TWI363092B - Biologically active peptides (4) - Google Patents

Description

1363092 IX. INSTRUCTIONS: TECHNICAL FIELD OF THE INVENTION C FIELD OF THE INVENTION The present invention relates to the field of immunology. In particular, the present invention relates to a peptide capable of modulating an immune response and a pharmaceutical composition thereof. L Prior Art 3 Background of the Invention Bioactive peptides are known in the art for use in the treatment of diseases and as pharmaceutical compositions. For example, US 6,191,113 discloses a peptide having inhibitory activity against smooth muscle cell growth 10, which can therefore be used for controlling pathological conditions associated with smooth muscle cell growth, such as arteriosclerosis, restenosis after angioplasty, and post-transplantation. The stenosis of the cavity, and leiomyosarcoma. Another peptide, disclosed in US 6,184,208, was found to regulate - some physiological processes, the weight gain activity of the epithelial growth zone and hair growth. 15 SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to identify bioactive peptides. A number of peptides were first synthesized by standard chemical methods and their biological activity was identified. The peptide number is indicated by the letter CMS followed by the digits. A total of 30 peptides were identified as having 20 biological activities in vivo. The sequences of these bioactive peptides and their corresponding sequence identification numbers (SEQ ID NO) are shown in Table A.

Table A Sequence Peptide Name Peptide Sequence 1363092 Collection · 1 CMS001 Pro Thr Thr Lys Thr Tyr Phe Pro His Phe 2 CMS002 Val Val Tyr Pro Trp Thr Gin Arg Phe 3 CMS008 Lys Ala Val Gly His Leu Asp Asp Leu Pro Gly Ala Leu 4 CMS010 Val Ala Pro Glu Glu His Pro Thr Leu Leu Thr Glu Ala ProLeu Asn Pro Lys 5 CMS012 Leu Gly Met Glu Ala Cys Gly lie His Glu Thr Thr Tyr 6 CMS013 Leu Arg Val Ala Pro Glu Glu His Pro Val Leu 7 CMS014 Ala Ala His His Pro Asp Asp Phe Asn Pro Ser Val 8 CMS015 Pro Ser lie Val Gly Arg Pro Arg His Gin Gly Val Met 9 CMS016 lie Gly Met Glu Ser Ala Gly lie His Glu Thr Thr Tyr 10 CMS018 Val Gly Met Gly Glu Lys Asp Ser Tyr 11 CMS019 Val Gly Met Gly Gin Lys Asp Ser Tyr 12 CMS020 Val Gly Met Gly Gin Lys Asp Ser Tyr Val 13 CMS021 Met Ala Thr Ala Ala Ser Ser Ser Ser Leu 14 CMS022 Tyr Ser Phe 15 CMS023 Ala Ala Phe 16 CMS024 Tyr Ser Leu 17 CMS026 Thr Thr Tyr Asn Ser lie Met 6 18 CM-S027 Phe Glu Glu Asn Met 19 CMS028 Phe Glu Pro Ser Phe 20 CMS029 Phe Asn Glu Glu 21 CMS030 Phe Gl u Glu Met 22 CMS032 Phe Glu Glu Glu 23 CMS033 Phe Glu Ser Phe 24 CMS034 Pro Glu Asn Phe 25 CMS035 Phe Val Asn Asp 26 CMS036 Phe Gin Pro Ser Phe 27 CMS003 Phe Asn Phe Val Pro Pro 28 CMS007 Ala Gly Asp Asp Ala Pro Arg Ala Val Phe 29 CMS009 Leu Arg Val Ala Pro Glu Glu His Pro Thr Leu 30 CMS011 Arg Val Ala Pro Glu Glu H-is Pro Thr Leu Accordingly, one aspect of the invention relates to substantially purified peptides having sequence recognition Number: 1 to sequence identification number: the sequence shown in 30. Accordingly, the present invention also relates to substantially purified peptides comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-30. The invention further relates to substantially purified peptide consisting essentially of an amino acid sequence selected from the group consisting of: Sequence Number: 1-30. In a specific embodiment, the peptides of the invention may modulate, but are not limited to, modulation, one or more of the following: immunological activity; hepatitis infection, including but not limited to hepatitis B infection; nephritis; growth of cancer, including but not limited to Sarcoma, liver cancer, leukemia and melanoma; and body weight. Another aspect of the invention relates to a substantially purified peptide which is a functional derivative of a peptide having the amino acid sequence of sequence identification number: 1-30. Accordingly, the present invention also relates to a substantially purified peptide comprising an amino acid sequence which is a functional derivative of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-30. The present invention also relates to a substantially purified peptide consisting essentially of an amino acid sequence which is a functional derivative selected from the group consisting of an amino acid sequence of SEQ ID NO: 30. In a specific embodiment, the peptides of the invention may be modulated, but not limited to, one or more of the following: immunological activity; hepatitis infection, including but not limited to hepatitis B infection; nephritis; growth of cancer, including but not limited to Sarcoma, liver 10 cancer, leukemia and melanoma, and body weight. Another aspect of the invention relates to a nucleic acid having the coding sequence for a peptide of the above sequence identification number: 1 to sequence identification number: 30. The present invention further relates to an expression vector comprising the sequence identification number: 1 to the sequence identification number: 30. i. Accordingly, this aspect of the invention also relates to a genetic vector comprising a nucleotide sequence encoding a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-30. The present invention also relates to a genetic vector comprising a nucleotide sequence encoding a peptide consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-30. The invention further relates to a genetic vector comprising a nucleotide sequence encoding a peptide of 20, said peptide comprising a functional derivative of a biologically active amino acid sequence selected from the group consisting of SEQ ID NO: 1-30. The invention also relates to a genetic vector comprising a nucleotide sequence encoding a peptide consisting essentially of a functional amino acid sequence which is a functional derivative of a biologically active amino acid sequence selected from the sequence identification number: 1-30. composition. A further aspect of the invention relates to a hybrid peptide comprising a leader or signal peptide adjacent to a peptide comprising an amino acid sequence selected from the group consisting of: 1-30. The present invention also relates to a hybrid peptide comprising a leader adjacent to a peptide comprising a functional derivative having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-30. The invention also relates to a genetic vector comprising a nucleotide sequence encoding a peptide comprising a leader amino acid sequence adjacent to a peptide comprising a functional amino acid sequence selected from the group consisting of: A functional derivative of a biologically active amino acid sequence of 1-30. The invention also relates to a genetic vector comprising a nucleotide sequence encoding a peptide comprising a leader amino acid sequence adjacent to a peptide consisting essentially of a functional amino acid sequence selected from the group consisting of a sequence Identification of J. Tiger: A functional derivative of a biologically active amino acid sequence of 1-30. In a specific embodiment, the peptide produced by any of the above genetic vectors can be modulated, but not limited to, one or more of the following: immunological activity, hepatitis infections including, but not limited to, hepatitis B infection; nephritis; cancer Growth, including but not limited to sarcoma, liver cancer, leukemia and melanoma; and body weight. A further aspect of the invention relates to a microorganism having a genome comprising a nucleotide sequence encoding a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-30. The present invention also relates to a microorganism having a genome comprising a nucleotide sequence encoding one month, the peptide consisting essentially of an amino acid sequence selected from the sequence identification number 1-30. + A further aspect of the invention relates to a microorganism having genetic material comprising a nucleoside 醆 sequence encoding a foreign peptide, the exogenous peptide comprising a function selected from the group consisting of a sequence number: a biologically active amino acid sequence $ Functional amino acid sequence of the organism. The present invention also relates to a microorganism having a genetic composition comprising a nucleotide sequence encoding a 'exogenous peptide, the exogenous peptide being substantially a species selected from the group consisting of the sequence identification number: Functional amino acid sequence composition of functional derivatives. As used herein, a foreign peptide refers to a peptide having an amino acid sequence that is different from any other peptide that is naturally expressed in the natural unmodified form. A further aspect of the invention relates to a microorganism having a genetic composition comprising a nucleotide sequence encoding an exogenous heterozygous genomic DNA, the exogenous hybrid peptide comprising a leader amino acid sequence adjacent to the sputum, the peptide comprising a Sequence Identification Number: An amino acid sequence of 1-30. The invention further relates to a microorganism having a genome comprising a nucleotide sequence encoding a hybrid peptide comprising a peptide consisting essentially of a gas-based acid sequence selected from the sequence identification number: Adjacent leader amino acid sequence. A further aspect of the invention relates to a microorganism having a genetic composition comprising a nucleotide sequence encoding an exogenous hybrid peptide, the exogenous hybrid peptide comprising a leader amino acid sequence adjacent to a peptide, the peptide comprising a A functional amino acid sequence selected from the functional derivative of a biologically active amino acid sequence of Sequence No.: 1-30. The present invention also relates to a microorganism having a genetic composition comprising a nucleotide sequence encoding an exogenous hybrid peptide comprising a leader amino acid sequence adjacent to a peptide, the peptide being substantially A functional amino acid sequence consisting of a functional derivative of a biologically active amino group 10 1363092 acid sequence selected from the sequence identification number: In a specific embodiment, the peptide produced by any of the above microorganisms can be modulated, but not limited to, one or more of the following: immunological activity; hepatitis infection, including but not limited to hepatitis B infection; nephritis; Growth, including but not limited to sarcoma, liver cancer, leukemia and melanoma; and body weight. Another aspect of the invention relates to a pharmaceutical composition comprising a substantially purified peptide comprising an amino acid sequence selected from the group consisting of: 1-30. The invention further relates to a pharmaceutical composition comprising a substantially purified peptide consisting essentially of an amino acid sequence selected from the group consisting of: 1-30. Another aspect of the invention relates to a pharmaceutical composition comprising a substantially purified peptide comprising a functional derivative selected from the group consisting of an amino acid sequence of 1-30. The invention further relates to a pharmaceutical composition comprising a substantially purified peptide consisting essentially of a functional derivative selected from the group consisting of an amino acid sequence of sequence number: 1-30. The invention further relates to a pharmaceutical composition consisting essentially of a substantially purified peptide consisting essentially of a functional derivative selected from the group consisting of an amino acid sequence of sequence number: 1-30. 2 In a specific embodiment, the peptide present in any of the above pharmaceutical compositions may be modulated, but not limited to, one or more of the following: immunological activity; hepatitis infection, including but not limited to hepatitis B infection; nephritis Cancer growth, including but not limited to sarcoma, liver cancer, leukemia and melanoma; and body weight. 11 Another aspect of the invention relates to a method of preparing a pharmaceutical composition comprising providing a substantially purified peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-30, and the substantially purified peptide The pharmaceutically acceptable carrier is mixed. The invention further relates to a method of preparing a pharmaceutical composition comprising providing a substantially purified peptide consisting essentially of an amino acid sequence of sequence number: 1-30. Another aspect of the invention relates to a method of preparing a pharmaceutical composition comprising providing a substantially purified lysate and mixing the lysine with a drug to form a substantially purified peptide An amino acid phase comprising a functional derivative selected from the group consisting of the amino acid sequence of the sequence identification number: The invention further relates to a method of preparing a pharmaceutical composition comprising providing a substantially purified peptide consisting essentially of a functional derivative selected from the group consisting of 1-3G amino acid sequences Amino acid sequence composition. The conditions described in any of the above methods may be adjusted, but not limited to, one or more of the following: immunological activity; hepatitis infection, including but not limited to hepatitis 8 infection; nephritis; growth of cancer, including but not limited to sarcoma , liver cancer, leukemia and melanoma; and weight. Another aspect of the invention relates to a method of treatment of a human comprising administering to a human a therapeutically effective amount of a substantially purified peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-30. The invention further relates to a method of treatment of a human comprising administering to a human a pharmaceutically effective amount of a substantially purified peptide comprising an amino acid sequence which is a functional derivative selected from the group consisting of an amino acid sequence of Sequence Identification Number: . In a specific embodiment, the peptide used to treat a human can be modulated, but not limited to, including, for example, a value of: ^: immunological activity: hepatitis infection, limited to sarcoma, X infection, nephritis; growth of cancer, including but not Escape!, leukemia and melanoma; and weight. Section, but not limited to, =, the nucleic acid sequence expressed in the moon too and / or heterozygous month too can regulate infection, self-twist / tone that 'as follows - or a variety of effects: immune activity; hepatitis but not limited to limited to hepatitis B Infection; nephritis; growth of cancer, including ... liver cancer 'leukemia and melanoma; and weight. Another aspect of the physical learning is related to the method of treating the disease, including administering the peptide of the 10 sequence of the sequence, the sequence identification number: 1 to the sequence identification number: the sequence of the substantially purified peptide of the sequence of 3〇 can be adjusted, but The invention is not limited to the administration of the following - or a plurality of effects: the immune life includes, but is not limited to, hepatitis B infection; nephritis; cancer includes, but is not limited to, sarcoma, liver cancer, white blood (four) _; as described above, the invention (4) The embodiment is a peptide or polypeptide substantially formed by the present invention. The term "consisting essentially of" as used herein refers to a peptide or multi-monthly comprising the amino acid sequence of the invention and the terminal end and/or amino ex-amino acid and (iv) the peptide of the invention provided herein. Activity. Thus, as a non-limiting example, when the activity of the peptide of the present invention is capable of modulating immunological activity, "substantially, by, the composition of the present invention, the peptide or polymorphism will have the properties provided herein. The activity of modulating immunological activity' does not have the ability to substantially reduce the immunological activity of the peptide or polymorphism or to constitute a substantial change in the substantially new feature of the peptide as a modulator of immunological activity. Thus, in the example of the former CS) 13 1363092, the full-length naturally occurring polypeptide whose main activity is not a modulatory immunological activity and which contains the amino acid sequence of the peptide of the present invention somewhere is not "mainly composed" of the peptide of the present invention. a peptide or polypeptide. Similarly, in the foregoing examples, the genetically engineered peptide or polypeptide whose primary activity is not a regulatory immunological activity but which comprises the amino acid sequence of the peptide of the present invention somewhere in 5 is not "consisting essentially of" the composition of the present invention. Peptide or polypeptide. In addition to the above-exemplified examples of immunological modulation effects, the foregoing definitions are also applicable to the various activities of all polypeptides of the present invention. In particular, the foregoing definitions are applicable to peptides of the present invention having activity to modulate viral infection by 10 degrees, to modulate the extent of hepatitis infection, to modulate the extent of nephritis, to modulate cancer growth, or to regulate body weight, as described in the detailed description below. Determination of the activity of a peptide or polypeptide by using the assays provided herein for specific peptides of the invention to modulate the extent of viral infection, modulate the extent of hepatitis infection, modulate the extent of nephritis, modulate cancer growth, or regulate body weight, etc. A person can readily determine whether the peptide or polypeptide conforms to the foregoing definition consists essentially of the peptide of the present invention. In a preferred embodiment, the term "consisting essentially of" refers to a peptide or polypeptide having less than 20 amino acid residues in addition to the peptides of the invention. In a more preferred embodiment, the term refers to a peptide having less than 15 amino acid residues in addition to the 20 peptides of the invention. In a further preferred embodiment, the term refers to peptides having less than 10 amino acid residues in addition to the peptides of the invention. In another preferred embodiment, the term refers to a peptide or polypeptide having less than 6 amino acid residues in addition to a peptide of the invention. In another preferred embodiment, the term is (S) 14 in addition to the present # Μ 耿 另外 另外 另外 另外 另外 或 或 或 或 或 或 或 或 或 或 或 或 或 或 或 或 或 或 或 或The term refers to a peptide or polypeptide having less than two amino acid residues in addition to one of the peptides of the present invention. 5 [" Γ 鲜 鲜 鲜 ^ 些 些 些 些 些 些 些 些 些 些 些 些 些 些 些 些 些 些 些 些 些 些 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Ίο —, biological activity model:: biological activity 'check 7 'The pre-clinical research guidelines for new drugs (Western medicine) issued by the Animal Bureau, the use of T lymphocyte transformation experiments, the detection of cell cytotoxic activity The L-2 and γ~lFN cells are associated with any possible effects on specific cellular immune function. Use small assays to detect any of these traits for non-specific cellular immune function. These peptides were tested for any possible effects of immune function using a sheep red blood cell (SRBC) hemolysis test. Using the weight of the immune organ 2 〇 she is in any of the possible materials. ^ In the study of nine, the peptide sample selected 4 arbitrary concentrations, the adjacent rich sound distance is 10 times 'so the highest and lowest concentration difference 诵 times. At the same time | water The group served as a negative control with the currently accepted immunological increase of IFW-νέΒ [called π and as a positive control. In addition, due to the lack of understanding of the dose response relationship 15 1363092', the body's immune response is quite complex* Therefore, any statistically significant difference between any dose group and negative control is considered to be biologically active. The conclusions of this study are as follows: 5 1 · Peptide CMS001, CMS002, CMS003, CMS007, CMS008, CMS009, CMS010, CMS011, CMS012 CMS015, CMS019, CMS0 2 CMS029, CMS034 can promote the transformation of T lymphocytes, which is significantly different from the saline group. The peptides CMS014 and CMS036 can inhibit the transformation of T lymphocytes, which is significantly different from the saline group. 2. Peptide CMS001, CMS002, CMS003, CMS008, CMS009, CMS010, CMS011, CMS012, CMS013, CMS015, GMS016, CMS020, CMS021, CM S022, CMS023, CMS024, CMS026, CMS027, CMS028, CMS029, CMS030, CMS032, CMS033, CMS034, CMS035, CMS036 have enhanced NK cell killing activity, 15 and there is a significant difference compared with saline group. Peptide CMS008 and CMS012 The appropriate concentration also has the effect of reducing the killing activity of NK cells, and there is a significant difference compared with the saline group. 3 · Peptides CMS001, CMS003, CMS007, CMS009, CMS010, CMS011, CMS012, CMS015, CMS020, CMS022, CMS034 can promote 20 T lymphocytes secrete interleukin-2 (IL-2), which is significantly different from the saline group. 4 · Peptides CMS001, CMS003, CMS009, CMS010, CMS011, CMS012, CMS013, CMS016, CMS021, CMS022, CMS028 can promote T Lymphocytes secrete IFN-γ, which is significantly different from the saline group by 16 CS ) 1363092. 5 · Peptides CMS001, CMS002, CMS003, CMS007, CMS008, CMS009'CMS010'CMS011'CMS012'CMS013'CMS014'CMS015' CMSO16, CMSO18, CMSO19, CMS020, CMS021, CMS022, CMS023, 5 CMS024, CMS026, CMS027, C MS028, CMS029, CMS030, CMS032, CMS033, CMS034, CMS035, and CMS036 promoted the synthesis of anti-SRBC antibodies upon antigen challenge, which was significantly different from the saline group. Peptides CMS002, CMS003, CMS009, CMS0ί〇, CMSOU, CMS013, CMS014, CMS015, CMS018, CMS019, CMS020, CMS026, CMS028, CMS029, 10 CMS030, CMS034, and CMS036 can also inhibit synthetic resistance during antigenic attack at appropriate concentrations. The SRBC antibody was significantly different from the saline group. 6·peptides CMS003, CMS008, CMS009, CMS010, CMS011, CMS013, CMS016, CMS018, CMS019, CMS020, CMS022, 15 CMS024, CMS027, CMS030, CMS035, CMS036 can enhance the phagocytic function of mononuclear phagocytic cells, compared with saline group There are significant differences. 7 · Peptides CMS001, CMS002, CMS008, CMS010, CMS012, CMS013, CMS014, CMS015, CMS016, CMS018, CMS019, CMS020, CMS021, CMS022, CMS023, CMS024, CMS026, CMS027, CMS028, 2〇CMS029' CMS030, CMS032, CMS033 CMS034, CMS035, and CMS036 were able to increase the weight of the thymus, which was significantly different from the saline group. 8 · Peptides CMS019, CMS020, CMS030 can increase the weight of the spleen, and there is a significant difference compared with the saline group. Peptides CMS001, CMS003, CMS007, CMS008, CMS009, CMS010, CMS011, CMS013, CMS014, CS) 17 1363092 CMS015, CMS021, CMS023, CMS024, CMS027, CMS029, and CMS036 reduce the weight and physiology of the spleen at appropriate There was a significant difference in the saline group. Materials and methods for analyzing the effects of peptides on mice are described below: 5 Materials 1. Experimental animals: Pure healthy BALB/C mice weighing i8-22 g, half male and half female, provided by the Experimental Animal Center of the Chinese Academy of Medical Sciences. 2. Administration method: Recombinant murine interferon-Y (rmlFN-y) group: 3xl〇5lu/kg/day ίο Recombinant human interleukin-2 (rhIL-2) group: 3xl〇5lu/kg/day saline group: 0.5ml/day/day CMS-like dose group I: 500pg/kg/day CMS peptide dose group II: 50pg/kg/day CMS peptide dose III group·· 5pg/kg/day 15 CMS peptide dose IV group: 0· 5pg Each group of drugs in /kg/day was dissolved in 0 ml of normal saline, and injected intraperitoneally once a day for 15 days. 1 · Main drugs and reagents Peptide: American Peptide Company 20 Recombinant murine interferon gamma (rmIFN-γ): Beijing Bonding Biotechnology Co., Ltd. Recombinant human IL-2 (rhIL-2): Shanghai Huaxin Biotechnology Co., Ltd.

RPMI-1640 cell culture medium, fetal bovine serum: American gibcO company tetramethyl azo salinium salt (MTT), concanavalin A (ConA): American SIGMA (S) 18 Lymphocyte separation solution: Blisters, Institute of Hematology, Chinese Academy of Medical Sciences Oral stomatitis virus (VSV), IFN_Y, IL2 standard: China National Institute for the Control of Pharmaceutical and Biological Products. HT-2 cells, L929 cells: Professor WFChen, Department of Immunology, Peking University. Method 1 · Effect of peptide on cellular immunity 1. Preparation of spleen cell suspension [1, 2] BALB/c mice were randomly divided into peptide group, ΙΡΝ7 group, IL_2 group 'saline group, 10 groups in each group . On the next day after the last administration, the mice were sacrificed by cervical dislocation, the spleen was aseptically operated, and the spleen tissue was gently crushed with an injection needle, and the single cells were passed through a 1 mesh stainless steel mesh (pore size 150 μm) into the cold Hanks solution. The cell suspension was transferred to a centrifuge tube, centrifuged at 200 g for 1 minute, and the supernatant was discarded. A volume of Tris-NH4C1 was added to the cell pellet, mixed, and allowed to stand at room temperature for 10 minutes and centrifuged at 150 g for 10 minutes. 'The cells were washed again with cold Hanks solution 2 to 4 times, and the number of cells was adjusted to the desired concentration using RPMI-1640 medium containing 10% fetal bovine serum. 1.2 Effect of peptide on T lymphocyte transformation [1'2] Add 1 X 106/ml spleen cell suspension to 96-well cell culture plate, ΙΟΟμΙ/well', set 3 replicate wells per sample. 100 μg/ml ConA of ι〇〇μι/well, plus RPMI-1640 of 100 μl/well as a control. Then, the 96-well culture plate was placed in a 37 C, 5% C〇2 incubator for 66 hours, and then the cells were cultured. The plate was centrifuged at 150 g for 10 minutes, the supernatant was taken, stored at -2 ° C, and the cytokines IL-2 and IFN were detected with 1363092. To the cell pellet of the cell culture plate after the supernatant was removed, 50 μl/well of 1 mg/m 1 MTT solution was added, and after shaking for 2 minutes, the culture was continued for 4 hours at 37 ° C in a 5% C 2 incubator. 150 g of the cell culture plate was further centrifuged for 10 minutes, and the supernatant was discarded. To 5 cell pellets - 40 mM hydrochloric acid-isopropanol oxime 20 μl/well was added, and after shaking for 3 minutes, the absorbance (〇D value) was measured on an ELISA-reader, and the measurement wavelength was 570 nm, and the reference wavelength was 630 nm. Calculation: Each mouse formed 3 assay wells and 3 control wells. The stimuli index (SI) of each mouse was obtained by first calculating the average 0D value of the three replicate wells and then dividing the value of the assay well by the value of the control well. 1.3 Effect of peptide on killing activity of NK cells [3'4] The spleen cells of mice were prepared into 4×l〇6/ml according to the method of 1.1, and YAC-1 cells which had reached the logarithmic growth phase were made into lxl〇. 5/ml. Spleen cells 1 μμ1/well and cell culture medium 1 μμ1/well were added to 96-well cell culture plates as effector wells; YAC-1 cells were added 1 μμl/well and medium 1 μμ1 /well, as a target cell well; add 1 〇〇μ1/well of YAC-1 cells and 1 〇〇μ1/well of spleen cells as experimental wells. There are 3 duplicate holes for each sample. The 96-well culture plate to which the sample was added was placed in a 37 ° C, 5% C 2 incubator for 4 hours. 2〇 Centrifuge the cell culture plate at 150g for 1 , minutes and discard the supernatant. 50 μl/well of lmg/ml MTT solution was added, and after shaking for 2 minutes, the culture was continued for 4 hours at 37 ° C in a 5% C 2 incubator. 150 g of the cell culture plate was centrifuged for 10 minutes, and the supernatant was discarded. After adding 40 mM hydrochloric acid-isopropyl alcohol 120 μl/well, after shaking for 3 minutes, the absorbance (0D value) was measured on an ELISA-reader, and the measurement wavelength was 570 nm, and the reference wave 20 1363092 was 630 nra. Calculations: There were 9 wells per small defeat: 3 controls containing only spleen cells, 3 controls containing only stem cells' 3 assay wells containing splenocytes and stem cells. The NK cell activity index of each petty 5 is obtained by first deriving the average 0D value of each of the three replicate wells, and then substituting this average (10) value into the following formula: NK cell activity index = [Bu (spleen cell and t cell pore mean 〇1) value _ only spleen cell hole average 〇D value) + (stem cell hole average (10) value)] χ1 discussion 10 1 · 4 peptide on Τ lymphocyte secretion il-2 activity effect experiment (5) The ΗΤ-2 cells in the logarithmic growth phase were centrifuged for 1 min at i5 〇g, the supernatant was discarded, the cold Hanks solution was resuspended and the cells were washed three times by centrifugation, and the collected HT-2 cells were resuspended in RpMI_164 ,, 37. (:, 5% C〇2 was incubated for 30 minutes, and then resuspended in RPMI-1640 and centrifuged twice, and RPMI-164 〇 was used to prepare a cell suspension of 2χ 15 105/ml. The supernatant was diluted with the rpmi-1640 cell culture medium to the following concentrations: 100%, 50%, 25%, 12.5%, 6.25%, 3.125%. rIL-2 was used in RPMI-1640 cell culture medium. Dilute to the following concentration: 500 IU/ml '250 IU/ml ' 125 IU/ml &gt; 62. 5 IU/ml ' 31. 25 IU/ml ' 20 15.5 IU/ml. In 96-well cell culture plates, set according to each combination below 3 parallel negative control wells: 100μ1ΚΡΜΙ-1640 + 100μ1ΗΤ-2 cell suspension rIL-2 standard wells: ΙΟΟμΙ rIL-2 solution + 1〇〇μ1ΗΤ-2 cells 25 suspension 21 analysis well: 1〇〇μ1 diluted well Cell supernatant + 1〇〇μ1ΗΤ_2 cell suspension. Add the sample cell culture plate to 37°C, 5%C〇2 incubator for culture 68], then centrifuge at 15°g for 15 minutes. The supernatant was removed, and 0. 5 mg/ml MTT, ΙΟΟμΙ/well prepared by phenol-free red RPMI-1640 was added to each well, and the incubation was continued for 4 hours. The cells were further centrifuged for 15 minutes at i5〇g for 15 minutes. go with On the last month, add isopropanol hydrochloride (1:3〇〇v/v), ΐ2〇μΐ/well, and mix by shaking for 3~4 minutes. Select the detection wavelength of 570mn on the microplate reader and measure the 0D value at the reference wavelength of 630nm. Calculation: Take the average 〇d of 3 parallel wells for each dilution and plot on the semi-logarithmic paper as the concentration of X. Obtain the concentration of the test supernatant and the saturation of rIL-2 at 5〇%〇d. Sample IL-2 activity = (dilution of the sample up to 50% of the maximum reaction IL-2 standard up to 50% of the maximum reaction dilution) xr IL-2 standard up to 50% of the maximum reaction dilution (IU /ml) 1. Effect of 5 peptide on T lymphocyte secretion of IFN activity [6] The supernatant obtained in 1.2 was diluted with RPMI-1640 cell culture medium to the following concentrations: 100%, 50%, 25%, 12. 5%, 6.25%, 3.125%. The rIFN standard was diluted to the following concentrations with RPMI-1640 cell culture medium: 500 IU/ml, 250 IU/ml, 125 IU/ml, 62.5 IU/ml, 31.25 IU/ Ml, 15. 5 IU/ml. Using RPMI-1640 cell culture medium, the [929 target cells in the logarithmic growth phase were adjusted to 2xl05/ml, and the attack intensity of the VSV virus stock solution was adjusted to 1363092 100TCID5. . In a 96-well cell culture plate, three parallel wells were set as follows. Negative control well: 150μ1ΙίΡΜΙ-1640 + 1〇〇μ1ί929 cells positive control well: 100plRPMI-1640 + 1〇〇μΐί929 cells + 5 50pLVSV virus rIFN active well: lOOplrlFN standard + ΐ〇〇μι [929 cells + 50pLVSV virus assay wells : ΙΟΟμΙ diluted cell supernatant + 1〇〇μ1ί929 cells + 50yLVSV virus 10 The 96-well cell culture plate in which the sample was added was cultured in a 37eC, 5% C〇2 incubator for 24 hours. Positive control wells were periodically observed under an inverted microscope to confirm cell lysis&apos; then 〇D was collected, washed and read for all wells as described in Section 1.4 above. Calculation: The concentration at 50% of the maximum reaction was obtained in the same manner as in Section 1.4. Calculate the IFN activity of the sample as follows: IFN activity of the sample = (dilution of the sample up to 50% of the maximum reaction 丨 FN standard up to 50% of the maximum reaction dilution) xr〗 FN standard up to 5 (10) dilution of the maximum reaction Degree (IU/ml) 2. Experimental study on the effect of antibody formation (7) 2〇 Sheep red blood cells (SRBC) were prepared as follows: Under normal conditions, blood was taken from the external jugular vein of healthy adult sheep and placed in a triangular flask with glass beads. Shake for three minutes, then mix the blood with Alsever solution (glucose 2. 〇 5g, sodium sulphate. 4g, sodium citrate 〇 8g, add distilled water to 1 〇〇 ml), and set aside 4 hrs for use. Samples were centrifuged at 130 g for 5 minutes before use to collect srbc. The cell pellet was collected by resuspending and centrifuging the cells in raw saline 23 1363092 for 2 times, followed by centrifugation at i8 μg for 1 minute. Dilute with 2% (v/v) in physiological saline to obtain the desired suspension of sheep red blood cells (SRBC). The fresh hamster serum was added to the centrifugal compaction group 5 at 10:1 (v/v), and gently shaken at 4 ° C for 30 minutes to prepare a complement. The supernatant was aspirated by centrifugation at 200 g for 10 minutes and diluted to 1:10 with physiological saline to prepare a desired complement working solution. BALB/c mice were randomly divided into peptide group, ifn group, IL-2 group and physiological saline group, with 10 rats in each group. The administration was carried out in accordance with the administration method described in Section 1.1, and on the 12th day after the administration, 0.2 ml of the SRBC suspension was intraperitoneally injected for immunization. After immunization for 4 1 day, the eyeballs were removed and blood was taken at room temperature for 1 h to allow the serum to fully exude. After centrifugation at 200 g for 10 minutes, the supernatant was taken and diluted 5 (9) times with physiological saline. In the reaction tube, firstly add the diluted mouse serum sample 1 (11 b and then add the sputum suspension 0. 5m 1 'cool the tube in the ice bath, add 1 ml of the complement working solution to each tube. Move the tube to a constant temperature water bath at 37 ° C Insulation for 1 〇 minutes, then placed in the I5 ice bath to terminate the reaction. Centrifuge at 200g for 10 minutes, take the supernatant for colorimetric determination. Determine the absorbance of the sample: take the above supernatant 1ml into the test tube Add Drabkin reagent (sodium bicarbonate l.〇g, potassium ferricyanide 〇. 2g, cyanide clock 0.05g 'steamed water 1000ml) 3ml, shake well, let stand at room temperature for 20 minutes, at 540nm wavelength Color: Record the absorbance. Calculation: Determine the absorbance value of SRBC hemolysis: Take the current suspension 0_25ml, add Drabkin reagent to 4ml 'shake well, let stand for 1 〇 at room temperature, and record the absorbance at 540nm wavelength. ; S ) 24 1363092 Sample serum index = (test sample ODsdOna · ^ reference 0 〇 540 ιιπι) χ 500 3. Experimental study on the effect of peptide on the phagocytic function of mononuclear phagocytic cells and the weight of immune organs [8'9] after the last administration The next day (day 16), each Tail vein injection 1: 55 dilution of saline India ink 0. lml / kg body weight. Blood was taken from the posterior tibial venous plexus 20 μl 1 minute and 5 minutes after the injection of the ink, using a blood collection pipette pre-wet with a heparin solution. The removed blood was mixed with 2 ml of 0·1% Na2C〇3 solution. Determination of ODesonm. Calculate the K value of the clearance index according to the following formula: 10 K = (IgAi - IgA) + (t2 - ti) where Al is 1 minute 0D68Qnm, A2 is 5 minutes 〇〇 680ηπι 9 ± 2 is 5 minutes, 11 is 1 minute 15 On the next day after the last administration (Day 16), the liver, spleen and thymus were separated and blotted dry with filter paper and weighed. The phagocytic index alpha value is calculated according to the following formula: a = (VK) (W + WLS) where W is the body weight and Wls is the liver spleen and weight. 20 Thymus index (%) = (thymus weight / body weight) χ 100%, spleen index (%) = (spleen weight / body weight) χ 100% Results Due to the large number of original data obtained, only the final statistical results were recorded, of which Compared with the saline group, the groups with no statistical difference of 25 (S) 1363092 were omitted. 1. The effect of peptide on the transformation of tau lymphocytes at 500pg/kg/day, CMS002, CMS007, CMS008, CMS009, CMS010, CMS012, CMS015, CMS019, CMS02, CMS029, can promote the transformation of T lymphocytes, and the saline group For comparison, there was a significant difference (p &lt; 0.05), in which CMS010, CMS015 was significantly different from the IFN-γ group and IL-2 group (P &lt; 0.05). The results are detailed in Table 1: Table 1 Number of animals in the group X±SD (stimulus index) CMS002 8 1.8±0.3 Wood CMS007 9 1.6±0.1* CMS008 9 1.7 soil 0.1* CMS009 10 1_ 7±0. 2 wood CMS010 9 2 0±0, CMS012 9 1.6±0.2* CMS015 9 1. 9±0.3^* CMS019 9 1.8±0. 3* CMS021 10 1.6±0.1* CMS029 9 1.7 ± 0.3 IFN-γ 10 1. 6±0.2* IL-2 10 1· 7±0. 2* saline 10 1 · 3±0.1 Note: Compared with the saline group, Ρ<〇· 05 β: compared with the IFN-γ group, Ρ&lt;0. 05 ~ : Compared with the IL-2 group, Ρ&lt;0. 05 26 1363092 When administered at a dose of 50 pg/kg/day, CMS〇〇1, cms〇〇2, CMS003 can stimulate the transformation of mouse T lymphocytes with normal saline. There were significant differences between the group, the IFN-γ group and the IL-2 group (ρ&lt;〇〇5 is statistically significant. CMS014 and CMS036 can inhibit the transformation of mouse tau lymphocytes, 5 compared with the saline group, with significant Sexual differences (ρ<0.05) were statistically significant. The results are detailed in Table 2: Table 2

Number of animals in the group X soil SD (stimulus index) CMS001 10 2. 2±0. 5*β~ CMS002 10 2· 6±0. 3, CMS003 8 2. 2±0. 5 Mub CMS014 9 1.0±0.1* CMS036 9 1. 0±0. 1* IFN-γ 9 1· 7±0· 2* IL-2 10 1· 8±0. 2 wood saline 10 1. 3+0. 1 Note: *: and physiology Comparison of saline group, ρ &lt; 〇. 〇 5 e : compared with IFN group, Ρ &lt; 〇 · 05 ίο —: compared with IL-2 group, Ρ &lt; 〇. 05 When the dose is 5 gg / kg / day, CMS0 (U, CMS003, CMS007, CMS034 can stimulate the transformation of mouse T lymphocytes, compared with the saline group, there is a significant difference (P &lt; 〇. 05), statistically significant. The results are shown in Table 3: 15 Table 3 Group number of animals X±SD (stimulus index) 27 1363092 CMS001 10 1. 7+0.2* CMS003 10 1. 6±0. 2* CMS007 8 1. 7±0.1* CMS034 9 1. 5±0. 2* IFN - γ 10 1. 6±0.2* IL-2 9 L6±0.1* Saline 10 1. 3±0.1 Note: *: Compared with the saline group, Ρ&lt;0· 05 when administered at a dose of p. 5pg/kg /day, CMS008, CMS010, CMS011 can stimulate the transformation of mouse T lymphocytes, compared with the saline group, there is a significant difference (P&lt;0.05), statistically significant. The results are detailed in Table 4: Table 4 Number of animals in the group X±SD (stimulus index) CMS008 10 1. 7±0. 3* CMS010 9 1. 7±0 3 wood CMS011 10 1. 6±0.4* IFN-γ 10 1.6±0.2* IL-2 10 1. 6±0.1* saline 10 1. 3±0.1

Note: *: Compared with saline group 'P&lt;〇. 05 2. Effect of peptide on NK cell cytotoxic activity When CMS peptide is administered at a dose of 500 pg/kg/day, CMS010, CMS013, 10 CMS016, CMS023, CMS024 , CMS026, CMS027, CMS028, CMS029,

28 1363092 CMS030, CMS032, CMS033, CMS034, CMS035, CMS036 can enhance the cytotoxic activity of NK cells, compared with the saline group, there is a significant difference (P < 0.05), among which CMS010, CMS016, CMS030 peptide group and IFN-γ group There was a significant difference between the IL-2 group and the IL-2 group (P&lt;0.05). The results are detailed in Table 5: Table 5

Number of animals in the group X±SD(°/〇) CMS010 9 91±4f CMS013 8 84±9* CMS016 9 91±7^ CMS023 10 79±12* CMS024 10 89±8* CMS026 10 89±7* CMS027 10 88 ±8* CMS028 10 90±5* CMS029 10 87±4 氺CMS030 10 91±5β CMS032 10 87±5* CMS033 9 89±8* CMS034 11 85±9* CMS035 8 90+10* CMS036 10 88±7* IFN-r 10 77±8* IL-2 10 77±8* saline 8 63±9 29 1363092 Note: *: Compared with the saline group, P &lt; 0, 05 0: compared with the IFN-γ group, P &lt; 0. 05 △: Compared with the IL-2 group, P&lt;0. 05 5 when the dose is 5 (^舀/1^/day, 0^00卜0"3003, 0^015, CMS021, CMS026, CMS035 The cytotoxic activity of NK cells was enhanced, and there was a significant difference compared with the saline group (P&lt;0.05). Among them, CMS021 was significantly different from IFN-γ group and IL-2 group (P&lt;0.05 CMS012 can inhibit the cytotoxic activity of NK cells, and has a significant difference (P<0.05) compared with the saline group. The results are shown in Table 6: Table 6 Number of animals in the group X±SD (%) CMS001 10 85±10 wood CMS003 10 85±6* CMS012 9 40±9* CMS015 8 78±8* CMS021 8 88±12 *§~ CMS026 10 76±9* CMS035 10 72+9* IFN-r 10 73±10* IL-2 10 74±8* Saline 10 56±8 Note: *: Compared with saline group, p<〇 05 e: Compared with the IFN group, P &lt; 0.05 ~ : compared with the IL-2 group, P <0. 05 30 15 1363092 when the dose is 5 pg / kg / day, CMS008, CMS009, CMS010, CMS011, CMS012, CMS020, CMS024, CMS034, CMS036 can enhance the cytotoxic activity of NK cells, compared with the saline group, there is a significant difference (P < 0.05), of which CMS008, CMS009 and IFN-γ group, IL-2 group ratio 5 There is a significant difference (P&lt;〇. 05). The results are detailed in Table 7: Table 7 Number of animals in the group X±SD (%) • CMS008 10 92 Soil 4 C CMS009 8 92±6 C CMS010 10 82±9* CMS011 10 76±10* • CMS012 10 85± 7* - CMS020 9 91±6* CMS024 9 78±3* CMS034 8 90±5* • CMS036 10 75±9 Wood IFN-γ 10 80±8* IL-2 10 80±8 Wood - Saline 10 60 ± 9 Note: Compared with the saline group, P &lt; 〇. 05 β : compared with the IFN-γ group, P < 0.05 -: compared with the IL-2 group, P &lt; 05. When the dose is 0. 5yg/kg/day, CMS002, CMS01, CMS012, CMS022, CMS028, and CMS035 can enhance the cytotoxic activity of NK cells, and there is a significant difference compared with the saline group (Ρ &lt;〇· 05). CMS008 was able to inhibit the NK cell cytotoxic activity of 31 1363092, which was significantly different from the saline group (Ρ &lt; 0.05). The results are detailed in Table 8: Table 8 Number of animals in the group X±SD (%) CMS002 8 76±9* CMS008 10 46±12 wood CMS011 9 79+3* CMS012 9 77±6* CMS022 10 73±11* CMS028 8 79±3* CMS035 10 76±10* IFN-γ 10 72 soil speak IL-2 10 74±10* saline 11 58±7 Note: *: Compared with saline group, p&lt;〇. 05 5 3. The effect of peptide on the secretion of IL-2 by sputum lymphocytes when the dose is 50 (^ cliff / 1 ^ / day, 0 ^ 007, 0 to 5009, 0 ^ ^ 010, CMS015 can promote the secretion of IL-2 by sputum lymphocytes Compared with the saline group, there was a significant difference (Ρ&lt;0.05), and the CMS007 and CMS015 groups were significantly different from the IL-2 group (P&lt;0.05) °CMS009, CMS010 and IFN-γ. There were significant differences between the 10 groups and the IL-2 group (P&lt;0.05). The results are detailed in Table 9: Table 9 Number of animals in the group X±SD (IU) CMS007 9 86±15** 32 1363092 CMS009 10 I14±13^~ CMS010 9 125±17 坎CMS015 9 85±17*~ IFN-γ 10 100±18 wood IL-2 10 70±13 wood saline 10 39±10

Note: *: Compared with the saline group, p&lt;〇. 05 e •• compared with the IFN~y group, P<0.05, compared with Jiang-2纰, P&lt;0.05, when the dose is 5〇Mg /kg/day, CMS0 (H, CMS003 can promote the secretion of IL-2 by T lymphocytes, compared with the saline group, there is a significant difference (P &lt; 0.05), of which CMS003 and IL-2 group, there is a significant difference, Statistically significant (P < 0.05). The results are detailed in Table 10: Table 10 Number of animals in the group X SD (IU) CMS001 10 60 ± 10 * CMS003 8 86 ± 9 * ' IFN-γ 9 99 ± 16* IL-2 10 72±12 wood saline 10 39±10 Note: Compared with saline group, p<〇, 05: compared with IL-2 group, P<0.05. When the dose is 5pg/ KMS007, CMS012, CMS020 33 1363092 can promote the secretion of IL-2 by T cells, which is significantly different from the saline group (P&lt;0.05). The results are shown in Table 11: Table 11 Number of animals X±SD(IU) CMS007 8 64±12 wood CMS012 9 65±16* CMS020 8 63±11 wood IFN-γ 10 96±14* IL-2 10 77±13* saline 10 37±9 Note ·· * ··Compared with saline group, p&lt;〇. 05 5 when given The dose was 0. 5Mg/kg/day, CMS010, CMSO Η, CMS012, CMS022, O!S034 can promote the secretion of IL-2 by sputum lymphocytes, compared with the saline group, there is a significant difference (p&lt;〇.〇5) There was a significant difference between the CMS034 and IL-2 groups (P<0.05). There was a significant difference between the CMS01 and CMS022 groups compared with the IL-2 1〇 group and the IFN-γ group. (P<0.05). The results are detailed in Table 12: Table 12 Number of animals in the group X Soil SD (IU) CMS010 9 66±11* CMS011 10 101+19*^ CMS012 8 59±13* CMS022 9 1〇 9±14*0' 34 CMS034 10 85 Soil 1 Of IFN-γ 10 87±15* IL-2 10 73±13* Saline 10 38±13 1363092 Note: Compared with saline group, P&lt;0.05 e: compared with the IFN~7 group, P&lt;〇. 05-: compared with the IL-2 group, P&lt;0.05. 4. The effect of peptide on the secretion of IFN activity by T lymphocytes when the dose was 500 pg/kg/day. CMS010, CMS013, and CMS016 can promote the secretion of IFN by T lymphocytes, which is significantly different from the saline group, IFN-γ group, and IL-2 group (P &lt; 0 Table 13.05). The results are detailed in Table 13. Number of animals in the group X±SD (IU) CMS010 9 167±13# CMS013 9 154115*0* CMS016 6 162±19e IFN 10 139±16* IL-2 10 120±13* Saline 10 65±11 10 Note: Compared with the saline group, P<0.05. • Compared with the IFN~y group, p&lt;〇. 05 \ compared with the group-2, P&lt;〇. 05 when the dose is 50pg /kg/day, CMS001, CMS003, CMS021 can promote the secretion of IFN by T lymphocytes, compared with the saline group, there is a significant difference (P &lt; 0.05). Compared with the IFN-γ group and the IL-2 group, CMS021 had a significant difference of 35 1363092, which was statistically significant (P<0. Table 14 05). The results are detailed in Table 1. Number of animals in the group X±SD (IU) CMS001 10 110±15* CMS003 8 106±16* CMS021 8 143±17^ IFN-γ 9 125±18 Wood IL-2 10 113+17* Saline 10 61+11 Note: *: Compared with the saline group, P <0_ 05 •• compared with the IFN-γ group, P &lt; 0.05 taken — compared with the IL-2 group, P &lt; 05. The dose was 5 pg/kg/day. CMS009 and CMS012 could promote the secretion of IFN by T lymphocytes, which was significantly different from that of the saline group (P<0.05). Compared with the IL-2 group, CMS009 has significant differences and has a statistical significance (P&lt;0.05). The results are detailed in Table 15: Table 15 Number of animals in the group X soil SD (IU) CMS009 10 121±15*~ CMS012 9 86±9* IL-2 9 105±14* Saline 10 66±10 Note: * : Compared with the saline group, p&lt;〇. 05: compared with the IL-2 group, PC0.05 36 1363092 When the dose was 0.5 yg/kg/day, CMS010, CMS01, CMS022, CMS028 could promote T lymphocyte secretion. IFN, compared with the saline group, had a significant difference (P &lt; 0.05). There were significant differences between CMS010 and CMS022 in the IFN-γ and IL-2 groups (P&lt;〇. 05) »Results 5 are detailed in Table 16: Table 16 Number of animals in the group X±SD (IU CMS010 9 142+18*®&quot; CMS011 10 89±18* CMS022 9 145±W CMS028 10 96±13 wood IFN-γ 10 124±16 wood IL-2 10., 107±13 wood saline 10 64 soil 13 Note: *: Compared with the saline group, P&lt;0.05 e: compared with the IFN group, P<0.05 # Λ: compared with the IL-2 group, P<0.05 10 5. The effect of the peptide on antibody formation. Dosage dose is 500pg/kg/day' CMS002, CMS003, CMS007, CMS0 08, CMS00 9, CMS010, CMS011, CMS012, CMS013, CMS014' CMS015, CMS016, CMS018, CMS019, CMS020, CMS022, CMS023, 15 CMS024, CMS029 , CMS033, CMS035 can promote the formation of anti-SRBC antibodies, compared with the saline group, there is a significant difference (Ρ &lt; 〇 · 05), of which CMS002, CMS003, CMS007, CMS008, CMS013, CMS019, CMS024, 37 1363092 CMS035 and IFN- The γ group comparison showed a significant difference (P &lt; 0.05). CMS009 'CMS010 'CMS011 'CMS012 'CMS014> CMS015 'CMS016' CMS020, CMS023, CMS029, CMS033 compared with IFN-γ group, IL-2 group, there was a significant difference, statistically significant (P < 0.05). The results are detailed in 5 Table 17:

Table 17 Number of groups of animals X±SD CMS002 10 87±18*® CMS003 10 96±18^ CMS007 10 69±17^ CMS008 10 82±15*® CMS009 10 113±22# CMS010 10 112±30^ CMS011 8 188 ±16^* CMS012 8 141121*^ CMS013 10 80±16*® CMS014 10 130±24*®&quot; CMS015 10 136±22*®&quot; CMS016 8 143±3, CMS018 10 66±16* CMS019 10 91± 26*® CMS020 6 155±35 C CMS022 8 68±31* CMS023 9 109±45轳~ 38 1363092 CMS024 8 75±29轳CMS029 8 115±22 浐CMS033 10 143±27^* CMS035 10 88±16*® IFN-γ 9 37±10 IL-2 10 71±11* saline 10 32+7 Note: * : Compared with saline group, ρ &lt; 〇 · 05 e : compared with IFN~y group, ρ &lt; 〇. 05 ~ : Compared with the IL-2 group, Ρ&lt;0.05 5 when the dose is 50 pg/kg/day, CMS003, CMS0U, CMS012, CMS013, CMS015, CMS021, CMS022, CMS023, CMS026, CMS027, CMS029, CMS030, CMS032, CMS033, CMS034, CMS035, and CMS036 were able to promote the formation of anti-SRBC antibodies, and there was a significant difference compared with the saline group (P < 0.05). Among them, the CMS011, CMS013, and CMS015 groups had significant differences compared with the IFN-γ group (p<〇.〇5). 10 CMS021 'CMS022 'CMS023 'CMS026 'CMS027 'CMS029 'CMS030 'CMS032 ' CMS033, CMS034, CMS035, CMS036 compared with IFN-γ group, IL-2 group, there is a significant difference, statistically significant (P &lt; 〇. 05). CMS009 was able to inhibit the formation of anti-SRBC antibody. There was a significant difference (P &lt; 0.05) compared with the saline group, which was statistically significant. The results are detailed in Table 18:

(S 15 Table 18 Group Number of animals X±SD(HG〇) CMS003 10 52±11* CMS009 8 13±5* 39 1363092

CMS011 9 67±9^ CMS012 8 50±14* CMS013 8 70 土9# CMS015 10 54土9*® CMS021 9 94±20 坎CMS022 9 110±16^ CMS023 8 84±11轳~ CMS026 9 98±9# CMS027 9 93±11*®&quot; CMS029 10 143113*®' CMS030 10 141±33氺CMS032 9 131±24 grave CMS033 8 112±15 坎CMS034 10 136±11^ CMS035 8 97±, CMS036 10 118±11 ^ IFN-γ 9 37±10 IL-2 10 71+11* saline 10 32±7 Note: *: Compared with the saline group, P<0.05 er: compared with the IFN-γ group, P&lt;0. 05 Λ : Compared with IL-2 group, P<0.05. When the dose is 5pg/kg/day, CMS01H, CMS003, CMS007, 5 CMS008, CMS009, CMS01, CMS012, CMS013, CMS015, CMS016, CMS019, CMS020 , CMS021, CMS023, CMS024, CMS026, CMS027, 40 1363092 CMS028, CMS029, CMS030, CMS032, CMS033, CMS034, CMS035, CMS036 can promote the formation of anti-SRBC antibodies, compared with the saline group, there is a significant difference (P &lt; 0.05) Among them, CMS003, CMS008, CMS009, CMS012, CMS015, CMS016, CMS020, CMS021 group and IFN-γ group 5 have significant differences (P&lt;;0. 05). CMS0 (H, CMS007, CMS0U, CMS019, CMS023, CMS024, CMS026, CMS027, CMS028, CMS029, CMS030, CMS032, CMS033, CMS034, CMS035, CMS036 compared with the IFN-γ group, IL-2 group, there was a significant difference, Statistically significant (P&lt;0.05). The results are detailed in Table 19 below: 1〇Table 19 Number of animals in the group X±SD(HCs〇) CMS001 9 110 soil 24*® CMS003 9 91+24^ CMS007 9 122±12炊CMS008 9 97±26轳CMS009 8 79±18^ CMS011 10 115±27^* CMS012 10 81±22轳CMS013 10 93±28*® CMS015 8 94±37轳CMS016 9 93±32轳CMS019 10 118±20浐CMS020 10 89±24*® CMS021 9 82±30*® 41 1363092

CMS023 10 Dirty 27 CMS024 7 171±39俨CMS026 9 191±17^' CMS027 9 117±45 CMS028 10 121+48*®' CMS029 9 147 Clay 23 Grave CMS030 9 Leg 37 CMS032 9 157±37俨CMS033 7 128±39 rose CMS034 8 172±37 CCMS035 9 176±39 轳a CMS036 8 3, IFN-γ 9 37±10 IL-2 10 71 ±11* saline 10 32±7 Note:: with saline group Comparison, P&lt;0_05: compared with the IFN-γ group, P&lt;0.05: compared with the IL-2 group, P&lt;0.05 5 when the dose was 〇·5 gg/kg/day, CMS02, CMS023, CMS024, CMS027, CMS033 can promote the formation of anti-SRBC antibodies, compared with the saline group, there is a significant difference (P &lt; 〇. 05), of which CMS023, CMS024, CMS027 compared with IFN-γ group, IL-2 group, there is a significant difference, with Statistical significance (P &lt; 0.05). Compared with the IFN-γ group, the CMS021 and CMS033 groups had significant differences (P&lt;〇_〇5). CMS002, CMS003, CMS009, CMS010, CMS011, 42 (S) 1363092 CMS013, CMS014, CMS015, CMS018, CMS019, CMS020, CMS026,

CMS028, CMS029, CMS030, CMS034, and CMS036 inhibited the formation of anti-srbc antibodies, which were significantly different from the saline group (Ρ&lt;0·〇5:N results are shown in Table 20: Table 20 Groups of animals X ±SD CMS002 9 4±1* CMS003 9 2±1^ CMS009 9 2±1* CMS010 10 10±3 Wood CMS011 10 5±3* CMS013 10 7±1* CMS014 10 15±6* CMS015 9 13±4 氺CMS018 9 3±1 wood CMS019 9 12±3氺CMS020 9 10±3 wood CMS021 9 57±9*β CMS023 10 108±21^ CMS024 10 98±6 坎CMS026 10 19±6 Wood CMS027 10 99±14*® ' CMS028 10 18±5* CMS029 9 18±7* 43 1363092 CMS030 9 I9±7* CMS033 9 78±12^ CMS034 10 20±2* CMS036 9 20±6 氺IFN-γ 9 37±10 IL-2 10 71±11* saline 10 32±7 Note: *: Compared with the saline group, P &lt; 0.05 • 0: compared with the IFN-γ group, P &lt; 0.05 -·· compared with the IL-2 group, P&lt;0. 05 6. Effect of peptide on phagocytosis of mononuclear phagocytic cells 5 When administered at a dose of 500 gg/kg/day, CMS003, CMS008, CMS020, CMS022, and CMS024 can enhance the phagocytosis and physiology of monocyte phagocytic cells. • Comparison of brine groups There was a significant difference (PC0.05), and there was a significant difference between CMS022 and IFN-γ group and IL-2 group, which was statistically significant (Ρ &lt; 0.05). The results are shown in Table 21: i ίο Table 21 Number of animals in the group X soil SD (phagocytic index) CMS003 10 6· 6±0_ 7* CMS008 10 6. 5±1.2* CMS020 10 6. 4±0. 6* CMS022 10 7. 4±0_ 6 Rose CMS024 10 6.4±1.0* IFN-γ 10 6. 4±0. 9* IL-2 9 5. 7±0. 8 44 1363092 Saline 10 5.1±0.6 Note: *: Compared with saline group, P&lt;〇. 05 e : Compared with the IFN-γ group, P<0.05> compared with the IL-2 group, PC0.05 5 When the dose is 50 pg/kg/day, CMS019, CMS024, CMS030 can enhance mononuclear phlegm cells. The phlegm function was significantly different from the saline group (P &lt; 0.05). There was a significant difference between the CMS019 and IL-2 groups (P&lt;0.05). The results are detailed in Table 22: Table 22 Number of animals in the group X±SD (phagocytic index) CMS019 9 6·7±0.9*&quot; CMS024 8 6. 6±0. 7* CMS030 10 6. 3±0.5* IFN- γ 10 6.4±0. 9* IL-2 9 5. 7±0.8 saline 10 5.1+0. 6 ίο Note: * ··Compared with saline group, Ρ&lt;0· 05 Λ : compared with IL-2 group ,Ρ&lt;0. 05 When the dose is 5pg/kg/day, CMS003, CMS008, CMS009, CMS010, CMS011, CMS013, CMS016, CMS018, CMS019, CMS035 15 can enhance the phagocytosis of mononuclear phagocytic cells, and the saline group Comparison, there was a significant difference (PC0.05). Among them, CMS003, CMS009, CMS010, CMS016, CMS019, CMS035 and IL-2 group have significant differences. &lt; S ) 45 1363092

(P<0.05). The results are detailed in Table 23 below: Table 23 Number of animals in the group X±SD (swallowing index) CMS003 9 6. 9±0. 9*' CMS008 9 6.4±0. 5* CMS009 9 6. 9±0. 9 *&quot; CMS010 10 7.1±0_ 7圹CMS011 10 6.4±1.1* CMS013 10 6. 7±0. 2* CMS016 9 6. 9±0. 8*&quot; CMS018 8 6. 7±1· 2* CMS019 8 6. 8±0. 6*' CMS035 9 6. 9±0. 9*' IFN-γ 10 6.4±0. 9* IL-2 9 5. 7±0. 8 saline 10 5.1±0. 6 Note ::Compared with saline group, p<〇. 05 ~ : compared with IL-2 group, P&lt;0.055 When the dose is 0.5 gg/kg/day, CMS024, CMS027, CMS036 can enhance single core The phagocytic function of phagocytic cells was significantly different from that of the saline group (P&lt;〇.〇5). There was a significant difference between the CMS024 group and the IL-2 group (P<0.05). The results are detailed in Table 24: Table 24 Number of animals in the group X±SD (population index) CMS024 10 6.7±0.5*^ 46 1363092 CMS027 10 6. 4±0. 6* CMS036 9 6. 2±0. 3* IFN-γ 10 6. 4±0. 9* IL-2 9 5. 7±0.8 saline 10 5.1 soil 0. 6 - Note: *: compared with saline group, P &lt; 〇. 05 ~ : and IL- Comparison of 2 groups, P &lt; 0.05 • 7. Effect of peptide on the weight of immune organs 5 When the dose is 500pg/kg/day, CMS008, CMS010, CMS016, CMS019, CMS020, CMS022, CMS026, CMS027, CMS028, CMS029, - CMS030, CMS032, CMS033, CMS034, CMS035, CMS036 can increase the weight of mouse thymus. Compared with saline group, it has significant difference w/· is different (Ρ &lt; 0.05), among which CMS027, CMS034 group and IL-2 Group comparisons have ίο significant differences (Ρ&lt;〇. 05). CMS008, CMS022, CMS029, CMS030, CMS032, CMS033, CMS035 compared with the IFN-γ group and the IL-2 group showed significant differences (Ρ &lt; 0.05). The results are detailed in Table 25: Table 25 Number of animals in the group X±SD (°/〇) CMS008 8 0.21±0.03 Wood 1 CMS010 10 0. 19±0. 04* CMS016 9 0. 1810. 05* CMS019 10 0· 19±0. 02* CMS020 10 0. 19±0. 04木47 (S) 1363092 CMS022 9 0. 26 soil 0. 05氺CMS026 10 0.20±0.03* CMS027 8 0. 20±0· 03** CMS028 10 0. 19±0. 02木CMS029 10 0. 22±0. 04*卜CMS030 8 0. 30±0. 03^ CMS032 8 0. 25±0. 03木^ CMS033 9 0· 25±0. 0, CMS034 9 0. 20±0. 05** CMS035 10 0. 21±0.03 木 i CMS036 9 0. 18±0· 02* IFN-γ 10 0. 15±0. 04 IL-2 9 0· 14±0.04 Normal saline 9 0. 12±0. 02 Note: Compared with the saline group, Ρ&lt;0·05 β: compared with the IFN-γ group, Ρ<〇. 〇5 &quot;: compared with the IL-2 group, Ρ&lt; 〇. 05 5 When the dose was 50 (^g/kg/day, CMS019 was able to increase the weight of the mouse spleen, compared with the saline group, there was a significant difference (P &lt; 0.05). CMS001, CMS003, CMS007, CMS009, CMS011, CMS013, CMS014, CMS015, CMS021, CMS023, CMS024, CMS027, and CMS036 can alleviate the weight of mouse spleen compared with saline group 10 having statistical significance (P <0 05.) The results detailed in Table 26: TABLE 26 (S) 48 1363092

Number of animals in the group X±SD(%) CMS001 10 0.43±0· 07* CMS003 8 0. 40±0. 04* CMS007 9 0.32±0_05* CMS009 9 〇.41±0. 03木CMS011 9 0. 41± 0. 04木CMS013 10 0. 44±0. 07木CMS014 10 0.04±03木CMS015 9 0. 36±0. 07木CMS019 9 0. 63±0. 08木CMS021 9 0. 36+0. 04* CMS023 9 0. 36土0. 06木CMS024 9 0. 34±0. 05木CMS027 10 0. 37±0. 03氺CMS036 10 0. 40±0. 03氺Normal saline 10 0. 53±0. 05 Note: *: Compared with the saline group, P&lt;〇. 05 when the dose is 50pg/kg/day, CMS002, CMS008, CMS012, CMS014, CMS016, CMS018, CMS019, CMS020, CMS022, CMS023, 5 CMS024, CMS026 , CMS027, CMS028, CMS029, CMS030, CMS032, CMS033, CMS034, CMS036 can increase the weight of mouse thymus, compared with the saline group, there is a significant difference (Ρ <0.05), of which CMS034 and 11&gt;-2 group For comparison, there is a significant difference (? &lt; 0.05). 0^002, 0^008, 49 1363092 CMSO12, CMSO14, CMSO16, CMS018, CMSO19, CMS020, CMSO22, CMS023, CMS024, CMS026, CMS027, CMS030, CMS032, CMS036 compared with IFN-γ group, IL-2 group, Significant differences were statistically significant (P&lt;0.05). The results are detailed in Table 27:

Table 27 Number of animals in the group X±SD(°/〇) CMS002 10 0. 21±0. 02*®&quot; CMS008 10 0. 20±0. 04*0' CMS012 10 0. 26±0. 02^&quot ; CMS014 10 0· 21±0. 02 坎CMS016 10 0. 20±0. 03*®' CMS018 10 0. 23±0. 02*^ CMS019 .10 0. 20±0. 03, CMS020 10 0· 27 ±0. 03玫CMS022 10 0. 30±0. 03*^ CMS023 10 0. 20±0. 02^ CMS024 10 0. 27±0. 02, CMS026 10 0. 27±0. 02*^ CMS027 8 0 21±0. 03*®&quot; CMS028 10 0.18±0. 04木CMS029 9 0.18+0. 05* CMS030 10 0· 25±0. 04 grave CMS032 10 0· 27±0. 03 rose CMS033 9 0. 18±0.03* 50 1363092 CMS034 8 0. Face.04木' CMS036 9 0. 22土0.02轳* IFN-γ 10 0.15±0. 04 IL-2 9 0.14士0. 04 Saline 9 0.12±0. 02 Note: *: Compared with the saline group, Ρ &lt;0· 05 β: compared with the IFN group, PC0.05 ~: compared with the IL-2 group, Ρ &lt;0· 05

5 When the dose was 50 pg/kg/day' CMS008, CMS010, and CMS029, the weight of the mouse spleen was reduced, and there was a significant difference compared with the saline group (P &lt; 0.05). The results are detailed in Table 28: Table 28 Number of animals in the group X soil SD (%) CMS008 10 0. 39±0. 08* CMS010 10 0. 38±0. 05* CMS029 10 0.42±0. 04* IFN-γ 10 0. 50+0. 04 IL-2 9 0. 62±0. 07 Saline 9 0. 53±0. 05 Note: *: Compared with saline group, Ρ&lt;〇· 05

10 When the dose is 5pg/kg/day' CMS001, CMS002, CMS010, CMS01, CMS012, CMS013, CMS014, CMS015, CMS016, CMS018, CMS019, CMS020, CMS021, CMS022, CMS023, CMS024, CMS026, CMS028, CMS029, CMS030, CMS032, CMS033, CMS034, CMS036 51 1363092 can increase the weight of mouse thymus, compared with saline group * significant difference (PC0.05), including CMS002, CMS014, CMS024, CMS030 group thymus index and IL- There was a significant difference between the two groups (P &lt; 0.05). CMS010, CMS012, CMS018, CMS019, CMS020, CMS022, CMS026, 5 CMS028, CMS032, CMS034, CMS036 were significantly different from the IFN-γ group and IL-2 group (P < lt. 〇 5 ). The results are detailed in Table 29: Table 29 Number of animals in the group X±SD(%) CS ) CMS001 10 0.22±0. 05* CMS002 9 0. 24±0_ 05木~ CMS010 9 0. 27±0. 05 坎CMS011 10 0. 22±0.04* CMS012 10 0. 27±0· 06俨CMS013 10 0.21+0.05* CMS014 10 0. 23±0.06K CMS015 9 0. 20±0.08* CMS016 10 0. 22±0.06* CMS018 10 0 24±0.0 expansion CMS019 10 〇. 24±0.02^' CMS020 10 0. 24±0. 07^&quot; CMS021 9 0. 20±0. 06 wood CMS022 9 0. 25±0. 045^ CMS023 10 0. 23±0.06木52 1363092 CMS024 9 0. 23±0. 06*~ CMS026 10 0. 31±0· 05俨CMS028 10 0. 28±0. 06 grave CMS029 10 0. 21±0· 03* CMS030 10 0 23±0. 07木~ CMS032 10 0. 29±0. 04^&quot; CMS033 10 0. 20±0. 02* CMS034 9 0_ 27±0. 06 坎CMS035 10 0. 21+0. 04* CMS036 10 0· 25±0. 0, IFN-γ 10 0.15±0. 04 IL-2 9 0.14±0. 04 Saline 9. 0.12±0. 02 Note: *: Compared with the saline group, p&lt;〇. 05 0: Compared with the IFN-γ group, P &lt; 0.05 ~ : compared with the IL-2 group, P &lt; 0.05 • 5 When the dose is 5 pg / kg / day, CMS030 can increase the weight of the mouse spleen With saline There was a significant difference between the group and the IFN group (P &lt; 0.05). - CMS015 was able to reduce the weight of the mouse spleen, which was significantly different from the saline group (P&lt;〇. 05). The results are detailed in Table 30: Table 30 Number of animals in the group X±SD (°/〇) CMS015 9 0. 38±0.15* CMS030 10 0· 64±0· 09* 53 1363092 0. 53±0.05 saline 〇 Note: Compared with the saline group, Ρ&lt;0· 05

When the dose was 0.5μg/kg/day, CMS002, CMS008, CMS010, CMSO12, CMSO14, CMSOl8, CMS020, CMS022, CMS026, CMS028, 5 CMS030, CMS032 could increase the weight of mouse thymus compared with the saline group. There was a significant difference (P&lt;〇.〇5), in which CMS008, CMS012 and IL-2 group had significant differences (p<0 05XMS002.CMS020, CMS030 compared with IFN group, IL-2 group, significant) Difference (p&lt;〇. 〇5). The results are detailed in Table 31 ··

Number of animals in the group X±SD(%) CMS002 8 0. 26±0.06*^ CMS008 10 0. 22±0. 07木~ CMS010 9 0. 21±0. 03* CMS012 10 0. 22±0. 06* Λ CMS014 10 0. 20±0. 04* CMS018 10 0. 20±0. 03* CMS020 9 0. 23±0. 05*®&quot; CMS022 10 0. 21±0. 06* CMS026 9 0. 21± 0. 05* CMS028 10 0. 20+0. 06* CMS030 8 0. 24±0. 05 grave CMS032 10 0. 21±0. 06 wood &lt; S ) 54 1363092 IFN-γ 10 0.15±0. 04 IL -2 9 0.14±0. 04 Saline 9 0.1210. 02 Note: *: Compared with the saline group, P < 〇. 05 0: compared with the IFN-γ group, P &lt; 〇. 〇5 ~ : and IL-2 Group comparison, P &lt; 〇. 05 When the dose was 0.5 pg / kg / day, CMS020 can increase the weight of 5 spleens of mice, compared with the saline group, IFN-γ group, and IL-2 group, significant • Sexual differences (P&lt;〇. 〇5). CMS001 was able to reduce the weight of the mouse spleen', compared with the physiological saline group (p<0.05). The results are shown in Table 32: Table 32 Number of animals in the group X±SD (%) CMS001 10 0.40±0. 05* CMS020 8 0_ 68±0. 09浐Normal saline 10 0. 53+0. 05 IFN-γ 10 0 50±0. 04 IL-2 10 0. 62±0. 07 Note: *: Compared with the saline group, Ρ<〇. 05 10 β: compared with the IFN-γ group, Ρ<0. 05 &quot;: Compared with the IL-2 group, Ρ &lt;0. 05 In summary, the peptides CMS001, CMS002, CMS003, CMS007, CMS008, CMS009, CMS010, CMS011, CMS012, 15 CMS013, CMS014, CMS015, CMS016, CMS018 were confirmed by animal experiments. CMS019, CMS020, CMS02, CMS022, CMS023, CMS024, CMS026, CMS027, CMS028, CMS029, CMS030, CMS032, CMS033, CMS034, CMS035, CMS036 55 1363092 are biologically active in animals. II. Antiviral effects of peptides in vivo In order to clarify whether these peptides have therapeutic value for viral infections, we used an animal model of duck hepatitis B infection to examine the in vivo effects of the above peptides on diseased animals. A model of hepatitis B infection in Chongqing Ma duck was established and intraperitoneal injection of peptide (50 μg/kg body weight/day, once a day) for 4 weeks. Serum duck hepatitis B virus DNA (DHBV-DNA) levels were determined by dot blot hybridization. At the same time, lamivudine and saline were used as positive and negative controls, respectively. It was found that the peptide CMS001 significantly reduced serum DHBV-DNA levels at week 4 of ίο (P&lt;0.05). It is concluded that CMS001 can be used alone or in combination to treat hepatitis B virus infection at the appropriate dosage level. Materials and Methods 1 · The peptide was synthesized from L-amino acid by American Peptide Company, Inc. 2·Animal model [1] One day old Chongqing duck was inoculated with 0.1 ml of DHBV-DNA positive serum by intraperitoneal injection, and the inoculum was 5×10 7 copies/ml. One week later, blood was taken from the external jugular vein of the duck, serum was separated, and the DHBV-DNA probe labeled with digoxin was spotted for hybridization, 20 to determine whether the infection was successful [2]. Infected ducks can be raised to 2 weeks before entering the experimental study. 3. Grouping and treatment DHBV-infected ducks were randomly divided into the following groups: (1) Negative control group (n=9): Each duck was intraperitoneally injected with 1 ml of physiological salt 56 丄 092 water once a day. (2) Lamivudine group (n=8): a positive control group. Each duck was orally (administered) with lamivudine (4) 50 mg/kg/d once daily. (5) The peptide group (n=9): Each duck was intraperitoneally injected with 5 〇g/kg/d 5 peptide once a day (adjusted to a final volume of 0.5-lml with physiological saline). The above groups were treated for 4 weeks, and then stopped for 1 week. On the third day of dosing, 7, 14 ' 21 ' 28 ' 35 days, 1 ml of blood was taken from the external jugular vein of the duck, and the blood was immediately separated and cleared to -20 ° C (3). 4. Serum DHBV-DNA titer assay The DHBV-DNA probe was labeled with fluorescent light according to the instructions provided by the kit manufacturer (Amershain Pharmacia Blotedl Co.). 4〇μι duck serum was spotted on the nitrocellulose membrane at a replication point per sample, and then hybridized with the DHBV-DNA probe of Lujiao [2]. After the hybridization is completed, the spot is scanned by the Vuego Scan (Brisa-620st) sweeper using the _51:31· 癸 疋 疋 s formula (RPN3690). ImageMaster TotalLab vl. 11· Ink soft% Quantitative analysis of the spots, statistical analysis using paired t test, using SPSS software. Results 'Table 11 _ 1 serum DHBV-DNA levels before and after treatment serum DHBV-DNA titer (103 units) Day 0 7th day Day 14 Day 21 Day 28 Day 35 Saline 26±12 45±31 49±23 102±66 60±38 50±43 Lamivudine 21±9 6±4 Wood 7±6 Wood 8±7 Wood 8±5* 20±19 CMS001 21 soil 18 11±13 20+18 14+14 5±3^ 18+16 57 (S ) 1363092. Paired t-test: compared with the same animal for the third day, 邛&lt ;0.〇5. Negative (saline) control group and positive (lamivudine) control group data showed successful establishment of an animal model of hepatitis A infection. Compared with before administration, peptide 5 CMS001 was 50 g/g. Serum DHBV-DNA titer (P< lt. 〇 5) was significantly decreased after 4 weeks of kg/d dose administration. The serum DHBV-DNA rebound was more obvious after stopping the drug, suggesting that the antiviral effect of CMS001 is reversible. It may be necessary to increase the dose and / or extend the course of treatment to eradicate the virus. Discussion The duck hepatitis B infection model (1) is an ideal and mature tool for studying human hepatitis 8 and screening for hepatitis B... The study found that peptide CMS001 medication 4 weeks can significantly reduce serum DHBV-DNA levels, indicating that CMS001 can be used alone or in combination with other drugs as a means of comprehensive treatment for human hepatitis B under appropriate dose and rational drug regimen. This study only tested intraperitoneal injection. The drug route, but does not exclude other routes of administration that may be effective. The peptide may be administered by intravenous injection, intramuscular injection, intra-abdominal left-shot, subcutaneous injection or subcutaneous implantation, and may be used or such as a monthly shell, sustained release. A drug delivery facilitating device such as a protective device. The tablet can be used for any oral administration, such as a tablet, a capsule, a suspension, a solution, etc., 20 in the form of a regular-form or sustained-release form with no or no gastrointestinal protection. The peptide can be further used for topical preparation, such as ointment, cream, colloid=, with or without transdermal facilitation device, or as inhalation powder a form of plastid protection of the agent, solvent or ruthenium. The peptide can also be translated into a gene sequence, cloned into an expression system, alone or in combination with other peptide sequences to produce a pure 58 1363092 purified or unpurified peptide molecule. The activities available are as described herein. Table II.2 Peptides effective for acetylated hepatitis C CMS code SEQ ID NO : CMS001 1 References 5 h Chen Congxi 'Guo Shuhua, Zhang Dingfeng' and so on. Establishment and application of hepatitis B model in Chongqing duck, Chinese Journal of Hepatology 1993 [1]: 89-91 2. Chen Jiangxi, Guo Shuhua, Chen Xuehua. Preparation and application of digoxin-labeled DHBV-DNA probe, Journal of Chongqing Medical University, 1994; 19(4): 295-297 10 3. Tang Xia, Huang Ailong, Guo Shuhua, et al. Duck hepatitis B virus humoral immunity System establishment and application of serological indicators, Chinese Journal of Hepatology, 2〇〇1; 9(1): 13-15 4. Chen Jiangxi 'Guo Shuhua, Qi Zhenyuan' and so on. Experimental study of lamivudine combined with famciclovir against duck hepatitis B virus, Chinese Journal of Hepatology, 2〇〇1; 15 9(4): 209-211 111 · The effect of peptides on nephritis is whether these peptides are likely to be nephritis For the therapeutic effect, we used an animal model of large milk Masugi nephritis to test the effects of the above-mentioned conditions on the body 20 of the diseased animal [3 4]. This study was designed to investigate the in vivo therapeutic effects of peptides on chronic glomerulonephritis in rats. By injecting rabbit anti-mouse kidney cortex into healthy Sprague Dawley rats 59 1363092

The IgG constructs the rat Masugi nephritis model, and then the intraperitoneal injection of each peptide is performed immediately. The dose was 50//g/kg/day' for 3 weeks. The glucocorticoid hydrocortisone is a positive control. The results showed that the proteinuria, serum creatinine level and spleen index of rats treated with CMS014, CMS018, CMS030, and CMS036 were significantly lower than those of the group 5 (P&lt;〇. 〇5). Microscopic examination of the kidneys after death suggests that the therapeutic effects of these peptides are similar to those treated with hydrocortisone. Therefore, 'CMS014, CMS018, CMS030, and CMS036 can be used as a means of chronic nephritis control. Materials 10 Sprague Dawley (SD) male rats were purchased from the Experimental Animal Center of Guangzhou University of Traditional Chinese Medicine and the Experimental Animal Center of the First Military Medical University, with an average body weight of 120 ± 2〇g. The cyan rabbit is provided by the Experimental Animal Center of Guangzhou University of Traditional Chinese Medicine with a body weight of about 3kg. The L-glycolic acid source was synthesized by American Peptide Company, 15 InC., USA, and diluted with physiological saline to give an injection of i〇#g/mi. The positive control drug hydrocortisone was produced by Yangzhou Pharmaceutical Factory. The serum creatinine level assay kit was purchased from Shanghai Rongsheng Biotechnology Co., Ltd. Method 20 (1) Preparation of rabbit anti-mouse renal cortex antiserum (1): 20 healthy SD rats after 3%

After intraperitoneal injection of pentobarbital sodium (4 〇mg/kg), the abdominal aorta was exposed to cold saline, and the kidneys were completely washed. The kidney was separated and the cortical part was removed. The volume of the cortex was increased by 5 0lM. After pH 8.1 Tris-HCl buffer, the second sentence was collected and filtered through a 140 mesh stainless steel sieve. The filtrate was collected and mixed with GIBC0-RPE 60 (S ) completely or incomplete Freund's adjuvant 1: 1 to obtain vaccination. Working solution. , -. Ten healthy rabbits were injected subcutaneously with this inoculum solution, first with complete Freund's adjuvant followed by incomplete adjuvant. Once every 10 days, every time 6 o'clock (〇·lml/point). From the 6th week on the rabbit ear vein blood, double the serum, determine the antibody titer (double diffusion method) ί2]» after 8 weeks, the rabbit can be used 3% pentobarbital sodium (20mg / kg) After intravenous anesthesia, the carotid artery was exposed and blood was taken for antiserum. Whole blood was also collected from 15 healthy SD rats. The green mouse erythrocytes were washed 3 times with physiological saline, and added to rabbit antiserum of about 25 〇mi, mixed with PC for overnight, and the red blood cells were removed by centrifugation, and the supernatant was 56. (Inactivated for 3 minutes, centrifuged to remove the precipitate, and then precipitated with ammonium sulfate (50%, 33%, 33%) three times (5), the supernatant was broken and partially purified to prepare crude IgG. Double-distilled water in 125ηιΐ, dialysis to remove ammonium sulfate. Double diffusion method to determine the anti-price of anti-xiancord. Attack, (2) induced glomerulonephritis in rats, 5 days before induction to the abdominal cavity of healthy rats = 〇. 25m 1 rabbit IgG - Complete Freund's adjuvant (containing non-specific 1 qi ~ ~) ^ force 0 迷 induced reaction. In addition to the following saline control group, other levels of mice were injected intraperitoneally with 1 0 ml of rabbit anti-mouse prepared as described in the method Kidney:: IgG' induces glomerulonephritis. He (3) grouping and administration: male SD rats were randomly divided into normal health versus Zhaodi (normal), nephritis placebo control group (placebo), and nephritis Pure,,,: inflammatory hydrocortisone treatment control group (hydrocortisone), 12 per group. 1 On the day of nephritis, the drug was administered intraperitoneally (4) _ 峨: the dose of hydrocortisone was 3.3 mg / Kg / day New salt, the course of treatment is 3 weeks. Ge 1363092 (4) observation index [4]: 1 urine protein level: each rat In a metabolic cage, free drinking water, 24 hours of urine collection, Coomassie blue method for protein content, once a week. 5 2 serum creatinine level: 3 weeks of treatment at the end of the rat, serum creatinine Level. Use the muscle needle kit provided by Shanghai Rongsheng Biotechnology Co., Ltd. 3 Spleen weight index: average spleen weight + average body weight is the spleen weight index. 4 Kidney tissue pathology: 6 groups of the largest kidneys in each group Sectioning and observation under the microscope. 10 Statistical analysis: The t test was used for comparison between groups. The wear value was set to p &lt; 0.05. To ensure the reliability of the model, the minimum value of 2 urine proteins was specified for each group of rats. Results 1. Effect of peptide therapy on urinary protein levels in rats 15 Table III. Effect of peptides on urinary protein levels (unit: mg) Number of group cases (η) Week 1 Week 2 Week 3 CMS014 group 10 5. 5+4. 0* 6. 3±6.8 wood 2.8+1.9* CMS018 group 10 7. 0+3. 7* 5. 5±7· 9* 3. 3+3.4* CMS030 group 10 5. 4+ 3. 4* 9. 5±16.2 2. 4±1.5* CMS036 group 10 7. 9+5. 8* 5. 0±7.1 wood 1.3+0. 9* Hydrocortisone 10 3.1±2. 0* 7 . 5+7. 7 7. 6+7.1 group 17. 2 soil placebo group 10 22. 9±22 14.5 20. 2+29. 0 62 1363092 1.7±1.3* 2.3+1.1* 0.4±0.2 wood note: compared with placebo group, *P<0.05. The peptides 0^014, 0^018, 0^〇3〇 and 0^036 were found to reduce the urine egg of the Masugi nephritis rat model at a dose of 50 μg/1^/day. Levels were statistically significantly different from placebo-treated controls, and s ^ ρ &lt; 0·05 〇 also noted that CMS014, CMS030, and CMS036, as well as hydrogenation, had a lower rate than the normal group. The growth rate of the hydrocortisone group decreased to the degree of self-sufficiency. After 1 week, the therapeutic agent had to be halved to avoid severe intolerance, and the effect of peptide on serum creatinine level. 10 Normal group Table III. Effect of 2 peptide on serum myocardium in rats Group number (η) Serum creatinine level UM) CMS014 group 10 159±1* CMS018 group 10 67+1* CMS030 group 10 93±1 Wood CMS036 group 10 80±1 Wood hydrocortisone group 10 239+107 placebo group 10 265+212 normal group 9 80+1* Danwen ninja treated nephritis rats comparison, *Ρ&lt;〇. 05 From the table, Masugi nephritis rat serum The creatinine level was significantly higher in the An-special treatment group than in the normal control group (P<0.05-0.11), suggesting that there were renal dysfunction in the 4 rats of the kidney-supplemented rats with 63 1363092. Peptide CM014, CMS018, CMS030, CMS036 at 50 μg/kg/day, once daily, reduced serum creatinine levels compared with placebo-treated nephritis rats, and was statistically significant in the placebo-treated group of nephritis rats. difference. 5 3. Effect of peptide on rat spleen index Table III. Effect of 3 peptide on rat spleen index Number of cases (η) Spleen index (χ1 (Γ3) CMS014 group 10 3. 6+1.3* CMS018 group 10 3 3±0. 8* CMS030 group 10 3. 0±0. 5* CMS036 group 10 3. 4+0. 8* Hydrocortisone 10 4. 5±1.4 group placebo group 10 4. 8+1.1 Normal Group 9 2. 4±0.1* Note: Compared with the placebo group, *P&lt;0.05. All the rats with induced nephritis had higher spleen index than the normal group, that is, there was obvious splenomegaly, suggesting that immune factors participate in nephritis. The formation of CMS014, 10 CMS018, CMS030, CMS036 at 50 μg/kg/day, once daily, significantly reduced the spleen index, which was statistically significantly different from the placebo treatment group, p &lt; 0.05. Peptides may exert anti-nephritis effect through immunosuppressive mechanism. 4. Effect of peptide on renal pathological changes in rats 15 Pathological examination showed that there was a little 64 1363092 cellulose exudate in the glomerular balloon cavity of rats in the placebo group. The epithelial cells of the sac wall layer proliferate, and part of the crescentic glomerular capillaries are expanded and congested. The proximal convoluted tubule epithelial cells have Degree of water degeneration 'farming small police and a small tube shape in the collecting tube, consistent with the pathological changes of chronic glomerulonephritis, indicating that the induction of nephritis was successful. Only 5 of the CMS014 group showed a cellulosic glomerular cavity Exudate, epithelial cells proliferating in the wall of the balloon. The other parameters of the rat and other rats in the same group have the same kidney-level texture as normal rats. All pathological parameters of all rats in the CMS018, CMS030, and CMS036 groups. All of them are normal. Conclusion 10 The experimental results show that 'peptide CMS014, CMS018, CMS030, CMS036 at a dose of 50/zg/kg/day' per day peritoneal administration has a therapeutic effect on the experimental Masugi nephritis rat model. These peptides can be corrected Proteinuria, creatinine levels, and renal histology in the treated rats were statistically significantly different from the placebo-treated control group. The effect of these peptides on the spleen weight index indicated that these peptides may provide a mechanism for the prevention of 15 epidemics, but not Exclude other possible pharmacological mechanisms. Discussion Peptides CMS014, CMS018, CMS030, CMS036 may or may not be used as part of the treatment of human nephritis, for example to reduce urine protein Improve or improve renal function. These peptides may be used alone, or in combination with two or more peptides, 20 or in combination with other drugs or food additives for the comprehensive treatment of nephritis. Table 4.4 peptides effective against nephritis —_________ CMS code SEQ ID N0 : 65 1363092 G1S014 7 CMS018 10 Q1S030 21 (1S036 26 References 1. The Ministry of Health of the People's Republic of China. New Drug (Western Medicine) Preclinical Research Guidelines, 1994: 96 (ie under the anti-nephritis medicine) 2. Xu Shuyun, etc. Pharmacological Experimental Methodology, People's Medical Publishing House, 5 Beijing, 2nd ed., 1991 : 1071 3. Chen Qi, et al. Methodology of pharmacology research of Chinese medicine, People's Medical Publishing House, Beijing, 1st edition, 1993: 390 4. Du Guanhua's main translation. Pharmacological Experimental Guide A New Drug Discovery and Pharmacological Evaluation, Science Press, Beijing, 1st edition, 2001: 598 10 5. Wang Shuxian. Nephrology, People's Medical Publishing House, Beijing, 11th edition, 1987 : 244 IV. Effects of CMS peptides on cancer To explore the possible anticancer therapeutic effects of these peptides, we tested them on diseased animals using various standard animal cancer models in this study 15 Was learning effect. Materials 1. Experimental animals: BALB/c mice, C57BL/6 mice, DBA/2 mice, weighing 18-22 g, provided by the Experimental Animal Center of the Chinese Academy of Medical Sciences. 20 2. Cell line: C S ) 66 1363092. Mouse sarcoma 180 (Sw) cells, mouse melanoma Bi6 cells, mouse Leukemia Lmo cells: provided by the Institute of Oncology, Chinese Academy of Medical Sciences. YAC-1 cells: Professor Yao Zhi from the Department of Immunology, Tianjin Medical University. 3. Main drugs and reagents: 5 Peptide: American Peptide Company Recombinant murine interferon gamma (rmIFN-γ): Beijing Bonding Biotechnology Co., Ltd. Recombinant human interleukin 2 (rhIL-2): Shanghai Huaxin Biotechnology Co., Ltd.

RPMI-1640 cell culture medium, fetal bovine serum: American GIBCO company 10 tetradecyl azozolium salt (MTT), concanavalin A (ConA): American SIGMA company lymphocyte separation solution: Institute of Hematology, Chinese Academy of Medical Sciences Amine (CTX): Shanghai Twelfth Pharmaceutical Factory Method 15 1. Administration Methods Various therapeutic drugs were injected intraperitoneally, except for the cyclophosphamide group, which was administered the next day after tumor inoculation. Dosing begins on days, with continuous administration for 30 days or until the animal dies. 2. The effect of peptide on the growth rate of transplanted S18e sarcoma in BALB/C mice and the effect on the immunological function of the host BALB/c mice were randomly divided into peptide group, cyclophosphamide group, rmifN-T group, rhIL-2 group and saline group, 20 animals in each group. The bundled mouse sarcoma Si8 cells were cultured in DMEM/F12 medium containing 1% fetal calf jk, 37. (:, incubated in a 5% C〇2 incubator for 72 hours, wash the cells 3~4 times with room temperature without 67 1363092 bacteria Hanks, adjust the cell number to 2χ109/L. Put 0.2ml cell suspension in the armpit. BALB/c mice were inoculated subcutaneously. S18 was inoculated and reared for 10 to 12 days. The tumor-derived mice were sacrificed by cervical dislocation, and the tumor blocks with strong growth and no ulceration were collected and washed with sterile physiological saline. In the physiological saline, 4 ml of sterile physiological saline was added per gram of tumor tissue to prepare a uniform cell suspension. The suspension was injected into the suspension of 0.2 ml to prepare a tumor-bearing mouse model [1], which was administered according to Method 1. 2.1 Peptide Loading The detection of the endocytosis function of the mononuclear phagocytic cells of the tumor mice [2, 3] was carried out by the injection of 1:5 diluted 10 Indian ink 0. lml/10g in the tail vein of the next day after the last administration. 1 minute and 5 minutes after the injection of the ink, 20 μl of blood was taken from the posterior iliac venous plexus using a blood suction pipe pre-wet by heparin solution. The extracted blood was mixed with 2 ml of 0.1% Na2C〇3 solution to measure ODesorm. Calculate the K value of the clearance index according to the following formula: K = (IgAi - lgA2) + (t2 - tl) 15 where A l is 1 minute 0De8() mi, A2 is 5 minutes OD680nm 't2 is 5 minutes, tl is 1 minute after endocytic index study, the mice are sacrificed by cervical dislocation, liver, spleen and 20 thymus are separated and blotted with filter paper, weighing Calculate the swallowing index α value according to the following formula: a = (3^K)(W ^ Wls) where W is the body weight and Wls is the liver and spleen and weight. 68 1363092 2.2 Calculating the tumor growth inhibition index according to the formula: Tumor growth inhibition Index = (average tumor weight of the control group _ average tumor weight of the treatment group) + average tumor weight of the control group

4. Peptide on BALB/C mouse ascites liver cancer Hu 将5 BALB/c mice were randomly divided into peptide group, cyclophosphamide group, rmiFN_Y group, rhIL-2 group and saline group, each group 20 only. The frozen ascites-type liver cancer H22 cells were cultured in DMEM/F12 medium containing 1% fetal bovine serum in a 37T, 5% C〇2 incubator for 72 hours, and washed with sterile Hanks solution. 4 times 'adjust the number of cells to 1~2xl〇9/L. BALB/c mice were intraperitoneally inoculated with ίο collected cells, 2 ml per mouse. The tumor-bearing mice inoculated for 6-8 days were sacrificed by cervical dislocation. After disinfection, the skin was cut and peeled off. The ascites was taken through the abdominal wall muscle layer with a sterile syringe and placed in a sterile vial. The collected ascites was diluted with Hanks' solution to lxl〇6/ml and aseptically operated. After the grouping, each mouse was intraperitoneally injected with 2 ml of ascites dilution, and administered according to the method. 15 Record mouse survival data. If the animal survives at the end of the experiment, the number of days of survival is calculated as the number of days of the experiment. Using the SPSS statistical software, the Kaplan_meier method was used to calculate the average survival time. The survival index was calculated according to the following formula: Survival private number - (average survival days in the test group - average survival days in the control group) / average survival days in the control group χ 1%. 2〇5. The effect of peptide on the cellular immunity of transplanted ascites in LB/e mice. 4. Preparation of spleen cell suspension Healthy BALB/c mice were randomly divided into peptide groups, rm I FN-γ Group, rh] L_2 group, saline group, 15 rats in each group, according to method 3, preparation of lotus root, ascites type 69 1363092 liver cancer mouse model "implantation of cancer cells, administration for 15 days, after the mouse cervical dislocation Lethal's aseptic operation to take the spleen, gently smash the spleen tissue with the needle core of the syringe, and let the single cell enter the cold Hanks solution through a 100 mesh stainless steel mesh (pore size 15 〇 μιη), and transfer the cell suspension to the centrifuge tube, 2 After centrifugation for 1 〇g, 5 discard the supernatant, add 1 〇 volume of Tris-NHKl to the cell pellet, mix well, let stand at room temperature for 10 minutes, centrifuge at 150g for 1 minute, then use cold Hanks solution. The cells were washed 2 to 4 times. The number of cells was adjusted to the desired concentration using rpmI-1640 medium containing 10% fetal bovine serum. 4.2 Effect of peptide on T lymphocyte transformation in tumor-bearing mice 10 The spleen cells of each group of mice were made into lxlOVml suspension and added to a 96-well cell culture plate, ΙΟΟμΙ/well, and each sample was set with 3 parallel wells. To the well, ΙΟΟμΙ/well of 100 pg/ml ConA in RPMI-1640 was added as an assay well, and ΙΟΟμΙ/well of RPMI-1640 well was added as a control well. Then, the 96-well culture plate was placed in a 37 ° C, 5% C〇2 incubator for 66 hours, and then the fine 15 cell culture plate was centrifuged at 150 g for 10 minutes, and the supernatant was taken and stored at -20 ° C for detection of cytokines. IL-2 and IFN. 50 μl/well of 1 mg/ml MTT solution was added to the cell pellet, and the cells were resuspended by shaking for 2 minutes and culture was continued for 4 hours. 150 g, centrifuge for 1 minute, discard the supernatant. 120 μl/well of 40 mM hydrochloric acid-isopropanol was added and shaken for 3 minutes. The absorbance (ODs" (10) value was measured on a 20 EUSA-reader with a reference wavelength of 63 〇 nm. Each mouse formed 3 assay wells and 3 control wells. The stimulation index (SI) of each mouse was calculated by first calculating The average 0D value of the three replicate wells was then obtained by dividing the value of the assay well by the value of the control well. 4.3 Effect of peptide on the killing activity of NK cells in tumor-bearing mice [5,6] 70 1363092. Mouse spleen cells, adjust the concentration of blister to 4χ 106/ml. YAC-1 target cells that have reached logarithmic growth phase are made into j χ 105/mle in 96-well cell culture plates, and ι〇〇μ spleen blister And adding cytotrophic ΙΟΟμΙ to the control well containing only spleen cells; adding 1〇〇μ1 target cells and medium ΙΟΟμΙ to the control 5 wells containing only the target cells; adding target cells ΙΟΟμΙ and spleen cells to the sputum activity assay wells小鼠μΙ. Each mouse was set up with 3 replicate wells as described above. The 96-well culture plate with the sample added was placed in 37. (:, 5% C0 stagnation incubator, cultured for 4 hours. Centrifuge sample 150g 10 Collect cells in minutes. Add μττ fluid ίο 50μ1/well' shake for 2 minutes, 37. (:, 5% C〇2 Incubate for 4 hours in the incubator. Centrifuge at 15 Gg for 10 minutes to 'discard the supernatant. Add 4 mM HCl-isopropyl alcohol 120 μl/well 'vibration for 3 minutes' and measure the absorbance (〇D value) on ELISA-reader. Wavelength 570nm, reference wavelength 630nm. Each mouse has 9 wells: 3 spleen cell controls, 3 target cell controls, 15 3 assay wells containing spleen cells and target cells. By first obtaining the average 〇D value of the three parallel wells of each combination, and then substituting this average 0D into the following formula: NK cell activity index = [1 - (spleen cells and target cell pore mean 〇d value - spleen cells) Mean average 0D value Μ target cell pore mean 〇d value)] χι〇〇% 20 5. Effect of peptide on survival of dba/2 mice with transplanted Lim leukemia DBA/2 mice, 6 to 8 weeks old , weight i8 ~ 22g, were randomly divided into peptide group, cyclophosphamide group, rmIFN-γ group, rhIL-2 group, saline group, each group of 20. (£) 71 1363092 will be the mouse leukemia Lm The cell strain was cultured in DMEM/F12 medium containing 1% fetal bovine serum in a 37 ° C, 5% C〇2 incubator for 72 hours with sterile Han. The cells were washed 3 to 4 times with ks, and the number of cells was adjusted to ixi〇5/mi. The lml cell suspension was implanted into the peritoneal cavity of several healthy DBA/2 mice, and the white squirrel mice were inoculated for 6 8 days. The cervical vertebrae were lethal to death, and ascites was aseptically taken. The collected ascites was diluted to ixl05/ml with sterile Hanks solution. 〇1 ml of the cell suspension was implanted into each test animal and the survival data of the animals were recorded. Test animals were administered according to method j. Using SPSS statistical software, apply j (apian-meier method to calculate the survival time of each group. If the animal survives at the end of the experiment, the survival and days are calculated according to the number of 10 experimental days. Calculate the survival index according to the following formula: Survival index = (experiment Group average survival days _ control group average survival days) + control group average survival days xl 〇〇% 6. Effect of peptide on humoral immunity of c57BL/6 mice carrying transplanted melanoma 15 C57BL/6 small gas, 6 ~8 weeks old, weighing 18~22g, randomly divided into peptide group, cyclophosphamide group, nnIFN-γ group, rhIL-2 group, normal saline group, each group of 20. The frozen mouse Bie melanoma The cells were cultured in DMEM/F12 medium containing 1% fetal bovine serum in a 37X, 5% C〇2 incubator for 72 hours, and washed with 20%-Hanks solution for 3 to 4 times to adjust the cell number to 2xl〇Vml. O.lnU tumor cell suspension was injected per tail vein to produce an animal model carrying βΐ6 melanoma [7,8]. Administration according to method 1. 6.1 Effect of peptide on humoral immunity of tumor-bearing mice [9 Sheep red blood cells (SRBC) are prepared as follows: self-health under sterile conditions 72 1363092 Blood was taken from the external jugular vein of the sheep, placed in a triangular flask with glass beads, shaken for three minutes, and then the blood and Alsever solution (glucose 2. 5 g, sodium chloride 〇. 4 g, sodium citrate 〇 8 g, plus Distilled water to i〇〇ml), set 4«&gt;c refrigerator for use. Samples were centrifuged at 13 μg for 5 minutes before use to collect SRBC. The cells were washed twice by resuspending in centrifugation saline and centrifuging twice. The cell pellet was then collected by centrifugation at i8 〇g for 1 。g. Diluted with 2% (v/v) in physiological saline to obtain the desired suspension of sheep red blood cells (SRBC). (v/v) was added to the centrifugally packed SRBC, gently shaken at 4 ° C for 30 minutes to prepare complement. 2 〇〇 g centrifugation for 1 〇 10 to take 10 supernatant, diluted with physiological saline 1:10 to prepare the preparation Replenishing working solution was required. Each group of mice was intraperitoneally injected with 0.2 ml of SRBC suspension to produce antibodies on the 27th day after administration. The next day after the last administration, the eyeballs were removed and blood was taken at room temperature for 1 hour to make the serum sufficient. Exudation. Centrifuge at 2 μg for 1 minute, take the upper serum 'diluted 5 times with physiological saline. 15 to 1 ml diluted mouse serum 0.5 ml of SRBC suspension was added to the sample, and cooled in an ice bath. Then, 1 ml of the complement working solution was added, and the mixture was incubated in a 37T constant temperature water bath for 10 minutes, and then placed in an ice bath to terminate the reaction. Centrifuge at 200 g for 1 minute, and take the supernatant. To 1 ml of the above supernatant, 3 ml of Drabkin reagent (hydrogen sodium carbonate 20 1. 〇g, potassium ferricyanide 0.2 g, potassium cyanide 0. 05 g, distilled water 1000 ml) was added, and the mixture was allowed to stand at room temperature for 10 minutes. D5_n. Take SRBC suspension 〇25ml, add Drabkin reagent to 4ml, shake well, and let stand for 1 〇 at room temperature, and measure reference 〇D54. (10). Hemolysis = number of samples = (sample 〇 D540nn value + reference OD540nm) Χ 500 73 1363092 6. 2 After humoral immunity study, the mice were sacrificed by cervical dislocation. Pulmonary tissue The pathological changes of the lungs were observed under the naked eye and under the microscope, and the number of nodules in the lung metastases was calculated. Results 5 1. The effect of peptide on the function of phagocytic cells in tumor-bearing mice (S18〇). (Method 2.1) Table IV. Effect of 1CMS peptide on phagocytic index of BALB/c mice carrying transplanted S18 sarcoma • _ Group drug dose animal swallowing index CMS001 50pg/kg 20 6. 24+0. 33*~ CMS001 5gg/kg 19 6. 67+0. 43*' CMS034 5gg/kg 19 6. 20±0. 44* * CMS034 0. 5pg/kg 20 6. 35±1. 02* IL-2 3xl05IU/kg 19 6.96±1_37* IFN-r 3xl05IU/kg 17 5. 45+0. 71 Cyclophosphamide 20mg/kg 19 5 92±2. 47 Saline 0.5ml 19 5. 38+0. 85 10 * : Significant difference compared with saline group. P&lt;0.05. ': There was a significant difference compared with the IFN-γ group. P&lt;〇. 05. CMS001 at 50 μg/kg/day and 5 μg/kg/day, CMS034 at 5 μg/kg/day and 0.5 μg/kg/day increased the swallowing index, which was statistically significantly different from the saline group. . 15 2. Effect of peptide on the growth rate of S18 sarcoma in BALB/C mice (Method 2.2) 74 Table IV, Effect of 2 peptides on tumor growth of transplanted S18 组 Group drug dose Animal weight (g) Tumor inhibition index 00 CMS010 500|ig/kg 20 0. 67+0. 35* 48.4 CMS034 0. 5^/kg 20 0. 83+0.48* 35.9 CMS035 _/kg 20 0. 71+0. 37* 44.6 rhIL -2 3xl05IU/kg 20 0. 69+0. 37* 46.2 rmlFN-γ 3xl05IU/kg 18 0. 96±0.45 25.3 Ring-filled guanamine 20mg/kg 20 0. 68+0. 32* 47.3 Normal saline 0.5ml 20 1.29±0.50 1363092 * : Significant difference compared with saline group. P&lt;0.05. CMS010 (500 gg/kg/day), CMS034 (0.5 gg/kg/day) and CMS035 (5 pg/kg/day) can reduce the growth of S180 sarcoma, which is statistically significant compared with the physiological 5 saline group, P&lt;;0. 05. 3. Effect of peptide on survival of BALB/C mice transplanted with ascites type H22 (Method 3) Table IV. Effect of 3 peptides on survival index of transplanted ascites type H22 in mice Group survival time of drug doses ( Day) Survival index (%) CMS008 5pg/kg 20 50.7 soil 20_ 9 wood & 67.8 CMS011 5pg/kg 20 36. 4+14. 8^ 60.2 CMS024 50pg/kg 20 36. 3±12. 7^$ 38.4 CMS024 5yg/kg 19 40. 6+14. 6*&$ 54.8 CMS024 0· 5pg/kg 19 46.4±14_8*~&$ 76.9 75 1363092 CMS032 0· 5|ig/kg 20 42.8±12·2* ... 63.3 rhI-2 3xl05IU/kg 18 13. 6±0.5 rraIFN-r 3xl05IU/kg 20 27.8±7· 5 6.1 Cyclohexylamine 20mg/kg 20 24. 7±10.2 saline 0. 5ml 19 26. 2±6 8 * : Significant difference compared with saline group. P<0.05. —: Significantly different from the IFN-7 group. P<0.05. &amp; : Significant differences compared to the IL-2 group. P&lt;0.05. $ : Significant difference compared to the cycloheximide group. P<0.05. 5 CMS008 (5pg/kg/day), CMS011 (5pg/kg/day), CMS024 (0.5pg/kg/day, 50pg/kg/day), CMS032 (0.5pg/kg/day), can significantly prolong ascites The survival time of H22 mice was significantly different from that of the saline group (P &lt; 0.05). It was also observed that more than 30% (n=6) of the mice in the CMS024 (0.5 gg/kg/heart ίο days) group survived for more than 90 days (2 months after the end of the experiment). A postmortum examination of the mouse did not show signs of tumor. Thus, CMS024 (0.5 pg/kg/day) can infect the growth of transplanted H22 by infecting tumors or inducing complete healing of established cancers. 4. Effect of peptide on T lymphocyte transformation of ascites type liver cancer H22. Mouse method (Part 15 method 4. 2) Table IV. Effect of 4 peptide on T lymphocyte transformation Group drug dose animal number stimulation index CMS010 500pg/kg 20 1.45±0.21*$ CMS019 0_ 5pg/kg 19 1.50±0.19*$ CMS024 0. 5pg/kg 19 1.46±0.19*$ 76 1363092 CMS024 5|ig/kg 20 1.45 Soil 0.210 CMS034 〇. 5ng/kg 20 1.37±0_ 10 wood* CMS035 〇· 5|ig/kg 20 1.40+0.13*$ CMS035 5jug/kg 20 1.46+0.16*$ rhIL-2 3xl05IU/kg 19 1.46+0.21* rmIFN-7 3xl05IU/kg 18 1.2715+0.1416 Cyclophosphamide Indoleamine 20 mg/kg 19 1.0051±0. 2317* normal saline 0. 5ml 20 1.2514+0. 0651 * : There was a significant difference compared with the saline group. p&lt;〇. 〇5. $:Compared with the cyclosporin group, 'has a significant difference β p<0.05. CMS010 (500 gg / kg / day), CMS019 (0. 5pg / kg / day), 5 CMS024 (0. 5pg / kg / day and 5pg / kg / day), CMS035 (0. 5pg / kg / day " and 5pg/kg/day) significantly increased the T-lymphocyte stimulation index, which was significantly increased compared with the saline group (P&lt;〇. 05). 5. Effect of peptide on the activity of NK cells in ascites-type liver cancer H22 mice (Method 4 3) • Table IV. Effect of 5 peptides on NK cell cytotoxic activity index Group drug dose Animals NK activity index (%) CMS003 500gg/kg 17 37.9±14.5圹$ CMS014 0. 5pg/kg 17 40. 7 ±19. 7木* CMS024 0. 5pg/kg 18 39. 3±18. 7*$ CMS024 5pg/kg 20 34. 9±12.1 Wood ^ CMS024 5〇ng/kg 20 43. 6土 13. 9*^ CMS032 5gg/kg 20 52. 6±12. 5 wood, CMS032 50pg/kg 19 41.0+18. 7*^ 77 1363092 CMS034 50|ig/kg 20 57. 3+17. rhIL-2 3xl05IU/kg 19 26. 0+9. 0 rmIFN-τ 3xl05IU/kg 18 20. 9+3. 3 Cyclophosphamide 20mg/kg 19 16.5±7.2 Wooden saline 0. 5ml 20 24. 0+8. 2 * : with saline group Comparatively, there was a significant difference. P < 0.05. Λ : There was a significant difference compared with the IFN-7 group. P < 0.05. &amp; : Compared with the IL-2 group There is a significant difference. P < 0.05. $ : Compared with the ring wall amine group, there is a significant difference. P &lt; 〇. 05. 5 CMS003 (500pg / kg / day), CMS014 (0. 5pg / kg /day), CMS024 (0. 5pg/kg/day, 5pg/kg/day and 50pg/kg/day), CMS034 (50pg/kg/day) can significantly increase NK cell cytotoxic activity, compared with saline group, There was a significant increase (P&lt;〇. 05). 10. Effect of peptide on the survival of DBA/2 mice with transplanted Li2i, white jk disease (Method 5) Table IV. 6 peptides against mouse transplantable Lmo leukemia Survival time effect group drug dose animal survival time (d) survival index (%) CMS019 0· 5pg/kg 20 21.1±5. 8* 26.8 CMS035 0· 5pg/kg 20 29. 3±15.4* 76.1 rhIL- 2 3xl05IU/kg 20 32. 0+13. 7* 92.3 RmIFN-r 3xl05IU/kg 20 15. 6+2.2 Cyclophosphamide 20mg/kg 20 24. 0±5. 3* 44.2 Normal saline 0.5ml 21 16.6± 5.6 * : Significant difference compared with saline group. P&lt;0.05. 78 1363092 CMS019 (0. 5pg/kg/day) and CMS035 (0.5 yg/kg/day) can significantly prolong the transplantable Lm. The survival time of leukemia DBA/2 mice was significantly different from that of the physiological saline group (p&lt;〇. 05). 7. Effect of peptide on body fluids of C^BL/6 mice with transplanted Β1β melanoma (Method 6.1)

Table IV. Effect of 7 peptides on hemolysis index of c57BL/6 mice with transplanted β16 melanoma Group Drug dose Animal number Hemolysis index CMS001 0. 5pg/kg 20 51.0+16.2*$ CMS001 5gg/kg 20 41.3+17 7*$ CMS001 50pg/kg 19 45. 0+31. 9*$ CMS001 500|ig/kg 20 36.0+10. 2*$ CMS003 0. 5ng/kg 20 61.6±26. 9氺$ CMS003 5pg/kg 20 37.2±15.9 wood* CMS003 50pg/kg 20 38.7+13.5*$ CMS003 500pg/kg 20 35. 9+13. 〇*$ CMS008 0. 5pg/kg 19 42.7±18.4氺$ CMS008 5pg/kg 20 38. 9 ±12. 0木* CMS008 50pg/kg 20 37.1±16.7 Wood* CMS008 500pg/kg 20 50.1+17.8*$ CMS010 0. 5yg/kg 18 34.9+10.5*$ CMS010 5pg/kg 20 51.0+14.6** CMS010 5 〇Mg/kg 20 39. 6±7. 7氺$ CMS010 500pg/kg 20 50.1+16.7*$ CMS011 0· 5pg/kg 20 32.0+14. 7* 79 1363092

CMS011 500jig/kg 20 34.4+19.4* CMS016 500|ig/kg 20 43. 3+30. 0* CMS019 0. 5pg/kg 20 42.0+12. 0*$ CMS019 5gg/kg 20 35.4±15.1*$ CMS019 5 (^g/kg 20 28. 3±7. 6* CMS024 0. 5pg/kg 20 43.0±10. 7*$ CMS024 5pg/kg 20 42.2±11_8*$ CMS024 50pg/kg 20 27. 8+9.1* CMS024 500pg/kg 18 30.1+10. 0* CMS034 0· 5pg/kg 18 50.8±18.4*$ CMS034 5gg/kg 19 43. 0±11.7*$ CMS034 5(Vg/kg 20 30. 2+10. 9* CMS035 5gg/kg 20 38.9±21.2*$ CMS035 50pg/kg 20 44. 7±22. 7*$ CMS035 500pg/kg 19 40.5+25.8* rhIL-2 3xl05IU/kg 19 49.3±24. 7* rmlFN-γ 3xl05IU/ </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; ~ : Compared with the IFN-7 group, there is a pathological difference. P < 〇. 05. &amp; : Compared with the IL-2 group, there is a difference in _. P &lt; 0.05. $: with cyclophosphamide Group comparison, there is a significant difference. P &lt; 0.05. CMS0 (U, CMS003, CMS008, CMS010, CMS0H, CMS016, CMS019, CMS024, CMS034, CMS035, given in Table IV. 7 At a dose of 80 1363092, the body fluid response (increased hemolysis index) was enhanced, which was significantly different from that of the saline group (P&lt;〇.〇5). 8. Peptide-inoculated b16 melanoma cells in C57BL/6 mice. Effect of viability (Method 6.2) Treatment with CMS008 (0_5pg/kg/day, 5pg/kg/day, 50pg/kg/day), CMS016 (5pg/kg/day, 50〇Mg/kg/day) There were no signs of B6 melanoma in the lungs of mice. Conclusion This study was based on the anti-tumor pharmacodynamics guidelines in the Guideline for Preclinical Research of New Drugs (Western Medicines) issued by the Ministry of Health of the People's Republic of China in 1993, to observe the therapeutic effect of peptides on transplanted tumors in mice. The following conclusions are drawn: 1. CMS010, CMS034, CMS035 can significantly inhibit the growth of transplanted Sl80 sarcoma in mice at appropriate doses; 15 2. CMS001 and CMS034 can enhance the phagocytic immune activity of Siso sarcoma small gas mites in appropriate dose; CMS008, CMS011, CMS024, CMS032 can prolong the survival time of ascites-type liver cancer H22 mice at appropriate doses; 4. CMS010, CMS019, CMS024, CMS034, CMS035 can promote T lymphocyte transformation in ascites-type liver cancer H22 mice at a suitable dose of 20 5 . CMS003, CMS014, CMS024, CMS032, CMS034 can enhance the NK cell cytotoxicity of mice transplanted with ascites-type liver cancer H22 at appropriate doses; 6 · CMS019, CMS035 can prolong the transplantation of L121 〇 white blood at a suitable dose

81 1363092 Survival time of diseased mice; 7. CMS008, CMS016 inhibits the development of transplantable Bie melanoma in mice at appropriate doses; 8 · CMS001 'CMS003 'CMS008 'CMS010'CMS011 'CMS016 ' 5 CMS019, CMS024, CMS034 CMS 035 can promote the humoral immune response of mice transplanted with B16 melanoma at a suitable dose. Discussion CMS001 'CMS003 ' CMS008 ' CMS010 ' CMS011 ' CMS014 ' CMS016 , CMS019 , CMS024 , CMS032 , CMS034 , CMS035 itself 1〇 or as part of it for human cancer control. For example, CMS001, CMS0 03, CMS0 08, CMS010, CMS01, CMS014, CMS016, CMS019, CMS024, CMS032, CMS034, CMS035 can be used to improve the immune function of cancer patients; CMS008, CMS010, CMS016, CMS034, CMS035 can interfere with tumor cells. Growth; CMS008, CMS011, CMS019, 15 CMS024, CMS032, CMS035 can be used to prolong the survival of patients with cancer. These peptides may be used alone or in combination or in combination with other drugs or food ingredients for the full control of the tumor. Table IV. 8 Peptides valid for cancer CMS code sequence identification number 82 1363092 CMS001 1 CMS003 27 CMS008 3 CMS010 4 CMS011 30 CMS014 7 CMS016 CMS019 11 CMS024 16 CMS032 22 CMS034 24 CMS035 25 References 1. 1. New drug (Western medicine) preclinical Guiding Principles of Research. Drug Administration Bureau of the Ministry of Health of the People's Republic of China. 1993, 7 : 137-143 5 2. Guiding Principles for Preclinical Research of New Drugs (Western Medicine). Drug Administration Bureau of the Ministry of Health of the People's Republic of China. 1993, 7 : 128-129 3. Zhang Yuanpei , Su Huaide. Pharmacological Experiment (Second Edition). People's Health Publishing House. 1998, 137-138 4. Xu Shuyun, Ru Ruru, Chen Xiu. Edited. Pharmacological Experimental Methodology. 10 1991, 1221-1234 5. New Drug (Western Medicine) Clinical Pre-study guidelines. Pharmaceutical Affairs Bureau of the Ministry of Health of the People's Republic of China. 1993, 7 : 140 83 1363092 6. He Jinsheng, Li Ruizhu, Zong Tingyi. Methodology for detecting NK cells/tongue by MTT reduction method. Chinese Journal of Immunology, Bile , Yiguang 356 358 7. Yang Yaoqin 'Yang Huchuan, Tao Ihong and other Tween for warming and anti-tumor effect $ enhancement effect small (9) melanoma experimental research on cancer prevention and treatment, 5 1999:26 (4):8-12 ' 8. Fu Jian' Zheng Jie, Fang Weishang, etc. Transfection of interleukin 12 gene inhibits the tumorigenicity and metastasis of 816 cells. Chinese Journal of Medicine, ^ 1998;78(8) : 627-629 9. Wang Qian. Experimental Methods of Modern Medicine. People's Health Publishing 1〇社·1998, 482-483 10. Pan Qichao, Yan Bin·Oncology and Chemotherapy (Second Edition) _ Henan Medical University Press. 2〇〇〇 , 66-69 • _ U· Zhang Yuanpei, Su Huaide. Pharmacological Experiment (Second Edition). People's Health Publishing House. 1998, 131 15 V · Effects on body weight • Feeding healthy rats with high-nutrient diet for 5 weeks, accompanied by or Simultaneous peptide therapy (intramuscular 300/zg/kg/day) was not included. Normal diet rats injected with normal saline served as a negative control group. After 5 weeks of treatment, stop the injection and maintain the same diet ^ week. Rat body weight was measured weekly and behavioral changes were observed. The results showed that during the treatment of peptide therapy, the weight gain of rats treated with CMS015 was significantly lower than that of the control group, and the trend of weight loss slowed down after withdrawal. Thus, the peptide CMS015 showing the appropriate dosage level has a reversible inhibitory effect on the body weight of the nutritionally obese rats. Material 84 1363092

Sprague Dawley (SD) rats, both male and female, weighing i45±l〇g, gambled at the Experimental Animal Center of Guangzhou University of Traditional Chinese Medicine. The peptide, a source of L-amino acid, was synthesized by American Peptide Company, Inc., USA, and was diluted with physiological saline to an injection solution of 1 〇 Ag/ml. Rats with high fat and high nutrition 5 feed and common feed formula refer to the guidelines for preclinical research of new drugs promulgated by the State Drug Administration [1], "Slimming drugs,". Methods Healthy rats were randomly divided into experimental group and positive control group. And the negative control group, 10 rats in each group, male and female. The rats in the experimental group were given high-nutrition diet for 5 weeks, 10 and the intramuscular injection peptide was given 300/zg/kg/day once a day. The positive control group gave the same high nutrition. The feed was injected with placebo saline, while the negative control group was injected with normal diet and placebo to verify whether the previous two groups were successful. After 5 weeks of treatment, the injection was stopped and the same feed was maintained for 3 weeks. The body weight and observed behavioral changes were measured weekly. 15 Statistical analysis The experimental data were expressed as mean soil standard deviation, and the paired t test and single factor ANOVA analysis were used to compare between groups and groups. The significant difference was ρ^〇〇5. Results 1 · Effect of peptide on body weight of SD rats Table V. Effect of peptide on body weight of SD rats Positive control group (g) CMS015 treatment group (g) Male n=5 _ sex n=5 Male n=5 Female n =5 145.6 ±13.6 1 before treatment 33.6 Soil 4.6 145. 6 ±8. 5 129.2 ±3.3 85 Week 1 after treatment 194.4 ± 14.5 164.4 + 8.7 183.8 ± 10.6 157.8 ± 8.3 after treatment 2 239.6 188.0 ± 6. 4 220.6 176.0 weeks ± 13.4 ± 12. 2 $ ±11.2* After treatment 3 265.0 208. 8 ± 8. 2 239.0 196.0 weeks +11.8 ± 16. (T ± 10. 5* after treatment 4 287.4 227. 2 ± 8. 2 258.2 212.0 weeks ± 17.7 ± 18 f ±13.5* After treatment 5 299.4 236.6 268.8 221.4 weeks ± 21.2 ± 10.9 ± 17.7 * ± 13. 2 * After treatment 6 333.4 249.4 299.4 235.6 weeks ± 27.1 ± 16.3 ± 21. f ± 16.3 after treatment 7 349.2 261.2 310.4 242.2., Week ± 28.9 ± 13.4 ± 25. 9* ± 18. 8* After treatment 8 374.4 255.6 337.4 252.8 weeks ± 37.2 ± 11.5 ± 30.6 ± 22.5 Compared with the positive control group: &quot;^&lt;0.05 1363092 Compared with the control group, the peptide CMS015 administered at 300 // g/kg/d significantly inhibited the weight gain of the obese rats (P&lt;0.05). The difference between the treatment group and the control group increased with the treatment time. After discontinuation of the CMS015 group, the difference between the treatment group and the 5 control group gradually decreased, and the difference was no longer statistically significant after 3 weeks, indicating that the effect of the peptide on the body weight of SD rats was reversible. The appetite and activity of the rats in each group were not affected during the whole experiment. Discussion 86 1363092 From the results of this experiment, it can be concluded that the appropriate dose of the peptide CMS015 can inhibit the weight gain of the nutritional obese rats, so it has the potential to be applied to human weight loss. The peptide can be used alone in the future, or in combination with two or more peptides, or in combination with other drugs or therapeutics for the combined treatment of obesity. 5 This study only examined the route of administration of intramuscular injection, but did not rule out other possible routes of administration. The peptide CMS015 can be administered by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection or subcutaneous implantation, and can be used with or without a drug delivery facilitating device such as liposome, sustained release protection. The peptide may also be administered orally in the form of a tablet, a capsule, a suspension, a solution, or the like, as well as a conventional form or a sustained release form which is modified without or without gastrointestinal protection. The peptide may further be used in the epidermis, such as ointments, creams, gels, and the like, with or without transdermal facilitation devices, or in the form of inhaled powders, solvents or liposomes. The peptide can also be translated into a gene sequence, cloned into an expression system, alone or in combination with other peptide sequences to produce a purified or unpurified peptide molecule, the available activity of which is as described herein. Table V. 2 Peptides effective for obesity CMS code Sequence Identification Number CMS015 8 References 20 1. State Drug Administration, Editor-in-Chief. Principles for Preclinical Research in New Drugs, 1993, 1st edition. 87 1363092 It is to be understood that additional amino acids may be added at the amino or carboxy terminus of the above peptides as another method of practicing the invention. For example, by adding one or two amino acids to the peptide without affecting its biological function, it is also possible to add three or four amino acids while still maintaining the function of the peptide. These are all referred to as variants of the peptide. Alternatively, one or two amino acids may be deleted from the peptide without affecting its biological activity, and three or four amino acids may be further deleted without affecting the biological function of the peptide. These are referred to as fragments of the peptides of the invention. In addition, derivatives of peptides such as conservative substitutions of one amino acid in the same functional region can also be used to practice another aspect of the invention. For example, a peptide having 10 non-polar ends or hydrophobic side chains can be substituted for one side group and its biological function is not diminished. As a further example, linkers/spacers can be inserted into peptide forming variants, but these variants retain the active portion of the original peptide used in this study. These are considered variants of the peptides of the invention. The term peptide analog as used herein includes 15 peptides having amino acid molecules that mimic natural amino acid structures, such as analogs having different backbone structures or D-amino acid substitutions. As a further example, although the amino acid used to synthesize the peptide is in the L-optical isomeric form, the peptide in which one or more amino acids in the sequence are substituted by the D-form may have similar biological activities. The term "functional derivative" as used in the claims of the present application encompasses fragments, variants, analogs and chemical derivatives of the peptides of the invention. The term "hybrid peptide" as used herein, refers to a peptide containing an additional peptide inserted into the original biologically active peptide having the sequence number: 1-30 or a functional derivative thereof while still maintaining substantially similar activity. The additional peptide includes a leader peptide containing, for example, an amino acid sequence recognized by one or more prokaryotic or eukaryotic cells as a signal for secretion of the hybrid peptide 88 1363092 to the extracellular. Secretion can be directly secreted or secreted indirectly by secretory vesicles. "Substantially purified peptide" means a peptide having a purity of at least 10% (w/w), preferably having a purity of 20%, more preferably 40%, more preferably 60%, further preferably 5 is greater than 90%. In a most preferred embodiment, the purity is greater than 99%. As described below, the substantially purified peptide can be used as a component of the mixture for the preparation of a pharmaceutical or nutritional formulation. The above peptides are useful in pharmaceutical formulations for the treatment of immune disorders as well as diseases which have a secondary effect on immunity such as cancer or infection or any of the above symptoms. In the pharmaceutical formulation of these peptides, in addition to the identified peptides, other active or inactive components may be mixed, including other peptides, for example two or several (for example, 3-5) Peptides can be added to the same formulation with or without other ingredients. Or... a list of peptides 15 15 can be used to prepare a formulation. Such a formula can be administered directly by intravenous '=H subcutaneous or intradermal administration' or arterial injection: S ' can also be sputum X powder, healthy way to breathe through the skin, percutaneous suction 2 minus other known delivery the way. The formula can also be taken orally, and can be used for the stomach of the anti-peptide after oral administration. It is known as a pharmaceutical carrier, a suitable carrier, but not limited to :何! The acceptable carrier of the quasi-learning material's tri-acid emulsion H, 7 money, such as oil-water mixture or glycerin, suitable for the filling, coating of tablet sacs, etc., according to the mode of administration select. 89 (S) 1363092. The form can be administered by intravenous injection, intramuscular injection, intraperitoneal injection and f-implantation. It can also be administered in a form of oral administration, such as tableting, sac, riding, and modification. The gastrointestinal or gastrointestinal tract is not administered in a protected form. The shape can be advanced into the form of any application such as ointment, cream, condensate or the like without the percutaneous promoting device. Months can also be converted to its genetic sequence and cloned into the expression system, or used in its own right or in combination with the rest (iv) to produce a morphological molecule that utilizes the activity described herein.

The dose to be administered in each form may be Ing-IGg/kg body weight. The administration agent 10 for injection may be 10 ng to 10 mg/kg, more preferably (4) to (4) / 。. The effective dose of the drug can be as low as 1 ng/kg, which may be caused by the binding of one or more peptides to the receptor or the peptide directly causing the body-series cascade reaction. For oral administration, the dose may be 1 ng to 1 g/kg body weight/day, preferably o.i/zg-ig/kg body weight/day, more preferably weight/day. 15 VI. Gene Therapy and Therapeutic Methods • Gene therapy of the peptide sequences found is carried out by designing a nucleic acid sequence encoding these peptides. The nucleic acid can be chemically synthesized and operably linked to a promoter and cloned into an expression vector. The expression vector is then administered to the human body as a gene therapy-H for expression in human cells. The term "base 20 vector," as used herein, includes these expression vectors. The vectors that can be used for gene therapy include adeno-associated viruses (Miz_, M. et al. (1998), Japanese cancer research miscellaneous

T489, 76-80) ^ LNSXmitCMiller, A. D 217, 58 599) and lentivirus (Goldman, μ, J. et al. (1997) Human Gene Therapy 8, 2261-2268) » 90 cs ) 1363092 . • &gt; Other vectors for peptide delivery include an expression vector encoding a desired month in an organism that is replicable in a host organism that is desired to be administered to the peptide without significantly affecting the health of the host organism. For example, the expression of 载 can be caught (four) material "the grant of bribes (4) is non-pathogenic 5 sex organisms. In certain embodiments, the expression vector produces a desired peptide in a fine or free organism that does not have a significant deleterious effect on the health of the host organism to which the peptide is administered. For example, the money vector encoding the desired peptide may be a monthly expression vector required for breeding in an organism such as lactic acid bacteria, colon g, or yeast. In one embodiment, the expression vector H) produces the desired peptide in a microorganism naturally present in the digestive tract of a mammal or in a mammalian digestive tract-resistant microorganism; can express a desired peptide • Some microbial species include But not limited to Lactobacillus species, such as L. acidophilus, L. amylovorus, L. casei, L. crispatus, L. gallinarum, L. gasseri, L. johnsonii, L. paracasei, L. plantarum, L. reuteri, 15 L. rhamnosus, 荨 'Difidobacterium species' such as B. adolescentis, B. animalus, B. bifidum, B. breve, B. infantis, B. lactis, β· longum, etc.; Enterococcus faecal is or Ent.facium Sporolactobaci 1 lus inulinus ; Bacillus subtil is or Bacillus cereus ; Escherichia coli i Propionibacterium freudenreichii ; 20 or Saccharomyces cerevisiae or Saccharomyces boulardii ° - Chemically synthesized or otherwise including, but not limited to, a gene generated by reverse transcription of mRNA to produce cDNA molecules; The nucleic acid sequences of the peptides of the invention are incorporated into expression vectors for gene transfer into the desired host by genetic engineering methods known in the art. The expression vector can be a DNA vector or an RNA vector. Example 91 (S) 1363092. If the expression vector can be based on a plastid or viral genetic element. The expression vector can be a vector for extrachromosomal reintegration or a vector-incorporated expression vector for integration into a chromosome, including initiation of operably linked to a nucleic acid encoding a peptide of the invention. The promoter may be a regulatable promoter, such as a seed promoter or a promoter. In some embodiments, the promoter may be selected to provide a peptide level of the genus level. In addition, expression vectors can include = sequence hybridization - production 'presentation and/or secretion if desired. In some embodiments, the nucleic acid encoding the peptide of the present invention and the nucleic acid sequence secreted by the guide peptide is effective. For example, a nucleic acid encoding the present invention can be operably linked to the nucleic acid sequence of the conjugated peptide. ▲In the actual scheme, the expression of the peptide which is engineered to encode the peptide of the present invention may be suitable for expressing the present invention in the bacterial species such as the (10) bacterial species and the Bacillus subtilis which constitute the normal digestive tract of the mammal. Expression vector. Examples of suitable expression vectors can be found in U.S. Patent Nos. 6, 〇〇, 388, and I5 5 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 It will be understood that any expression vector that facilitates expression of the invention in a healthy organism that does not impair the host organism in which the peptide is desired to be administered can be used. In some embodiments, an expression vector engineered to encode a peptide of the invention may be a yeast species suitable for being digested by a mammal, such as omyces cerevisiae or more preferably Saccharomyces boulardii (which may be in the human digestive tract) Hosting and used to treat certain forms of abdomen/write) expressed in this expression of the dragon. A yeast expression vector constituting a silky heterologous protein and a genus can be used because it is very stable and thus can be delivered to progeny during mitosis and meiosis, and the vector can include 92 1363092 including a signal peptide or directing a recombinant protein. The coding sequence of the horizontally secreted peptide. An example of such a yeast carrier is described in U.S. Patent No. 6,391,585, the disclosure of which is incorporated herein by reference. An expression vector encoding a peptide of the present invention can be introduced into an organism in which the peptide is to be expressed by techniques known in the art. These techniques include traditional methods of transforming bacteria, yeast or other microorganisms, such as those using chemically competent bacterial cells, electroporation or lithium acetate conversion (for yeast), and transformation methods for some bacterial strains that are ineffective with these programs. progress. In some embodiments, the expression vector is introduced into a lactic acid bacterium that has been known to be untransformable by the method disclosed by Leer et al. (W095/35389), which is incorporated herein by reference. The introduced sequence can be incorporated into the chromosomal DNA of the microorganism or in the form of an extrachromosomal DNA element. This genetically engineered microorganism containing an expression vector can then be inoculated with a digestive tract, a vagina, a trachea or the like to effect immunotherapy. In some embodiments, the organism expressing the peptide of the invention is ingested in an inactive form or, preferably, in an active form. In the digestive tract, these microorganisms produce the peptide, which is released into the cavity by secretion or microbial lysis, or otherwise presents the peptide to the host, whereby the peptide produces its intended effect on the host organism. In other embodiments, the 20 peptide is presented to the host organism at the nasal passage, vaginal or intestinal mucosa. Another treatment is the use of liposomes as a means of delivering specific nucleic acids to human cells. Nucleic acids (eg, expression vectors containing nucleic acids encoding peptides of Sequence Identification Number: 1-30) are delivered in an environment conducive to cellular uptake and chromosomal incorporation, eg, Gao, X. and Huang, L. (1995) Gene Therapy 93 1363092 2, 710-722 and U.S. Patent 6,207,456. Alternatively, using the method of US 6,245,427, the peptide itself can be packaged in liposomes and delivered directly. All of the above scientific publications and patents are incorporated herein by reference. Nucleic acid sequences useful in the above gene therapy and therapeutic methods include sequences encoding 5 of these peptides and functional derivatives thereof. Based on the degenerate cryptosystem, any of a number of nucleic acid sequences can be used to encode these peptides and their derivatives. 0 The following references are incorporated herein by reference: 1 · New Drugs (Western Medicine) Preclinical Research Guidelines. People's Republic of China 10 Health Department of Pharmaceutical Affairs. 1993, 7 : 134_135 2·Xu Shuyun, Ru Ruru, Chen Xiu, ed. Pharmacological Experimental Methodology. 1991, 1221-1234 3 · New Drug (Western Medicine) Preclinical Research Guidelines. Pharmaceutical Affairs Bureau of the Ministry of Health of the People's Republic of China. , 7 : 140 4 · He Jinsheng, Li Ruizhu, Zong Tingyi. Methodological study on detection of NK cell activity by MTT reduction method. Chinese Journal of Immunology, 1996: 1(6): 356-358 5. Wang Qian. Modern Medical Experimental Methods ·People's Health Press·1998, 482-483 6 ·Guidelines for preclinical research of new drugs (Western medicine). 2nd People's Republic of China 2 Department of Health and Drug Administration · 1993, 7 : 141 7 · New drug (Western medicine) preclinical research guidelines. Drug Administration Bureau of the Ministry of Health of the People's Republic of China. 1993, 7: 132-133 8 · Guiding Principles for Preclinical Research of New Drugs (Western Medicine). Drug Administration Bureau of the Ministry of Health of the People's Republic of China. 1993, 7: 128-129 94 1363092 9 · Zhang Yuanpei , Su Huaide. Pharmacological Experiment (Second Edition). People's Health Publishing House. 1998, 137-138 1〇·Li Jiatai. Clinical Pharmacology (Second Edition). People's Health Publishing Du·1998, 1338-1339 5 Example 1 Delivery of Peptide by Genetically Engineered Lactobacillus The following provides an exemplary method of delivering a peptide of the invention to a host as described above. The DNA sequence encoding one of the peptides listed in Table A was chemically synthesized and introduced into an expression vector by standard genetic engineering techniques well known to those skilled in the art. The selected expression vector contains a constitutive promoter that is functional in Lactobacillus, a multiple cloning site for introducing a DNA sequence in a specific 5, to 3' direction, and a selection marker that confers resistance to antibiotics. The gene (to aid the cloning procedure) and may also contain other sequences that contribute to the production and/or secretion of the peptide, such as signal peptide sequences. An example of such a carrier 15 is described in U.S. Patent No. 5,592,908, the disclosure of which is incorporated herein by reference. Briefly, the patent discusses several known promoters that are functional in Lactobacillus species, and methods for discovering new promoters in such bacteria, any of which can be associated with a nucleic acid encoding a peptide of the invention. Efficient ligation to express the peptide in R. ssp. A nucleic acid encoding a signal peptide, such as a peptide consisting of 16 to 35 mostly hydrophobic amino acids active in Lactobacillus lactis, as described in U.S. Patent No. 5,592,9,8,20, is inserted into a promoter and encoded by the present invention. The nucleic acid between the nucleic acids, and thus the signal peptide, is in frame with the nucleic acid encoding the form of the invention. In addition to the coding sequence of the form, the 'sense-employment sequence may also include a phase that helps 95 1363092: material gram_money_. (d), corresponding to the multiple cloning site of the vector (four) __鄕 can be "human synthetic" so that the sequence can be cloned into the vector in the correct orientation. The carrier and synthetic circle specific restriction enzymes were purified. The vector and the conjugate are transformed into a suitable strain of Escherichia coli after the ligation reaction. The transformed bacteria are plated on a medium containing an antibiotic to which the carrier confers resistance. Colonies that transform bacteria are selected for growth culture (4) pellet preparation. Confirmed the correct DNA formation.

The expression is then transformed into a bacterial host cell of the mast® _M.aeid〇philus 1〇. The transformed cells are selected by a selection marker in the vector sequence, and secretion of the peptide can be confirmed by performing Western blotting, performing gel electrophoresis in the form of a sputum, and other standard techniques. A fine-grained culture of genetically engineered bacteria was selected and used. The culture of the genetically engineered bacteria expressing the desired peptide is cultured and at least a portion thereof is administered to the digestive tract, vaginal, trachea of the host organism, or the other region in which the bacteria can replicate. If desired, the bacterial culture can be treated in a variety of ways to supplement the supplement absorbed by the host's gut. These treatments include drying or other methods of preserving the bacteria, and the bacteria may be combined with carrier agents such as solutions, solvents, dispersion &quot;quality release agents&apos; emulsions and the like. The use of these agents to prepare supplements is well known in the art. For example, bacteria can be used to prepare yogurt products or other foods for human consumption so that the microorganisms expressing the peptide reside in the human digestive tract. Various methods of incorporating a particular strain of lactic acid bacteria into foods such as yoghurt, kimchi, cheese and cream are described in U.S. Patent No. 6,036,952, the disclosure of which is incorporated herein by reference. After ingesting the bacteria by either route, the engineered bacteria can reside in the digestive tract 96 1363092 such that the mucosal layer of the digestive tract presents and/or absorbs the peptide of the invention. Example 2 Bacillus subtilis delivery peptide in genetically engineered form An exemplary method of delivering a peptide of the invention to a host as described above is provided below. The dna sequence encoding one of the peptides listed in Table A was synthesized by chemical method 5, and this DNA sequence was introduced into an expression vector using standard genetic engineering techniques well known to those skilled in the art. The expression vector of choice includes a shuttle vector, such as pTZ18R (Pharmacia, Piscataway, NJ), which is capable of breeding in both E. coli and B. subtilis and contains an antibiotic resistance gene to select for transformed colonies. This vector may contain a promoter active in Bacillus subtilis, such as a promoter derived from the Bacillus subtilis SacB gene, and a nucleotide sequence encoding a signal or leader peptide active in Bacillus subtilis. The signal or leader peptide directs the expression of the heterologous protein for efficient export from bacterial cells. An example of such a carrier is described in U.S. Patent No. 6,268, 169, incorporated herein by reference. Briefly, as described above, DNA encoding a peptide of the present invention having a restriction enzyme site and/or other sequence that promotes DNA cloning is synthesized by the technique 15 well known in the art. The transformant was transformed into Bacillus subtilis after transformation into E. coli, plating, selection and propagation of the plasmid to produce a stock solution, and transformants were selected based on resistance to antibiotics in the plating medium. The production and secretion of the peptide by the genetically engineered subtilis was confirmed by radiographically using SDS-PAGE analysis or _ _ _ 20 traces after radiographs. The culture of the genetically engineered bacteria expressing the desired peptide is cultured and administered to at least a portion of the host organism's digestive tract, vagina, trachea or other area in which the bacteria can replicate. 97 1363092

10

The sputum yeast strain selected for transformation is a mutant auxotrophic strain which requires a gene on the plasmid to grow on minimal medium. The transformed yeast colonies are selected by plating the yeast on a medium lacking the gene provided on the vector. Only the recipient vector and its selection gene and the yeast expressing the gene product can grow into colonies on minimal medium. Peptide expression can be confirmed by Western blotting, gel electrophoresis of peptides present in the culture medium, or other standard techniques. Example 3: Delivery of peptides by genetically engineered Saccharomyces cerevisiae to provide an exemplary method for transporting a peptide of the present invention to a host as described above. The skilled person is familiar with (4) standard-based I-way technology to introduce this-wake sequence into the expression vector. The selected expression vector comprises a stably maintained yeast protein expression vector, which is packaged to form a yeast promoter such as Π, such that the vector can replicate at both the yeast and the large intestine (4), and the auxotrophic yeast mutant is protected. One or more genes of choice for the purpose of selection, polygreen (tetra) (MCS), and the sequence of the code that encodes 1 Lu. Such vectors are commercially available and are well known in the art or can be constructed using standard techniques. The vector is prepared by inserting the synthetic DNA into a yeast vector, transforming into Escherichia coli: cutting the transformed sputum on the selection medium, selecting the transformed bacterial colonies, and preparing the f-particles from the bacterial culture of the (4). Techniques such as B conversion or electroporation are converted into phase sugar yeast. Yeast transformed colonies were selected and used to prepare large scale cultures. A culture of genetically engineered yeast expressing the desired peptide is cultured and at least a portion thereof is administered to the digestive tract, vaginal, trachea of the host organism or other areas in which the yeast can replicate 98 1363092. If desired, the yeast culture can be treated in a variety of ways to produce a supplement that is absorbed by the intestinal tract of the host. These treatments include lyophilization or other methods of preserving yeast, and yeast may be combined with carrier agents such as solutions, solvents, dispersion media, sustained release agents, emulsions and the like. The use of these agents to prepare supplements is well known in the art. In another embodiment, the transformed yeast can be used to prepare food products such as fermented milk products such as yogurt and kefir using techniques known in the art. Like the live lactic acid bacteria cultures in these foods, the transformed yeast resides in the digestive tract for at least a short time and provides the peptide to the host via the digestive tract. t Schematic description 3 10 Schematic description 4 »»&gt; [Main component symbol description] None 99

Claims (1)

13,63092. • Patent Application No. 97118145 Applicable Patent Revision Amendment This revision period: 100.11.2 X. Patent application scope: Identification of the live abortion of cancers identified 1 · A substantially purified peptide It consists of the amino acid sequence of the sequence number _1. 2. The substantially purified peptide of claim 1, wherein the peptide modulates at least one of the following symptoms: immunological activity, hepatitis infection, hepatitis B infection, and growth of the disease. 3. The substantially purified peptide of claim 1, wherein the peptide modulates at least one of the following: T lymphocyte transformation, NK cell cytotoxic activity - T lymphocyte secretion of IL-2, T lymphocyte fraction. The weight of the thymus and the weight of the spleen formed by the anti-SRBC antibody when IFN, antigen is stimulated. A pharmaceutical composition comprising a substantially purified peptide mixed with a pharmaceutically acceptable carrier, the peptide consisting of the amino acid sequence of SEQ ID NO: 1. 5. A method of preparing a pharmaceutical composition comprising a substantially purified peptide consisting of the amino acid sequence of Sequence Number 1; and a substantially purified peptide and a pharmaceutically acceptable Body mixing. 1 1363092 Patent Application No. 97118145 Scope of Application Patent Revision This Revision: 100.11.2 6. A method of preparing a pharmaceutical composition comprising providing a substantially purified peptide consisting of an amine of sequence number 1 The base acid sequence is composed. 7. Use of a substantially purified peptide for the manufacture of a medicament consisting of the amino acid sequence of SEQ ID NO: 1, which is useful for modulating at least one of the following symptoms: immunological activity, hepatitis infection, B Hepatitis infection and cancer growth. 8. Use of a substantially purified peptide for the manufacture of a medicament consisting of the amino acid sequence of SEQ ID NO: 1, which is useful for modulating at least one of the following activities: T lymphocyte transformation, NK cells Cytotoxic activity, secretion of IL-2 by T lymphocytes, secretion of IFN by T lymphocytes, formation of anti-SRBC antibodies upon antigen stimulation, weight of thymus and weight of spleen. 2