis factor mutein with higher activity and / or lower toxicity is also produced.

In an embodiment of the present invention, mutations are performed on the 32nd and 157th positions of human TNFa to form a mutant protein with the Arg32Trp / Leul57Plie mutation. The mutant protein is named MTNF1 (abbreviated as M1), and its amino acid sequence As shown in SEQ ID NO: 1.

In another embodiment of the present invention, mutations at positions 2 and 32 of human TNFa are also performed, thereby forming a mutant protein having the Arg2Lys / Asn30Ser / Arg32Trp / Leul57Phe mutation, and the mutant protein is It is named MTNF2 (abbreviated as M2), and its amino acid sequence is shown in SEQ ID NO: 2.

The pharmaceutical composition of the present invention comprises an effective amount of one or several novel T Foc muteins of the present invention, and at least one pharmaceutically acceptable carrier, diluent or excipient. In preparing these compositions, the active ingredient is usually mixed with, or diluted with, an excipient, or enclosed in a carrier that can be in the form of a capsule or sachet. When an excipient functions as a diluent, it can be a solid, semi-solid, or liquid material as an excipient, carrier, or medium for the active ingredient. Therefore, the composition may be a tablet, a pill, a powder, a solution, a syrup, a sterile injection solution, or the like. Examples of suitable excipients include: lactose, glucose, sucrose, sorbitol, mannitol, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, and the like. The formulation may also include: wetting agents, emulsifiers, preservatives (such as methyl and propyl hydroxybenzoate), sweeteners, and the like. The composition can be made into unit or multiple dosage forms. Each dosage form contains a predetermined amount of active substance calculated to produce the desired therapeutic effect, as well as suitable pharmaceutical excipients.

The pharmaceutical composition can be administered according to the disease to be treated and in a dose beneficial to the patient (depending on weight and other considerations), which can be determined by the medical staff.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows the construction of a novel human tumor necrosis factor mutant protein M1 and M2 expression plasmid of the present invention.

Figure 2 is a 1.5% agarose electrophoresis image of the Over-Lap PCR product. Among them, lane 1, mutant gene Ml; lane 2, mutant gene M2; lane 3, molecular weight marker (pGEM7Zf (+) DNA / HaeIII).

-Figure 3 is the EcoRI / BamHI digestion identification map of each mutant protein gene expression plasmid. Lane 1, molecular weight marker (pGEM7Zf (+) DNA / Haeni); lane 2, EcoRI + BamHI digestion result of the expression plasmid pSB-MTNFl; lane 3, EcoRI + BamHI digestion result of the expression plasmid pSB-MTNF2; lane 4. EcoRI + BamHI digestion results of the expression vector pSB-92.

Figure 4 shows the corresponding Western-Blot results after high expression of each mutant protein gene expression plasmid in E. coli. Lane 1, the mutant gene M1 is induced to express for 4 hours; lane 2, the mutant gene M2 is induced to express for 4 'hours; lane 3, the wild-type gene hTNF-α is induced to express for 4 hours; lane 4, protein molecular weight standard; lane 5, wild-type Protein imprinting results of gene hTNF-ct; lane 6, protein imprinting results of mutant gene M2; 'lane 7, protein imprinting results of mutant gene M1.

Figure 5 shows the SDS-PAGE results of each mutant protein after purification. Among them, lane 1, Ml; lane 2, M2; lane 3, hTNF-a; lane 4, protein molecular weight standard.

Figure 6 shows the full sequence of plasmid PSB-92.

Figure 7 is an amino acid sequence diagram of natural human TNFa. The present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods in the following examples are not marked with specific conditions, usually according to conventional conditions such as Sambrook et al. Molecular cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

'' Examples

I. Materials:,

The expression vector pSB-92 _: see Figure 6 for the complete sequence.

Expression plasmid pSB-TR: The expression vector pSB92 containing the human TNFa gene.

pSB-TK: An expression vector pSB92 containing a mutated human TNFa gene, in which the arginine at position 2 is mutated to lysine (high longevity, Mao Shenlan, Yu Yue, etc.), a novel high-efficiency, low-toxic human tumor necrosis factor Efficient expression of mutein in E. coli, Journal of Biochemistry and Biophysics, 1996, 28 (1): 49-55) pUC-19 plasmid was purchased from Huamei Bioengineering Company.

E. coli YK537 (supE44hsdRhsdMrecAlpho48LeuB6thilacYrpsL20galK2ara-

14xyl-5mtl-l) and JM109 (recAlsupE44endAllisdR17gyrA96relAlthiA (lacproAB) F '[traD36proAB ⁺ lad¾acZ ΔM15〗) were purchased from the Chinese Academy of Sciences strain collection center.

Various restriction endonuclease, 'T ₄ DNA ligase and Taq DNA polymerase were purchased from Promega Hekou Biorhinger Mannheim Corporation company.

The DNA sequencer was determined by the American ΑΓΓ automatic tester. .

The human laryngeal cancer cell line HEp-2 was a gift from Professor Pedersen of Aarhus University in Denmark. The human gastric cancer cell line MGC was Professor Liu Quanhai of the Shanghai Pharmaceutical Industry Research Institute of the State Medical Administration. The human breast cancer cell line MCF-7 and the human ovarian cancer cell line 3AO. A gift from Professor Jiao Binghua of the Second Military Medical University, and a human liver cancer cell line HepG-2 was given by Professor Wen Yumei of Shanghai Medical University. '

Mouse anti-hTNF-α antiserum was purchased from the Immunology Laboratory of the Fourth Military Medical University. Method

(1) Determination of biological activity of expression products

With 0.25% trypsin L929 cells in logarithmic growth phase, the cell concentration adjusted to 2-2.5 X 10 ⁵ / ml, added to 96-well culture plates, lOOul per well were incubated for 18 hours, serially diluted TNF sample, Continue to incubate for 18 hours. The dilution solution is Actinomycin D containing 2ug / ml (Actinomycin D is a product of Sigma), ELPMI1640 with 5% NCS. Each dilution is made into 3 replicate wells, and a blank control is set at the same time. Cell control, actinomycin] control, the sample titer is set to the corresponding dilution factor that causes 50% of the cells to survive.

(2) Western Blot

The induced expression products were separated by SDS-PAGE, and then transferred to a nitrocellulose membrane.

Block 5% skimmed milk in PBS (pH 7.2) at 37 ° C for 1 hour, add mouse anti-hTNF-α hybridoma ascites (1: 1000 dilution) and react at room temperature for 2 hours, wash 3 times with PBS, and add goat anti-mouse IgG- After HRP (1: 1000 dilution) was reacted for 1 hour, the substrate diaminobenzidine was added to develop a color. .

(3) Determination of killing effect of hTNF-α mutant protein on human tumor cells

Digest human tumor cells in logarithmic growth phase with 0.25% trypsin and adjust the cell concentration to 4 X 10 ⁵ / ml, add to a 96-well culture plate, 100ul per well, and incubate in a 37 ° C incubator for 24 hours. Dilute the TNF sample with RPMI1640 medium containing 30-50 ug / ml actinomycinone and 5% NCS. Then, add it to the plated cell plate and put it in a 37Ό incubator. Place it for 24 hours, stain it with crystal violet, measure the OD value with a microplate reader at 595nm, and use the sample dilution factor as the abscissa and the OD value as the ordinate. The pure live curve of tumor cells was set with blank control, cell control, and D control. The killing titer of the sample to human tumor cells was determined as the corresponding dilution factor that caused 50% of cell survival.

(4) Determination of hT F-α mutein LD50

Six Kunming mice were randomly selected from each group, male and female, weighing 18-22 g (provided by the Animal Room of Tianjin Pharmaceutical Research Institute, animal certificate: Jin Shidong Facility Standard No. 001 :), and intravenous injection of different doses For protein samples, 0.4ml / 20g body weight, the LD50 of each sample was obtained by the Bliss method.

(5) Toxicity test of rTNFa mutant protein M2 by intravenous injection in rhesus monkeys for 10 consecutive days

8 Rhesus monkeys were randomly divided into two groups. Each group consisted of 4 males and half females. One group was injected with wild-type hTNFo intravenously and the other group was injected with hTNFa mutant protein M2. The dose was calculated based on the body surface area of the animal. The daily injection dose was 200 μ ₈ / η ² , and the drug was administered continuously for 10 days to observe the death of the animal. For the hTNFa mutant protein M2 injection group, weigh each time before and after the administration; before and after the end of the administration, the hind limbs are quiet: pulse blood, coagulate at room temperature and separate the serum, and perform the following with a clinical biochemical analyzer Measurement of indicators: total protein (TP :), albumin

(ALB), blood glucose (GLU), 'total bilirubin (T-BIL), creatinine (Crea), total cholesterol (T-CHO),' urea nitrogen (BUN), alanine aminoconverting enzyme (ALT), sky door Aspartate aminoconvertase (AST) and alkaline phosphatase (ALP). Check for serum biochemical indicators. (6) Effect of hTNFa mutant protein M2 on mouse sarcoma S180 and liver cancer HAC solid tumors

Take well-growing S180 and HAC ascites, and dilute 1: 4 with normal saline. 0.2ml of each mouse is subcutaneously axillary, randomly divided into groups of 10 mice. Set normal saline group, CTX group, wild type hTNFa group (0.025mg / kg M2, there are 3 dose groups, 2.5, 0.25 and 0.025mg / kg, respectively, and the dose is 0.5ml / head. The next day after inoculation, 7 consecutive days of injection. 10 days after inoculation The animals were sacrificed by neck dissection, tumor masses were dissected, and the tumor weights of each dose group were compared. The results were determined according to the following formula:

_l00%

(7) Effect of hTNFa mutant protein M2 on human bladder cancer CP-3 transplanted in nude mice

CP-3 ascites of healthy human bladder cancer were separately taken, diluted 1: 4 with normal saline, 0.2ml subcutaneously axillary in each nude mouse, randomly divided into groups, and 10 nude mice in each group were provided with a normal saline group, a CTX group, In the wild-type hTNFa group (0.025 mg / kg), M2 was set in three dose groups of 2.5, 0.25, and 0.025 mg / kg, respectively, and the dose was 0.5 ml / head. The next day after the inoculation, the injection was continued for 7 days. The animals were sacrificed 10 days after inoculation, and tumor masses were dissected out to compare the tumor weight of each dose group. The formula for judging the results is the same as the tumor suppression experiment in mice. Example 1 The expression vectors pSB-TR (containing the natural hTNF-α gene) and pSB-TK (containing the hTNF- DK2 gene) were constructed. The coding sequence shown in SEQ ID NO: 4 was artificially synthesized based on the amino acid sequence of natural human TNFa, and then. The human TNFa gene hTNF-TR was then cloned between the EcoRI and BamHI sites of the expression vector pSB_92 (see Figure 6 for the complete sequence) to obtain the expression plasmid pSB-TR, which contains the coding sequence SEQ ID NO: encoding natural human TNFa: 4 expression vector pSB92.

According to Gao Changshou et al., "A new type of highly efficient and low toxicity human tumor necrosis factor mutant and its high expression in Enterobacter", Chinese Journal of Biochemistry and Biophysics, 1996, 28 (1): 49-55 The method described above mutates the arginine codon at position 2 of the natural human TNFa to a lysine codon to obtain the coding sequence hTNF-TK shown in SEQ ID NO: 5. The hTNF-TK gene was cloned between the EcoRI and BamHI sites of the expression vector pSB-92 (see Figure 6 for the complete sequence) to obtain the plasmid pSB-TK, the PSB92 expression vector containing the human TNFa gene with the Arg2Lys mutation.

Construction of expression vectors and engineered bacteria expressing hTNF-a mutant proteins M1 and M2

Based on the expression plasmids pSB-TR (containing the natural hTNF-a gene) and pSB-TK (containing the hTNF-DK2 gene) constructed in Example 1, expression vectors expressing hTNF-mutants M1 and M2 were constructed.

(1) Design and synthesize the following primers:

No. 1: a sequence of the vector pSB-92 (upstream primer)

5 '-GATACGAAACGAAGCATTGGTTAA-3'

No. 2: Change arginine at position 32 to tryptophan (Over-Lap PCR downstream primer)

5, -AGAGCGTTAGCCCAACGGTTC-3 '

No. 3: Change arginine at position 32 to tryptophan (Over-Lap PCR upstream primer)

5, -GAACCGTTGGGCTAACGCTCA-3 '

No. 4: Change asparagine at position 30 to serine and arginine at position 32 to tryptophan (Over-Lap PCR downstream primer)

5, -AGCGTTAGCCCAACGGGACAGCCATTG-3 '

No. 5: Change asparagine at position 30 to serine and arginine at position 32 to tryptophan (Over-Lap PCR upstream primer)

5 '-CAATGGCTGTCCCGTTGGGCTAACGCT-3'

No. 6: Change leucine 157 to phenylalanine (downstream primer)

5, -TCAACTTAGCGATAATACCGAA-3 '

(2) conventional PCR

100ng of template was added to the 100ul reaction system, and 50pmol of each of the two primers were denatured at 95 ° C for 5 minutes to enter the cycle. The temperature of the cycle was 94 ° C for 40 seconds; 52 ° C for 50 seconds and 72 for 1 minute. A total of 35 cycles , Then 72 ° C for 10 minutes.

(3) Over-Lap PCR

Referring to Figure 1, 'Take pSB-TR as a template, mix primer 1 and primer 2 of downstream primer No. 2 of Over-lap PCR with primer 1 and primers 3 and 6 of upstream primer of Over-lap PCR lb. After denaturing at 94 V for 5 minutes and then naturally cooling to 30 ° C, Taq enzyme and άΝΤί 37 were added. After 30 minutes at C, primers No. 1 and No. 6 were added to perform the above-mentioned conventional PCR amplification to obtain 480 bp amplified fragment I.

See Figure 1 ', using' pSB-TK as a template, the primer i and the amplified fragment 11a of the downstream primer No. 4 of the Over-lap PCR and the amplified fragment lib of the upstream primers No. 5 and 6 of the Over-lap PCR After mixing, the above process was repeated to obtain a 480 bp amplified fragment ^ Π.

(4) Construction of mutant gene expression plasmid and expression in E. coli:

• Referring to Figure 1, a PCR product (amplified fragment I or II) of about .480bp was digested with EcoRI and BamHI, and inserted into the pSB-92 expression vector digested with the same enzyme to transform E. coli YK537. The transformants containing the inserted fragments were screened to obtain the mutant genes pSB-MTNF1 and pSB-MTNF2 (Fig. 1). Each expression plasmid was digested with EcoRI and BarnHI to cut out a 480 bp fragment (Figure 3). This fragment was inserted into Puc-19 to extract double-stranded DNA, and sequenced on the automatic sequencer of ABI Corporation in the United States. The results were as expected (results not shown).

From the transformed E. coli YK537, which respectively contained the expression plasmids pSB-MTNF1 and pSB-MTNF2, the expression plasmids pSB-MTNF1 and pSB-MTNF2 were isolated and transformed into E. coli BL21, respectively, to obtain transformed engineered bacteria. The engineered bacteria obtained from E. coli BL21 transformed with the expression plasmid pSB-MTNF2 is named E. coli BL21 / pSB-T4; the engineered bacteria obtained from E. coli BL21 transformed with the expression plasmid pSB-MTNF1 is named E. coli BL21 / pSB-T2.

Escherichia coli BL21 / pSB-T4, which expresses the mutant protein M2 of human tumor necrosis factor TNFa, was deposited at CCTCC (Wuhan, China) on March 3, 1999 under CCTCC M99004.

'

Example 3

hTNF-α mutein expression plasmid in E. coli for expression and protein blotting method. When expressing, pick a single colony in LB medium at 30. ° C overnight and incubate at 1:50 ratio. X In YT, 3ΰ Ό was cultured with vigorous shaking for 2 hours, and culture was continued for 4 hours after induction at 42 ° C, and the cells were collected.

After the collected bacterial cells were broken, the bacterial cell lysate was transferred to nitrocellulose membrane by SDS-PAGE after electrophoretic printing, and then reacted with murine anti-hTNF-α monoclonal antibody ascites and secondary antibody goat anti-mouse IgG- HRP Then, the substrate diamino 'benzidine was added. As a result, each mutant protein was able to bind to the anti-wild-type monoclonal antibody, and developed the color at the same position as the wild-type TNF (Figure 4).

From Escherichia coli YK537 transformed with the expression plasmids pSB-MTNF1 and pSB-MTNF2, a single clone was picked and grown to a logarithmic growth phase and induced by temperature. After induction of expression, the expressing cells were subjected to 15% SDS-PAGE. After bright blue staining, a 17KD size band appeared (Figure 4), and the expression of each mutant protein was scanned by laser grayscale. The amounts accounted for more than 50% of the total protein of the bacteria. Compared with the wild type, each mutant protein was It was also highly expressed in E. coli. Example 4

Isolation and purification of human TNFa mutein M1 and M2 expression products

After inducing expression, the bacterial cells were sonicated and purified according to the following purification method: Take the sonicated centrifugal supernatant 40-60% of the ammonium sulfate component by DEAE-Sepharose FF, collect the TNF peak, and CM-Sepharose FF After further purification, the TNF peak was collected and precipitated by dialysis with ammonium sulfate precipitation, and the pure TNF product was obtained by Sephacryl-100 (Fig. 5). The purity was more than 95% by laser grayscale scanning. Example 5

Activity and Toxicity of Human TNFa Muteins M1 and M2

A. Activity on mouse fibroblastoma cell L929.

The activity was measured using wild-type hTNF-a as a control. The activities were: wild-type hTN a was 8. lxl0 ⁶ U / mg, MutTNF-1 was 7.94xl0 ² U / mg, MutTNF- 2 The activity was 4. OxlO mg. The results showed that the activity of the mutant protein M1 on mouse fibroblast tumor cell L929 decreased by 4 orders of magnitude, and the activity of the mutant protein M2 was almost completely lost, indicating that the 30-position Asn → Ser, 32-position Arg -Tyr modification severely reduced the killing activity of each mutant protein on L929 cells.

B, hTNF- ex muteins on the killing effect of various human tumor cells

Human tumor cells: laryngeal cancer Hep-2 cells, liver cancer HepG-2 cells, gastric cancer MGC cells, and ovarian cancer 3A0 cells were plated, and then serially diluted mutant protein samples (more than 95% purity) were added, and purified wild-type The sample was used as a control, and the killing activity of the sample on various human tumor cells was measured (Table 1). It is known from the table that although the activity of each mutant protein on mouse fibroblast cells L929 has been severely reduced, the killing of several human tumor cells is similar to that of the wild type. Among them, the activity ratio of Ml to liver cancer HepG-2 cells The wild-type hTNF-a is 6 times higher, indicating that the results of hTNF-a on mouse tumor cells and human tumor cells are not parallel, that is, the biological activity on L929 cells does not represent killing human tumors. Cell results.

Table 1. Determination of killing activity of each mutant protein on four human tumor cells

C, hTNF-a mutant protein LD50 determination The mice were injected with different doses of mutant proteins Ml and M2 at one time, and wild-type hTNF-a was used as a control. The LD50 value of each mutant protein was determined by the Bliss method (Table 2). The mutant protein M2 could not be restricted by the concentration of the sample. The LD50 value was obtained, but according to the pre-experiment results, it can be known that the 30% lethal dose to mice is 107 mg / kg. The results showed that the LD50 of the mutant protein M1 was 300 times lower than that of the wild type, and the 30% lethal dose of M2 to the mice was 700 times lower than that of the wild type LD50. As for the gender death difference after the injection of MTNF-1, it remains to be seen 'For further research. ,

Determination of LD50 value of each mutant protein

D. Preliminary toxicity observation of rTNFoc mutein M2 by intravenous injection in rhesus monkeys for 10 consecutive days

The results of a long-term pretoxicity test of rhesus monkeys by continuous intravenous injection for 10 days showed that when the injection dose was 200 _U g / m2 / d, 4 monkeys in the wild-type hTNFa administration group died and 3 in the hT Fa mutant protein M2 group. All survived (Table 3). In addition, the hTNFa mutant protein M2 group's body weight decreased slightly after the drug administration, but the average body weight did not change significantly after administration of Yu (Table 4); except for the ALT drug, the blood biochemical indicators of this group had a significant decrease. There was no significant difference in each index before and after administration in other cases (Table 5). Table 3 Toxicity experiments of wild type hTNFcc and mutant protein M2 on rhesus monkeys

Table 5. Intravenous injection of hTNFa mutein M2 (200ug / m ² / dx lOd) in rhesus monkeys Impact on blood biochemical indicators

的 The efficacy of hTNFa 'mutant protein M2 on mouse sarcoma S180 and liver cancer HAC solid tumors. At a dose of 0.025 mg / kg, the inhibitory rate of M2 on mouse sarcoma S180 is about 23% higher than that of wild-type hTNFa. At this concentration, the inhibitory rate of M2 on liver cancer HAC solid tumors is slightly lower than that of wild-type hTNFa. At the injection doses of 0.25 mg / kg and 2.5 mg / kg, the tumor inhibition rates of M2 on S180 reached 59.35% and 69.06%, respectively, while the tumor inhibition rates of M2 on HAC reached 62.41% and 75.94%, respectively. The experimental results are shown in Tables 6 and 7.

Table 6 Antitumor effect of hTNFa mutant protein M2 on mouse sarcoma S180

* P <0.01, compared with the normal saline group Table 7 hTNFa mutant protein M2 antitumor effect on mouse liver cancer HAC Number of animals administered by dose Animal weight tumor weight ω Tumor inhibition rate group (mg / kg) X + SD% at the beginning of the plan Blank control 10ml / kg ivx7 10 10 19.6 30.5 2.66 + 0.57

(Saline)

Wild type hT Fa 0.025 ivx7 10 10 19.5 28.8 1.45 + 0.51 * 45.49

0.025 ivx7 10 10 19.1 29.5 1.63 ± 0.50 * 38.72

M2 0.25 ivx7 10 10 19.4 25.5 1.00 ± 0.49 * 62.41

2.5 ivx7 10 10 19.2 23.1 0.64 ± 0.21 * 75.94

* P <0.01, compared with saline group

F. Effect of TNFa mutein M2 on human bladder cancer CP-3 transplanted in nude mice

The experimental results of hTNFa mutant protein M2 on human bladder cancer CP-3 transplanted in nude mice are as follows: At a dose concentration of 0.025 mg / kg, the tumor inhibition rate of M2 is 45.50%, while that of wild-type hTNFa is only 15.50%. Tumor inhibition rate. And M2 also achieved high tumor inhibition rates of 52.10% and 64.10% at the injection doses of 0.25mg / kg and 2.5mg / kg (Table 8). hTNFa mutant protein M2 on nude mice

Antitumor effect of human bladder cancer CP-3

* P <0.01, compared with the saline group

The TNFct mutant protein of the present invention can efficiently kill human tumor cells and the LD50 of the acute toxicity experiment is much lower than that of the wild type, but the biological activity of the mouse fibroblast cell L929 is almost lost. Specifically, the mutant protein Ml had an activity of 157 amino acids and decreased L929 activity by four orders of magnitude compared with the wild type, but its activity against human laryngeal cancer cells Hep-2 was consistent with that of the single 32-bit mutant protein, which was similar to that of the wild type. ; Mutant protein M2 After increasing the N-terminal 2 and 30 mutations, the activity on L92Q decreased by five orders of magnitude compared with the wild type, but the killing activity on tumor cells was still maintained. The most unexpected is the LD50 of the mutant proteins M1 and M2. The results show that they have extremely low toxicity, of which M2 is even less toxic than Ml.

The results of a long-term toxicity pre-injection test for rhesus monkeys for 10 consecutive days showed that when the injection dose was 200 ug / m2 / d, 4 monkeys in the wild-type hTNFc administration group died and 3 in the hTNFa mutein M2 group. After the second drug, there was no chilling symptom commonly seen in intravenous tumor necrosis factor. The weight of the animals decreased slightly after the treatment, but there was no significant change in the average body weight before and after the administration. There were no significant differences in blood biochemical indicators of animals except for the ALT drug before and after administration. The hTNFa mutant protein M2 is less toxic to rhesus monkeys than wild-type hTNFcc.

The in vivo curative effect test of the hTNPa mutant protein M2 of the present invention on mouse sarcoma S180 shows that at a concentration of 0.025 mg / kg, the tumor inhibition rate of M2 is about 23 percentage points higher than that of wild-type hTNFa, and at this concentration, The inhibitory rate of M2 on liver cancer HAC solid tumors is slightly lower than that of wild-type hTNFa. However, because M2 mice have very low toxicity in vivo, higher concentrations can be used. At 0.25 mg / kg and 2.5 mg / kg injection doses, the tumor inhibition rates of M2 on S180 reached 59.35% and 69.06%, respectively, while the tumor inhibition rates of M2 on HAC reached very high 62.41% and 75.94%, respectively. In addition, the efficacy of hTNFa mutant protein M2 on human bladder cancer CP-3 transplanted in nude mice further demonstrated the antitumor effect of M2. At a concentration of 0.02,5 mg / kg, the tumor inhibition rate of M2 was 45.50%, while that of wild-type hTNFa was only 15.50%. And M2 also achieved high tumor inhibition rates of 52.10% and 64.10% at the injection doses of 0.25mg / kg and 2.5mg / kg.

The results of this experiment differ from previous reports of protein-engineered TNF muteins. Internationally, mouse fibroblast L929 was used as a cell model to test the biological activity of hTNFa. When the activities of two hTNFa mutant proteins, Ml and M2, were measured, their results on L929 cells and their effects on human tumor cells were found. The results are not parallel; in addition, the hTNFa mutein M2 tumor inhibition experiments in mice further prove that the mutein has a good effect on tumors of mouse origin. This shows that there is a certain limitation on the biological activity of hTNFcc measured by murine L929 cells alone '. -All documents mentioned in the present invention are cited in this application as if each document was individually cited as a reference. In addition, it should be understood that after reading the above-mentioned teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application. Sequence Listing

(1) General information:

(i) Applicant: Shanghai Center for Biological Engineering, Chinese Academy of Sciences

(ii) Name of invention: Novel human tumor necrosis factor mutant protein, and preparation method and application thereof

(iii) Number of sequences: 5

(2) Information of SEQ ID NO. 1

(i) Sequence characteristics:

(A) Length: 157 amino acids

(B) Type: Amino acid

(C) Chain: single chain

(D) Topological structure: linear

(ii) Molecular type: peptide

(ix) Sequence description: SEQ ID NO. 1:

Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 16

Val Ala Asn Pro Gin Ala Glu Gly Gin Leu Gin Trp Leu Asn Arg Trp Ala 33

Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gin Leu Val Val 50

Pro Ser Glu Gly Leu Tyr Leu lie Tyr Ser Gin Val Leu Phe Lys Gly Gin 67

Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr lie Ser Arg lie Ala 84

Val Ser Tyr Gin Thr Lys Val Asn Leu Leu Ser Ala lie Lys Ser Pro Cys 101

Gin Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro lie 118

Tyr Leu Gly Gly Val Phe Gin Leu Glu Lys Gly Asp Arg Leu Ser Ala Glu 135 lie Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly Gin Val Tyr Phe 152

Gly lie lie Ala Phe 157

(2) Information of SEQ ID NO. 2

(i) Sequence characteristics:

(A) Length: 157 amino acids

(B) Type: Amino acid

(C) Chain: single chain

(D) Topological structure: linear

(ii) Molecular type: peptide

(ix) Sequence description: SEQ ID NO. 2:

Val Lys Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 16 Val Ala Asn Pro Gin Ala Glu Gly Gin Leu Gin Trp Leu Ser Arg Trp Ala 33 Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gin Leu Val Val 50 Pro Ser Glu Gly Leu Tyr Leu lie Tyr Ser Gin Val Leu Phe Lys Gly Gin 67 Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr lie Ser Arg lie Ala 84 Val Ser Tyr Gin Thr Lys Val Asn Leu Leu Seru Ala lie Lys Ser Pro Cys 101 Gin Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro lie 118 Tyr Leu Gly Gly Val Phe Gin Leu Glu Lys Gly Asp Arg Leu Ser Ala Glu 135 lie Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly Gin Val Tyr Phe 152 Gly lie lie Ala Phe 157

(2) Information of SEQ ID NO. 3

(i) Sequence characteristics-

(A) Length: 157 amino acids

(B) Type: Amino acid

(C) Chain: single chain

(D) Topological structure: linear

(ii) Molecular type: peptide

(ix) Sequence description: SEQ ID NO. 3:

Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val 16 Val Ala Asn Pro Gin Ala Glu Gly Gin Leu Gin Trp Leu Asn Arg Arg Ala 33

Asn Ala Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gin Leu Val Val 50

Pro Ser Glu Gly Leu Tyr Leu lie Tyr Ser Gin Val Leu Phe Lys Gly Gin 67

Gly Cys Pro Ser Thr His Val Leu Leu Thr His Thr lie Ser Arg lie Ala 84

Val Ser Tyr Gin Thr Lys Val Asn Leu Leu Ser Ala lie Lys Ser Pro Cys 101 Gin Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu Pro lie 118

Gly lie lie Ala Leu 157 (2) SEQ ID NO. 4

(i) Sequence characteristics:

(A) Length: 471bp

(B) Type: Nucleotide

(C) Chain: single chain

(D) Topological structure: linear

(ii) Molecular type: Nucleotide

(ix) Sequence description: SEQ ID NO. 4:

GTA AGA TCT AGC TCT CGC ACT CCA TCT GAC AAA CCA GTT GCT CAT GTT 48

GTT GCT AAC CCA CAG GCT GAA GGT CAG CTG CAA TGG CTG AAC CGT CGT GCT 99

AAC GCT CTG CTG GCT AAC GGT GTT GAA CTG CGT GAC AAC CAG CTT GTG GTA 150

CCG TCT GAA GGT CTG TAC CTG ATC TAC TCC CAG GTT CTT TTC AAA GGT CAG 201

GGT TGC CCA TCT ACA CAC GTT CTG CTT ACC CAC ACT ATC TCT CGT ATT GCT 252

GTT TCC TAC CAG ACT AAA GTT AAC CTG CTG TCT GCG ATC AAA TCT CCG TGC 303

CAG CGT GAA ACC CCA GAA GGT GCT GAA GCT AAA CCA TGG TAT GAA CCG ATT 354

TAC CTT GGT GGT GTT TTC CAA CTG GAG AAG GGT GAC CGT CTG TCT GCT GAA 405

ATC AAC CGT CCA GAC TAC CTT GAC TTC GCT GAA TCT GGT CAG GTA TAC TTC 456

GGT ATT ATC GCT CTG 471 (2) Information of SEQ ID NO. 5

(i) Sequence characteristics:

(A) Length: 471bp

(B) Type: Nucleotide

(C) Chain: single chain

(D) Topological structure: linear

(ii) Molecular type: Nucleotide

(ix) Sequence description: SEQ ID NO. 5:

GTA AAA TCT AGC TCT CGC ACT CCA TCT GAC AAA CCA GTT GCT CAT GTT 48

GTT GCT AAC CCA CAG GCT GAA GGT CAG CTG CAA TGG CTG AAC CGT CGT GCT 99

AAC GCT CTG CTG GCT AAC GGT GTT GAA CTG CGT GAC AAC CAG CTT GTG GTA 150

CCG TCT GAA GGT CTG TAC CTG ATC TAC TCC CAG GTT CTT TTC AAA GGT CAG 201

GGT TGC CCA TCT ACA CAC GTT CTG CTT ACC CAC ACT ATC TCT CGT ATT GCT 252

GTT TCC TAC CAG ACT AAA GTT AAC CTG CTG TCT GCG ATC AAA TCT CCG TGC 303

CAG CGT GAA ACC CCA GAA GGT GCT GAA GCT AAA CCA TGG TAT GAA CCG ATT 354

TAC CTT GGT GGT GTT TTC CAA CTG GAG AAG GGT GAC CGT CTG TCT GCT GAA 405

ATC AAC CGT CCA GAC TAC CTT GAC TTC GCT GAA TCT GGT CAG GTA TAC TTC 456

GGT ATT ATC GCT CTG 471

Claims

Claim

A human tumor necrosis factor mutant protein, characterized in that it contains an Arg32Trp / Leul57Phe mutation.

The human tumor necrosis factor mutant protein according to claim 1, further comprising an Arg2Lys / Asn30Ser mutation.

The human tumor necrosis factor mutein according to claim 1 or 2, characterized in that it has the amino acid sequence shown in SEQ ID NO. 1 or 2.

A pharmaceutical composition, characterized in that it contains an effective amount of the human tumor necrosis factor mutant protein according to claim 1 and a pharmaceutically acceptable carrier and / or excipient.

5. An isolated DNA sequence, characterized in that it encodes the human tumor necrosis factor mutant protein according to claim 1.

6. An expression vector comprising the DNA sequence according to claim 5.

7. A host cell characterized by being transformed with the expression vector according to claim 6.

The host cell according to claim 7, wherein the host cell is E. coli BL21 / pSB-T4 CCTCC No. M99004.

A method for producing a human tumor necrosis factor mutant protein according to claim 1, wherein the method comprises the steps of:

(a) operably linking a DNA sequence encoding the human tumor necrosis factor mutant protein according to claim 1 to an expression control sequence to form a human tumor necrosis factor mutant protein expression vector;

(b) transforming the expression vector in step (a) into a host cell;

(d) isolating and purifying the human tumor necrosis factor mutant protein.

10. The use of the human tumor necrosis factor mutein according to claim 1, characterized in that it is used for preparing a pharmaceutical composition.