TW466117B - Pharmaceutical compositions of the retinoblastoma susceptibility gene product - Google Patents

Abstract

This invention provides a method of preventing or inhibiting the proliferation of a pathologically proliferating cell, wherein the pathological proliferation of the cell is the result of the absence of a functional retinoblastoma protein or polypeptide in the cell. The method requires contacting the cell with an effective amount of retinoblastoma protein or polypeptide. This method also is useful to prevent or treat retinoblastoma or a secondary cancer to retinoblastoma by administering to a patient a functional retinoblastoma protein or polypeptide.

Description

466117 Α7 Β7

Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs. 5. Description of the invention (1) 'It was indeed completed under the government's support. Degree of right. In this patent specification 'references to publications are incorporated by reference. The publications of this type of publication are incorporated herein by reference for a more detailed description of the techniques related to the present invention. In summary, the present invention is a cell therapy for preventing the proliferative effect of pathologically proliferating cells, that is, the administration of effective preventive or therapeutic doses of cancer suppressor gene proteins such as R Β protein · white matter or more than 呔 M to achieve swelling. Tumor inhibition. BACKGROUND OF THE INVENTION ‘. There is considerable progress in understanding the function of genes in maintaining biological health. The general rule can be said to be because the cells' genes cannot produce appropriate proteins, which causes many pathological phenomena in the body. The loss of tritium function may be due to the loss of the entire gene, or because the gene itself is damaged for various reasons. The identification of such factors has led to significant advances in gene therapy. For example, renal embryonal sarcoma, a childhood kidney cancer, is thought to be due to the inactivation of genes on chromosome 11. Using single-chromosome microcell fusion-media-mediated transfer technology, it has been shown that tumor cells that contain normal chromosomes 11 to kidney embryoblastic sarcoma can inhibit tumor formation. On the other hand, * human chromosomes X and 1 3 do not have this effect. Although the whole human chromosome transfer may have some value on the basis of experiments, it is not practical to consider the use of this kind of transfer for the treatment of genetic defects. Because of this, the preparation of appropriate chromosomes for holistic applications is very laborious, time consuming, costly and expensive. The results show that this technology is used in many applications -4, this paper is shattered and praised * Guo Lisai Tablet Standard (please read the notice on the back before filling in this page)] _ 4 661 1 7

A7 B7 V. Description of the invention (2. It is suitable to use wood. Due to the inappropriateness of the entire chromosomal therapeutic use, another consideration is to deliver all or at least operable parts of the appropriate gene to the patient. Although this method is more than It is practical to transport the entire chromosome, but gene therapy is only suitable for certain applications. In this regard, isolation, sequence analysis, and selection of appropriate nucleic acid materials are very expensive and time consuming. In addition, there are very few places in the world that have This type of technology requires highly precise molecular genetic technology. Furthermore, this type of technology has not yet been able to mass-produce materials suitable for use in holistic therapy. To sum up the above points, if there is a specific medical method in the cell, At the level, it is ideal to use biotechnology technology and to use materials that are relatively cheap, reliable, more widely available and have specific biochemical effects. Furthermore * more ideally, there is a treatment method • The treatment product K can be delivered on the girl's cell surface to cause changes, such as inhibition or tumor suppression. Of course, it is more desirable to have a product that can be a large amount, one It can be manufactured with a method of determining purity, and can be used for the germinal membrane. Netview is based on the therapeutic formula If _ β β cells, this bundle of mitochondrial diseases is suitable for the diseases that are often sent to the non-disease. The life-threatening part of the tumor is sensational in the layers. The membranous reticulum group is a thin tumor membrane S and the oblique is thin. The retinal membrane is the retina of the tumor. The Central Bureau of Standards Consumer Cooperatives printed the nuclear bureau ’s dyeing and deep dyeing. Zhengzheng. Extra fine long tumor and spores of the somatophyte Weiwei Qiang rose into the network. And the side 'two cells in the fine life The last thing about the shape of the round and the small ones is 0. One of them is the type and the symptoms of the post-generation generation, and the sexual transmission of cancer is inherited. The thing is that it can and can be especially combined with tumors. A tumor dead cell swollen dead fine endoblasts " ball osteoplasty eye human net's invasion of the seeing section often looked at by God ---------- V equipment ----- 1T --- --So --------- (please listen to the notice on the back first # '· Fill in this page), I. pm country_ > 21 / ^-..., · u. ·. Λ1 | 466 彳 彳 7 A7 B7 Shellfish, Central Bureau of Standards, Ministry of Economic Affairs Printed by Fei Cooperative Society 5. Description of the Invention (5). The epiphenomenon-type sign is an early-onset and multiple tumor disease nest. Acquired patterns occur in the later stages of life and are unilateral swelling (Proc. Hatl. Acad. S ci · (1971 丨 68: 820-823; Hum, Genet. (1 979) 5 2: 1 -54: S ei ft η ne (1984) 223: 1028-1033. The susceptibility to hereditary retinal germ cell 遗传 is inherited to the offspring with a positive chromosomal dominant trait at a frequency of 90¾ genetic traits. This tumor has thus become and remains a prototype model for studying genetic factors in cancer (Am. J. Hum Genet. (1 978) 30: 406-4 1 0; Cancer (1 975) 35: 1022-1025 °-Regarding the tumorigenic effect of retinoblastoma, there are at least two kinds of false sharp births. The first hypothesis It is suggested that the swelling is caused by two mutations (Proc. Natl. Acad, Sci. (1971) 68: 820-823; Cancer (1 975) 35: 1 022-1026. Another false sharp proposed positive Chromosomal dominant hereditary tumors, such as retinoblastoma, represent a group of transforming genes that are regulated under normal circumstances, most likely the defect-regulating genes of the proto-tumor genes or the remnants of suppressor genes. When retinal cells develop, it is assumed that keratinocytes cause Kβ The loss of the second copy of the gene. Therefore, the ϋΒ protein > was not produced at the timing of the fishermen and could not be used for differentiation. In the development of higher organisms, such as animals and humans, there is an orderly-cell cycle evolution is One The key factor. Due to the orderly and systematic cell differentiation of this evolution, it eventually developed from a single undifferentiated cell into a highly structured organism with various special structures. In short, physiologically normal cell cycle evolution It is very important for living things, not just in the early growth stage * but also in the whole life cycle of living things. Therefore, even " 6----------...------- 1 ; Install ------ 11 ------------ (Please read the note on the back before filling out this page) i's name '夺 ^^ 4Use Chinese country rubbing ^ CNS) ίΑ4 ^ (210 × 297 mm) 4661 1 7

Α7 Β7 Μ Printed by the Central Laboratories of the Ministry of Economic Affairs of the Central Bureau of Standards and Labor Cooperatives Fifth, the description of the invention (4) is at the maturity period of -sheng # 1, and normal cell evolution is still a very important factor for health. This puppet, for example, is clearly seen in the importance of proper blood regulation of the blood-forming and reproductive organs. In some cases, when normal cell cycle evolution cannot be carried out, it often causes a devastating film to the organism. This abnormality, for example, can be seen in various forms of cancer, because undetected and uncontrollable cell evolution occurs through the cell cycle, from the completion of mitosis to the period between cell divisions and back to mitosis, In many cases, tumorigenesis is a life-threatening event. Therefore, sometimes attempts have been made to limit the cyclic evolution of uncontrollable cells to treat or control tumorigenesis. According to some of these hypotheses, hereditary retinoblastoma may be caused by the retina_ germ cell precursor, which carries a defective dual gene that is also trapped by the survivor of the somatic mutation, but the non-hereditary case is Two individual mutations are required in the same cell. The latest indirect evidence supports the existence of such cancer suppressor genes in retinal embryocytomas and K. pelvic sarcomas, also known as renal blastomas, neuroblastomas, and osteosarcomas. The frequent abnormalities of chromosome 13 in the chromosome sister of tumors and the attenuation of esterase D activity in tumors have shown that the RB restriction involves non-hereditary retinoblastoma cells (Cancer Genet. Cytogp. Net. (1983) 10: 3 1 1 -333; Cancer Genet. C.ytogfinp.t., (1 982) 6: 213- 221. It has been proposed that the inactivation of the two antigenes of the RB gene located in the region I3ql4 'can cause retina Germ cell tumors. This proposal—partially—is based on the case of a hereditary retinoblastoma, two of which are presumed to be non-existent based on dual genes (Science (.1983) 219-973-975). But (谙 先Note on the back of the reading. The gray world fills in this page.) Paper selection, the standard is applicable to the national standards of country X. ": .. 210X.297 mm.) 4661 彳 7 A7 B7 V. Description of the invention (Central Bureau of Standards, Ministry of Economic Affairs) Printed by Industrial and Consumer Cooperatives. Yes,-the assumption on which this proposal is based, that is, the loss of acetase D activity means that the loss of esterase D and RB genes has been overturned (Hum. (1987) 76: 3 3- 40) However, other findings show that chromosomes that are heterozygous in somatic cells are labeled 3 * in the retinal embryo. Tumor tumors often become homozygous or hemizygous. Therefore, isozygous deletions occur in regions 13 <? 14 of 3/3 7 retinoblastoma cell sources (Mature (1983) 305: 779-784; Proc. Hatl, Acad. Sci, < 19δ6) S3: 739 1-7394. These experiments provide evidence that the RB gene mentioned does indeed act in a sensible manner on the cell surface (Science (1987) 235 : 305-311; Cancer Res. Π 9 8 6) 46: 157 3-〗 580), which is different from the "dominant" activity of the Fu Tuo tumor genes measured by the M-migration infection test (S cie in Ce (1985) 223: 669-B76; Nature (1985) 315: 190-195). Both forms of retinoblastoma are now treatable, and most patients reach adulthood. However, patients with hereditary retinoblastoma have a high risk of developing a second non-globus malignant tumor (Ophthalmology (1984) 91: 1351 ~ 1355; Hew Engl. J. Hed (1971) 2δ 5: 307-3 Π; Cancer (1 974) 34: 2077-2079, with an incidence rate as high as 90¾ within 30 years of initial diagnosis (0 ph t. K a] mn] 〇gv (1984) 91: 13 136 136). Most Frequent secondary cancers are usually osteosarcomas. Rather, cured non-hereditary retinoblastoma patients show the same chance of cancer as ordinary people. This finding is very interesting because it suggests that the RB gene may be regulating Other tumors have a critical task .. Human retinoblastoma genes have been successfully cloned, identified, and sequenced. 8 _Feng Su ^ Beiguo Jianjia Kneading Rate (CNS) (210X 297), read first Note on the back Page 4 6 61 1 7 A7 B7 V. Description of the invention (6) Printed by the Central Ministry of Economic Affairs of the Ministry of Economic Affairs and Consumer Cooperatives 4 (?) (Science (1987) 23 5: 1394-1399). Retina The blastoma gene is located in a region very close to the esterase D gene 1 3 q 1 4: Chromosome 1 3 | has also been identified, selected and sequenced (Proc. K ati. Sci. (1986) 8, 3: 6790-6794: Pore. Hat 1. Acad. Sci. (1 986 ) 83: 6337-634 1; Proc, Nat ί, Acad, Sci, (1986) 83: 6 337-6341). Through the migration of chromosomes from the esterase D gene, the deletion of the same zygote according to the position of the chromosome Tumor-specific mutations during expression • Identified retinoblastoma (1ΪΒ) gene. The RB gene shows a messenger RNA (mRNA) with 47 23 nucleotides and a code of 4.8 kilobases (kb). It is found that the transcription of RB DNA to RB mRNA is not normal in blastoblastoma patients. It is not completely impossible to measure the transcription, indicating the absence or complete inactivation of the RB gene * or the size of the transcribed mR NA molecule is reduced by about 4,0 kb, which indicates a defective RB gene. Sequence analysis of RB complementary DNA (cDH A) clones * yields a single long open code, which indicates that it can encode 816 amino acid hypothetical proteins. Computer-assisted protein sequence data Search for Inventory Proteins That Did Not Show Close Correlations The unique limbic acid sequence (Science (19 8 7) 2 3 5 · 1 3 9 4-1 3 9 9. The predicted protein appears to have several proline-rich regions, similar to those observed in other nuclear tumor gene proteins such as the proteins "in yc" and "myb " (RH A Tumor Vircuses, Cold Spring Harbor Laboratoyr , Cold Spring Harbor, New York (1985)) ° Also relevant to the present invention, in the development of higher organisms, such as moving 9 wheat paper standards are applicable, Zhongguo Jiajia standard: India NS) :: A4 size (210X297 mm:

4661 17 A7 B7 _ ^ _ V. Description of the invention (7) Object and-man-made, orderly cell cycle evolution is a key factor. Due to this evolution, sequential and systematic cell differentiation occurred, and eventually developed from a single undifferentiated cell into a highly structured organism with various special tissues. In a nutshell, the cyclical evolution of a physiologically normal girl cell is very important to the organism, not only during the early growth period, but also throughout the life cycle of the organism. Therefore, even when the organism has reached maturity, normal cell evolution is still a very important factor for health. This puppet, for example, is clearly seen in the importance of blood formation and proper cellular regulation of the reproductive organs. + Under certain circumstances, when normal cell cycle evolution cannot be performed, it often causes devastating effects on living things. This abnormality, for example, can be seen in various forms of cancer, because unhydroxylated detection and uncontrollable cell evolution occur through cell cycle and cycle, from completion of mitosis to cell division and back to mitosis In many cases, tumorigenesis has become a life-threatening fact. Therefore, sometimes attempts have been made to limit the uncontrolled evolution of the cell cycle to treat or control tumorigenesis. Printed by the central standard of the Ministry of Economic Affairs, the Shellfish Consumer Cooperative. Under the current circumstances, violent treatment measures such as radiation therapy and chemotherapy are used in areas where tumors are developing. Both of these methods are very expensive and in some cases___ cause great damage to living things. Chemotherapy, for example, can cause patient death, and when chemotherapy successfully controls tumor formation, it can have long-term side effects on the body. This kind of side effect can cause early heart problems in patients. Therefore, although traditional treatment methods, such as radiation and chemotherapy, can effectively destroy some uncontrollable cells, they can also have the undesired effect of destroying useful cells and weakening the organ system-10- Use Shi Guoguo to raise fCNS : :) 囔 4 ^ (110X297 mm. V, 4 Guang No. 83 ^ 1 1748 patent application 6 6 彳 Ί * 7 in 1T sharp bright nuclear TFW fS5i 钜 11 members 1 A7 B7

V. Description of the invention (Printed by the Shellfish Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs. Looking at the foregoing points, it is very necessary to have the ability to regulate malaise, such as tumorigenicity | and also to hold the cell life composition at the same time. In addition, it is also very much needed This kind of technology and sister compound have little or no side effects, and do not cause the development of tumour formation that cannot be stopped at the cellular level. The obvious advantages of this novel technology and sister compound are that it can regulate undesired cells in the case of swelling. Proliferative effect, and other cancer treatment procedures are occasionally applied. Therefore * use this type to avoid the destructive effects of futon radiation and chemotherapy but can work. Therefore, patients can maintain physical strength and vitality κ caused by medical surgery, can be used more safely Summary of the Invention The present invention provides the ability to prevent or inhibit the proliferation of diseased proliferating cells due to the lack of a functional cytoma protein or polypeptide in the cell. In this method, the cell needs to be contacted with a membranous blastoma protein or polypeptide. The method penetrates functional retinoblastoma protein or polypeptide, and also treats retinoblastoma or secondary cancer Brief description of the diagrams Figure 1 is a historical analysis showing that K rabbits identified proteins by immunoprecipitation in various cell sources. Human 塍 cells such as the god LAN-1 (lines 1 and 2) * Alexander Hepatocellular carcinoma (3 Line U20S (line 4) · Normal fibroblasts (line 5). The technology of Wang Zidi's evolutionary ability and the change of the cell to change the cell. Under the terminal tumor formation effect, the technology can reduce or discontinue when sufficient. Swelling technology * such as exogenous effect, in which a small effective dose of retinal retinal embryos can be administered to the patient can prevent or prevent anti-RB IgG (blastoblastoma), osteosarcoma and 5 kinds of retina—— ,,--V (Please read the notes on the back # .Fill in this page first) 11 The paper size is applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 4 6 61 1 7 Ministry of Economic Affairs Printed by the Consumer Standards Cooperative of the Central Bureau of Standards i: · A7 .__ B7___ V. Description of the invention (9) Embryo fines (lines δ to 10) Right "S-Methionate labeled rabbit IgG immunized in childhood (line 1 ) Or rabbit anti-rb. [GG (lines 2-10) for rabbit epidemic sinking. M 7 * 5% SDS-PP Gel electrophoresis and autoradiography analysis-immunoprecipitates. Figure 2 is the complete RB c D M / \ nucleotide sequence and the putative amino acid sequence of the RB protein. This protein is designated as ppRB110 or pllORB. At most The 5 ′ about 240 nucleotides are cDNA clones derived from retinoblastoma cell source Y79. The nucleotide sequence obtained from this clone is aligned with the original normal rb clone M sequence. The first and second starting positions are framed, and the alanine and proline groups are underlined. Figure 3 is a chromatogram showing the modification of the RB protein. LAN-1 cells M 3SS-methionine ( Lines [[-3]) or 32f > -phosphoric acid (0.5 taci / ml) (lines-4 and 5) are marked for 3 hours. The lysates of the cells were precipitated in rabbits immunized as a child 1§0 (lines 1 and 4) or anti-! ^ 180 (lines 2, 3 and 5). An entire portion of the 36S-methionine-labeled RB protein was digested with endonuclease H (ICH ImmunoBiologicals) overnight (line 3). Such immunosuppressants were analyzed on a 7,5¾ SDS-polyamidamine gel as described in Figure 1. It was found that the R B gene product was acidified but not glycosylated. Drawing 4 is a crumb analysis spectrum, explaining the retention of RB gene production in different vertebrate species. Human neuroblastoma, LAN-5 (lines 1 and 2), monkey COS, (lines 3 and 4), quail fibroblasts, QT6, (lines 5 and 6) (mouse fibroblasts, NIH73T3 ), (Lines 7 and 8), and mouse fibroblasts' rat-2, (lines 9 and 10) The cell source is labeled with 32P-phosphate and then-ISG (odd line) or anti-immune -RB UG (even-numbered row) immunoprecipitation -12-: .p Liyan, 1661 1 7 5. Invention Description (i〇) A7 B7 Printed by the Shellfish Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs and analyzed according to f. Similar but different sizes of RB proteins were found among different spinal animal species. Figure 5A is a chromatogram showing the localization of proteins. 3SS-Methionine-labeled UN-1 cells. (Line 4) fractionated to cells_ (line 1), cytoplasm (line 2) and nucleus (line 3). 'Protein M anti-pRB110 IgG immunoprecipitation. Immunoprecipitate SDS-P AGE analysis as described in Figure 1. Fig. 5B is a chromatogram showing the results of immunofluorescence studies of RB protein localization in osteosarcoma-derived U20S. The cells reacted with U) anti-ϋΒ I sG and (Π) immunized rabbit IsG in childhood. The most fluorescence is found in the nucleus. A column chromatography photo of the RB gene phosphoprotein in single-stranded cellulose «Protein lysate M 32P-phosphate metabolism marker of human neuroblastoma cell LA Ν-1 and passed through a single-stranded DNA column 'KNaC 丨Gradient analysis (line 3 = 0.05; line 4 = 0_1; line 5 = 0.2; line 6 = 0, 3; line 7 = 0.5; and line 8 = 1.0 concentration NaCl). The first line shows the total cell lysate of immunoprecipitated with anti-IsG. Figure 6B is a column chromatography picture of the RB gene phosphoprotein in double-stranded DNA cellulose. Human neuroblastoma cell LAN-ΐ protein lysates are labeled with 32P-phosphate metabolism and passed through a double-stranded DNA column and then M NaCI to increase gradient isolation (line 3, line 4 · τ = 0.1; line 5 = 0.2; line 6 = 0.3; line 7 = 0, 5; and line 8 = 1.0 浓度 concentration NaCl). The first line shows the total marital lysates immunoprecipitated with anti-RB UG. Figure 7A is a diagram illustrating the production of a TRP E-RB fusion protein. EcoRl -13- The size of this paper is free from the Chinese Gu family, and the quasi-body secretary is jealous from 21 × X to 97 mm) srl — I n ---- ______ τ__ (# 先 闻 读 的 后 事事 ## (This page) I. 鲫

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I 4 6 611 A7 B7 Printed by the Shellfish Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5 'Inventory (u) -EcoRl cDN / \ RBH (0.7 kb) fusion constructs to the E co R 1 position of the pATH3 vector. From the detailed restriction map of each position, its arrangement was confirmed. The plastids were re-transformed into Escherichia coli mm294. Fig. 7B is a photograph of a polyacrylamide gel electrophoresis of a heavy body TRP E-RB fusion protein. The major plastids were transformed into E. coli m 2 9 4 and grown in M9 minimum nutrient requirements medium supplemented with 20 mg / 20 ml tryptophan. The culture was diluted to 1: 100 in M9 plus prion amino acid and ampicillin. Enzyme. At a light density of 0, 2 S00 nra, a 10: 1 mg / ml indolyl · acrylic acid 1: 1000 dilution in 100Si ethanol was added to induce TRP E gene expression. Bacterial cells were pelleted, and the bacterial cells were boiled in Laemmli gel sample buffer for 15 minutes, and then analyzed by polyacrylamide gel electrophoresis. The gel was stained with Cox's blue. The 58 kD protein was found in the induced culture (line 2) 'but not in the control culture (line 1). Figures 8A and 8B show immunosuppressants of RB protein from various cellular sources. Figure 8A is a chromatogram * showing immunosuppressants of RB protein / anti-RB protein U * D from various human cell sources. Prepared from human hepatocellular carcinoma Alexander cell source (line 丨, line), human osteosarcoma cell source, U 2 0 S (line 2_), normal-ordinary human dystrophin (line 3), human neuroblast Cell tumor source, LAN-5 (line 4), and rabbit IsG immunized in childhood immunized with 35S-methionine-labeled details of human neuroblastoma lysate (line 5) · Cell extracts were immunoprecipitated with passivated rabbit anti-RB IgG. One pair of bands with a surface molecular weight of about 110-114 kD was observed in lines 1-4. -14- --------- Install ------ Order ---- (谙 Read the note on the backϊπ #, fill out this page) M: 466117 A7 B7 V. Description of the invention ( 12) Figure B Lu chromatogram showing immunoprecipitation of RB protein / anti-RB protein UG from several retinoblastoma cells. Cell extracts from 5 different optic retina embryos and cell tumor cells were labeled with 3 2 S -methionine, and then immunoprecipitated from purified rabbit anti-O I g. One pair of bands appeared in one of the cell sources from liver cancer, osteosarcoma, fibroblastoma, and neuroblastoma, but not in all five retinoblastoma cell sources, RB 355 (line 1), Y79 ( (Line 2), WERI-1 (line 3), WERI-24 (line 4 >, and WERI-27 (line 5), human orthoblasts and cell-derived LAN-5 (line 6) And neuroblastoma lysates precipitated from rabbit UG immunized by K in childhood. The RB protein was identified based on these results. Figure 9 is a chromatogram showing the biochemical fractionation of K. The effect of RB protein was demonstrated by 3SS "A All thioic acid-labeled LAN 5 cells (line 4) were fractionated into the cell membrane (line 1), cytoplasm (line 2) and nucleus (line 3) and then K rabbit anti-rb igG immunoprecipitation. Large Most of the RB11〇_114 proteins are found in the nucleus, and a few appear in the cell membrane or cytoplasm. Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs--^ ϊ. M-n ^-II. 窣 (Please read Note on the back Mi write this page) Figures 10A and 10B describe the South and West 〇ΝΛ knot Tested. 6 micrograms of purified TRPE-RB fusion protein, M and purified baculovirus-expressed pp110rb were added to 10 «SDS-PAGE. Figure 10A illustrates the addition to 10 ¾ SDS-PAGE, Coomassie blue staining The fusion protein and baculovirus watch gauge p110m south-westward DHA binding test. Coomassie blue was cultured with 3 2 P-labeled DNA fragments and analyzed by autoradiography. The display is as follows : Line 1: RB19-22; Line 2: -1 5-, ί «£ " Weng Γ .: 4 6 6117 A7 B7 Printed by the Shellfish Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs ), RB2 3 ~ -27; Line 3: RB19-27; Line 4: Purified RB protein from AcNPV_Y4 RB-infected insect cells. Figure 10B is from the parallel gel to the coagulation used to generate Figure iOA. The self-explanation spectrum of the glue smear. 巳. It is proved that the fusion protein RB19-27, which contains the main functional site that interacts with DNA, has a 20-fold higher affinity for DNA than the two subregions RB19-22 and RB23-27. In this regard, compare line 3 and lines 1 and 2 of Figure 10B, and also show the purified full-length protein (Figure 10B; line 4 ). The DNA-binding activity of purified RB protein from insect cells can also be confirmed by the retention of protein by DNA- fulvicin and subsequent isolation from the column with about 400 millimolar NaCl plus M. Figure 11 is the layer The analysis showed the complex formation of RB protein (ρ110βB) and SV40 I-antigen expressed by baculovirus. In a test tube, i.e. in vitro, the inactivated full-length RB protein is mixed with purified T-antigen. The same mixture was whole and then immunoprecipitated with PA Β 41 9 (line 2) or anti-i? B0,47 (line 3), and analyzed by western blotting. Lines 1 and 4 show purified SV40 T- Antigen and PAB419 immunoprecipitation * RB protein expressed by purified baculovirus and anti-RB0.47 antibody immunoprecipitation ° * Co-immunoprecipitate of protein and PAB419 produced by mixing protein and SV40 T antigen in vitro (line 2), Co-immunoprecipitates of M and T and anti-RB0.47 antibodies (line 3). Figure 12 shows the translocation of the nucleus of the purified protein after microinjection into the cytoplasm of Soas-2 cells according to M. MSDS-PAGE analysis of protein preparations used for microinjection. Line 1: From the HBsAg virus susceptibility to RB 16- ---------- U ^-(谙 First read the note on the back Fill out this page). P.

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V. Description of the invention (u) .. The stained cell is named p 110 R β; line 2: biotinylated rabbit anti-sheep antibody; line 3: anti-RB.495 antibody; line 4: Anti-RB R2 antibody; line 5: anti-RB.47 antibody; line 6: sister weaving HI (histone); line 7: from the large intestine. Bacillus? 56〃. One microliter of each sample was applied to a 15¾ acrylamide gel. Coomassie stained gel. The position showing the quasi molecular weight is measured in units of M thousand channels. Figure 13 SDS-PAGE analysis of the concentration and purity of protein preparations used for microinjection. Figures 14 to 14F are micrographs; microinjection and immunostaining of labeled Saos-2 cells. Figure 15 shows the percentage of labeled cells up to 4% after injection. Figure 16 shows the relationship between the percentage of labeled cells and the concentration of β> 56 in Figure 16K. Figure 17 > 561 ^ Effects of injection on cells in phase 3. Figures 18A to 18C are graphs showing the dependence of the cell cycle on the period of truncated protein injection. Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs J -------- install-- (please read the notice on the back of this page first) Defective small cells and non-small small cells. Growth of lung cancer cells. What is the result of this experiment? 11〇1 * 8 can inhibit the proliferation of NCL-H596 (HSCLC; Rb negative). Mouth 11〇 * ^ has weak activity on A5 49 (NSCLC; Rb-donor) tumor cell source. P110rb used in the present invention is derived from baculovirus (B-Rb; modified as described by Cell Growth (1990) supra) or from E. coli (E_Rb). The control protein used was bovine serum albumin (BSA). Appears in the growth medium 4 6 61 1 7 Α7 Β7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs. 5. Description of the invention (15). The above-all grandfather proteins are 25 μg / ml. Figure 20 is the result of the time course study of the inhibitory effect of R B -media. 3 Η-thymidine incorporation is manifested as buffer control + /-S.D. percentage. Figures 2A through 2D show cell uptake and cell nucleus localization at 12 s I-ρ 1 10 β 8. Figure 2 Α shows the time-dependent localization effect of 12 M-ρ 1 1 0 R Β on each cell fraction. Figure 2 Β shows the immune killing and identification of 1 2 5 I-ρ 11 0 R Β in the nuclear part of N C 丨 -'H 5 9 6 cells. Shows molecular size standards. Line 1: 0 hours; Line 2: 1 hour; Line 3: 48 hours; Line 4: 12SI-protein standard. Figure 2C shows the immunodepletion and identification of 12 M-p 11 0 R B in the nucleus of lung cancer cells. Line 1: NC Η 5 9 6 NSCLC (R Β ηβ *); Line 2: Δ549 HSCLC (RBP ° S): Line 3: MRC-9 normal lung epithelial cells (RB p ° 3); Line 4 Row: 1 2 5 p 1 1 0 RB protein standard. Figure 21D shows nucleus-localized quantification of 1251 in NC1-H596 KSCLC cells. _ Cells were harvested at 0, 8, 24, 48, and 72 hours to quantify the nucleus 12S. [-P110SS (_) or 125I-p56rb (click). Figure 22 shows the injection of RB protein around the tumor on the growth of subcutaneous lung tumors in nude mice. Fig. 22A shows the NC-H596 tumor size shown as the mean ± SD of 3 animals in each experimental group. The tumor size (mm2) on day 87 was: pllORB. Processor: 169, 13 0., 100; p56RB-Handle L -------- ": :) Packing-(Please read the notes on the back first and fill in this page privately) Order _ Paper size applicable to China National Standards (CNS) A4 Specification {210X297 mm) V. Description of the invention (16) A7 B7 Central Standards Bureau of Ministry of Economic Affairs-Industrial-Consumer Cooperation Du Maker: 400, 360, 252: Buffer processor: δ58, 420, 770 (ρρ 11 0 κ Β, (Yu) -P 5 6 R Β, (▲)-Yuan rinse solution control sister. Figure 22B shows the A549 (RBP ° S) tumor size shown, which represents the mean SD of 3 animals in each experimental group. The tumor size (mm2) of individual animals on day 87 was: p ll0RB-processor: 120, 136, 1 18; p56rs- processor: 18, 132, 121; Subject: 11〇, 1 39, 126. (·) -ρ110κβ, (Lu > -p56Re, (▲) -buffer control group. Figure 23 shows that the injection of p 110 RB into the veins of human lung tumors in nude mice Effect of cell growth. Active P 1 10 RB, 50 μg / dose (significant); active p110rb, 200 μg / dose (Δ); or inactivated p110RB 200 μg / dose (0 1. The values are average values (± SEM). The arrows show the number of days of scavenging. Figure 24 shows that p 110RB can inhibit the growth of non-small cell lung cancer cells in vitro, but has no effect on the proliferation of normal cell sources. PllKB It is prepared from E. coli strain BL21 and contains a T7 promoter gene (described in S. Huang et al., Infra-) that drives the full-length PnORB epitope. It is described according to Cell Growth and Diff. (Γ990) 1: 429-437 The experimental shovel was pure Pi 10RB. In the experiment shown in Figure 24, p110rb was added every 8 hours during the test period to culture the cells. 3H-thymidine was added overnight in the last 8 hours to measure cell DNA synthesis The type of cells used for this test was purchased from American Type C Collection ulture C ο 1 1 e c t i ο η. Including: Η 5 9 6 (R b -negative; non-small cell lung cancer), MRC-9 (Rb-positive; normal lung epithelial cell source), wi-38 (Rb-yang 19 + paper size applicable to Chinese theorists 々 参 々} A4 size (21〇χ297 public waste 1- nn pm ^^^ 1 Bn tn ^ v ^^ 1, 1, 豸 (please read the note on the back first, fill out this page) I. Order P + —Γ 4661 1 7 A7 B7 V. Description of the invention (17) Sexual, ugly lung epithelial cell source> CCD 597 SK (Rb-positive, normal foreskin epithelium cell source), FS Η 7 3 8 BL (R b-positive; normal bladder epithelial cell source), .HI Η 3 T 3 (R b-positive, non-transformed mouse cell source). Figure 2 5 shows that p 110 R β inhibition is the smallest defect in R b protein expression Growth of cell and non-small cell lung cancer cell sources. The results of this test showed that p 11 〇β β can inhibit Η 1 2 8 and Η 6 9 cell sources (both R b, negative) and Η 5 9 6 (NSCLC; R b-negative). ρ I 1 0 s Β has weak activity on A 5 4 9 (R b-normal) tumor cells. p110rb used in this experiment is derived from baculovirus ( B-R b; according to this patent specification (Purified) or from E. coli (E-Rb). The control protein used is < Bovine Serum Albumin (& SA). All proteins appearing on the growth medium are 25 μg / ml. The test otherwise follows FIG. 24 The description proceeds. Figure 26 shows that p U 0 κ B can enter human cells and localize to the nucleus. The 11 2 s-labeled p U 0 R β shown enters the cell in an inter-M-dependent manner and Localized in the nucleus. Cells were fractionated as described in Nature (1 9 8 7) 3 2 9: 6 4 2-6 4 5. Figure 27 shows that intact p110R3 can be used in H596 tumor cells treated with Ila5-labeled pllOfcB® Measured in the nucleus. This figure shows that when the nucleus part (according to the system I bid) was detected by denaturing SDS gel electrophoresis and autoradiography technology * full length p 11 0 was seen in the nucleus after 4 8 hours of culture RB. Iodized p 11 0 RB is used as a marker for autoradiography. Figure 28 shows that p 1 1 0 RB and p 5 6 RB are biologically active for subcutaneous treatment of non-small cell lung cancer in animal phantoms. Two types Proteins were prepared from E. coli BL21 as described above using the purification method described in this patent specification. , 100 -20- W This paper is used for half a country (CNS) A4 size (210X297). Order

C Consumers' cooperation with the Central Bureau of Standards of the Ministry of Economic Affairs of the People ’s Republic of China Du Yinzhuangdi 83111748 Patent Application 4 6 6 Ί U Specification Revised Page (August 15, 1990, Description of Invention () · _ YEAR " ff1 Microgram? 110 * ^ Or? 56111 »is administered subcutaneously to the tumor cell area daily. Both proteins have biological activity as demonstrated by circle. Although p56rb has weaker activity than? 110 * 8, this experiment did confirm that the p110rb fragment was biologically active. Figure 2-9 shows non-intestinal therapies for lung cancer using pllORB in an appropriate chess body. 200 micrograms of pllORB was injected via the tail vein to Balb / c nude mice with subcutaneous H596 human NSCLC tumors, which were injected three times a week. This dose is at a dose of about 1 g per kilogram of human radon. The experiment was performed over a period of 1 month as shown. During the treatment period * the untreated fish was growing rapidly and the treated tumor did not grow or K Very degraded growth rate. Figure 30 shows the amino acid sequence of the retinoblastoma protein produced in E. coli. Compared with circle 2, when white matter is produced in E. coli, it is convenient for colonization. 2 Amino acid (proline) was changed to alanine. Draw 31 to show the structure of a baculovirus expression vector for pp110rb synthesis-> Building. 'Pumps 32A and 3 2B are westward smears of ppRB infected insect cells (Figure 32A) and smearing of the identified cell extract 0 The cell extract is from infected cells 72 hours after infection. Printed by the Shellfish Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling out this page) ) 圔 33 is the result of self-emission spectrum analysis of RB protein release and dephosphorylation analysis of RB protein released in insect cells. Detailed description of the invention The main purpose of the present invention is to provide a universally safe but specific treatment method and product, Can be used to control the evolution of cell cycle in vivo and control the inhibition of cancer 0 ... -21-This paper size applies to Chinese national standards (CNS > A4 size (210X297 mm) 4 661 1 7 A7 B7 9 IT / V Explains that the combination of the hair and the French side of C. Meihua has merged the whole cycle of the Anxun donor to refine the internal objects and objects i. . Can be used for its technical skills and legal prescription Can be used as a supply to produce a tumor is the tumor's swelling-the internal and external body material is stagnation of hair growth%, and the law of France and France and the physical and chemical properties used to make the system is combined with cancer 'system In addition to the control of root supply and control, the tumor is swollen and cancerous to Ming Dingfa. This is the disease or cancer for thin interstitial cells, and the treatment of sexual pitting and defect is provided by the supply layer. The cell is a fine confession. It should be clearly stated in the list of items that are / are the other parties of the I matter method. The essence of the issue is ^, the hair substance is the same as the drug 0. The combination of the physical and chemical properties of the physical and chemical properties of the physical and chemical properties of the physical and chemical products is based on the basic ingredients. The white egg is inhibited by the basic ingredients. The cancer system has a special feature of the tumor, which is the confluence of the cells, and the method to complete the formula M. The treatment of the staining cells " sense is included in the package of Mingyu hair products. Confession of the cause of the need to provide the basis of the current method of the development of the treatment of the current indications of cancer, and the early release of the radioactive period is often transmitted to the non-low-lived patients can reduce their tumors-Technical Health Law Mingfa Fadan has been used as a price-cutting cancer-prevention system based on the use of the formula system. The facet is ten layers and the cells are thin and fine. The square is finished. The quality of the product is stupid and white, and the foundation of the egg is clear. For suppressing cancer (please read the notice on the back to fill in this page) Printed by the Consumer Standards Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs Although the opposite is excellent, I produced a whitening egg hair cause + base. The enlistees who entered Fana Fang are right, but they do n’t have the following points: “The new point is that there are some tumors that are swollen.” Mingsheng, a book made by the malignant cancer management department, said that for Li Yi, but the book is changed. The advantage is that "there is a completeness of the facial therapy layer cell carcinoma and the fineness of the cell therapy in the poisonous cell therapy system"

-T State China National Materials-r 'public 97 2 ~ x A7 B7 4661 17 V. Description of the invention (20)-It is the most traumatic. The present invention is a method of cell therapy, wherein a fixed tumor suppressor gene protein product is delivered to infected cells to complete tumor suppression. It is known that the defect of the cell may be the loss or the presence of a defective gene, resulting in a lack of protein in the cell. The method of the present invention relates to the use of a gene protein product related to a defective gene. Hydroxypurified protein products are delivered to infected cells to complete, for example, tumor suppression. The product is delivered in a suitable carrier on the exogenous drug, thus allowing the protein product to act on the cell, or sub-cell surface. As an example of this method | The RB gene protein has been delivered to cells that have lost or have a defective RB gene, which is a cancer suppressor gene. In the preferred case with SI cells, retinoblastoma, a rare childhood cancer of retinal development, is a prototype model that studies the role of old tumors. Based on the location of genetic elements associated with chromosome 13ql4 and evidence of its recessive nature, the putative cancer suppressor gene, the retinoblastoma tumor susceptibility gene (RB.), Was cloned. The gene contains 27 expression sequences, which are dispersed in a DNA gene of 2 million bases D N A, and in all normal tissues tested, it expresses transcripts of ibRN A of 4, 7 bases. Sequence analysis of complementary DNA-selected strains revealed a long opening code that encodes 928 amino acid hypothetical proteins. Use-against antibodies induced by a selected epitope, which is predicted from the RB cD NA sequence, the RB gene product has been identified as a nuclear phosphoprotein with a relative molecular mass (Mr) of 110,000-114,000 and has been named PPllORB ° In addition to retinoblastomas, the loss of RB gene function is also related to other -23- ~ This paper is used in Chinese National Standard (CNS) A4 (210 ^ 297 mm) (please read the back first) (Notes for filling in this page)

G Printed by the Shellfish Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs — ^^ -------- ^, equipment -------- Order ----- V ^ --------- Α7 Β7 466117 V. Description of the invention (21). Several kinds of preparations are related, including breast cancer, osteosarcoma, and prostate cancer. Small cell lung cancer. Recently, it has been shown that the retrograde transfer of human RB gene to the retinoid blastoblastoma, osteosarcoma or prostate cancer cells via retrovirus-mediated base transfer significantly inhibits several characteristics of its tumor phenotype, including ten tumors in nude mice. Generating effect, providing a direct proof of the tumor suppressive function of the RB gene. However, the molecular basis of this biological activity has not yet been defined. Until now, because this kind of protein is very scarce in cells, it is easy to get enough. Although this kind of protein hinders the interpretation of the biochemical properties of cancer control gene products, the mouth ’s biological function is blocked, and a cell therapy method has been invented to deliver Gene-specific protein products to cells with defective or lost genes. With the present invention, a gene product protein which is purified, intact, and biochemically active can be delivered to a defective cell with a therapeutically effective agent. The cell therapy of the present invention has a wide range of applications. The purpose of this protein product is not only to treat defective cells, but also to explain the gene functions that interact between genes at the cell level. A specific case is provided here, in which the retinal gene protein product, pp110rb, can be widely used in the treatment of eukaryotic cells with defective or lost RB genes. __ It was found that the purified protein can bind to DNA and form a specific complex with SV40 T antigen in a similar manner as real human 臈 pp110rb Μ. The immediate nuclear translocation effect of the microinjected protein further suggests the active nature of the purified gene product protein and its medical application. Another object of the present invention is to provide a cell cycle control composition and method. 5- / Install-(Please read the notes on the back first, fill out this page}-Order

Q Printed by the Central Laboratories of the Ministry of Economic Affairs, Shellfish Consumer Cooperative, 4661 1 7 A7 B7 Printed by the Central Laboratories of the Ministry of Economic Affairs, Male Workers Consumer Cooperative, V. Description of Invention (22). Its composition and method are similar to those used to control living organism Evolution of the inner cell cycle. Another object of the present invention is to provide `` a safe and effective method and composition that can reversibly terminate the evolution of the cell cycle in living organisms. Another object of the present invention is to provide two technologies that can be combined with medical methods Use M to arrest tumorigenesis in vivo. Therefore, the present invention includes a method and a composition for controlling the cyclic evolution of cells, which are adapted to regulate the human circulation to the cells to be controlled during the mitosis of the cells. This sister compound is selected from the gene protein product and the segment of the protein to reversibly reward the evolution of the cell cycle of the cell, but still maintain its viability. The hairpin protein fragments are unexpectedly and surprisingly soluble in low concentrations of glycerol, thus promoting their wide range of medical applications. The silly point of the present invention is that the evolution of the stagnant cell cycle can be reversed in a convenient and safe manner without causing harm to the organism. Therefore, the tumorigenic effect, for example, * can be controlled. Another advantage of the present invention is that the composition for controlling cell cycle evolution using M is very cheap and readily available. Another aspect of the present invention is that the sister compound used therein has little or no effect on healthy cells and can be used in conjunction with other cancer treatment methods. The compound and the method of the present invention can be used with regulating capacity and are physiologically compatible with other methods and equipment for regulating certain physiological processes in the body, such as the production of blood cells and the production of gametes. Detailed description of the invention Therefore, the present invention provides the effect of preventing or inhibiting the proliferation of pathologically proliferating cells 25 " L -------- U ^-(Please read the notes on the back first ^ write this page) Order

G ---- This paper Αdegree freely uses China's national ladder standards: —— ~ 661 1 1 Ί A? B7 Printed by the consumerism cooperation of the Central Bureau of the Ministry of Economic Affairs of the People's Republic of China 5. Production Description (25), of which i Morbid proliferation is the result of a lack of intracellular peptides or proteins. This includes contacting cells with omental blastoma tumor peptides or proteins. This is used to predict the proliferation effect. As used in this patent specification, the term "'suppressive effect of inhibiting the proliferation of the cell's replication effect. In a specific case, the absence of colony proliferation (apoptosis) or the target of another specific case of the cell, the reducing effect The special feature is the transformation of the target phenotype into differentiation. The loss of the pyrogenic or benign phenotype or polypeptide may occur in the retinal germ cell tumor (defined below), or the mutant insufficiency gene is insufficiently expressed. Pond degree._ The term " mutation " as used in this patent specification is defined as a different form or forced to be designated as normal. Gene deletion, single point deletion, addition, replacement and shift. All of this should Gene-like mutations are associated with retinoblastoma and retinopathy. The present invention also provides RB protein related persons with non-ocular malignancies, cancer, or swollen lesions. Treatment and prevention, for example, Patients with retinoblastoma with genetically susceptible diseases or cured have a higher risk than normal. They are the major cancers of the eye. This type of non-ocular major cancer is named by Mingte as "continuation" Retinoblastoma-related cancer, "tumor cell renewal "., As the disease prevention or inhibition of cell with an effective dose of retinoblastoma can -26-" means fine indicators, which slows for cell death. The cells in are self-malignant. Functional protein genes or dual genes are present, so they appear naturally common. The changes can be patients with secondary tumors of germ cell tumors that are generally at 1 3 q 1 4 positions, and their treatment methods. But I have never touched it. It is not suitable for the hair or retinal embryo.

Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs •, c :: i '• 4661 1 (A7 ______ B7_____ V. Description of the Invention (24) "Secondary retinoblastoma-associated cancer" includes cases with secondary testing, of which Two separate mutations occurred in each of the dual genes of 13ql4, causing pathological mutations in RB protein. Examples of secondary retinoblastoma or tumor spring include, but are not limited to, osteosarcoma, bone cancer, synovial sarcoma, breast Cancer, small cell and non-small cell lung cancer, bladder cancer, polycellular carcinoma, gastric cancer, prostate cancer, leukemia, neck cancer, fibrosarcoma, glioblastoma, swan neuroma, chronic lymphocytic leukemia, and acute epiphyseal Leukemia. At the clinical level, the present invention intends to provide preventive treatment and / or treatment of cancer, tumor or malignant disease, which is the result of RB protein lacking or pathological mutation in cells of tumor or cancer tissue. As known to the skilled artisan * cancer is defined as a disease of animals, and its special emblem is the uncontrollable growth of thin beer, such as leukemia, lymphoma, sarcoma, cancer, sarcoma, Metastatic growth, and extraneous diseases. Tumors are a cluster of cells due to abnormal proliferation. Therefore, the present invention provides treatment to stop uncontrolled cell growth in patients, and thus alleviate the symptoms of the disease or the disease. Cachexia. The effects of this treatment or preventive treatment include prolonging the patient's survival, reducing the number of tumor cells in the mass or reducing or reducing circulation. It is also included within the scope of this H is a combination of protein therapy and traditional treatment For the purpose of this invention, the terms "patient" and "patient" are synonymous, and are intended to include human patients as well as vertebrate patients such as mammals At the cellular level, the present invention provides a method for preventing or inhibiting uncontrolled cell growth associated with a lack of normal or wild-type R.B protein in the cell. -27-: Ben Qiao ^ 4¾Using Kang S Home Rubbing Rate ( CMS〇A4 ^ (210 × 297 mm) 46 61 1 7 Λ7 A / B7 V. Description of the invention (25) In other words, the RB protein loses the blastoma protein. Therefore, the present invention provides The method, in which the diseased omental germ cell tumor polypeptide or functional or biological activity * can be used to prevent or inhibit the disease when it is used for prevention. • The pathological proliferation of the party. Other cells that are in contact with the RB protein are also inhibited at the same time, and the cells that are called "pathologically proliferating cells" * are also present and the cells are diseased because of the metamorphosis. The solid line cells of this type of cells, psoriasis Cells, lung cells, meat cells, with signs of cancer. Tumor cells such as tumors, glioblastomas, small cell lung cancer, non-small cells, for example. The method of the present invention requires the contact of a tumor cell protein or a peptide. Functional retina caused by stalk dysfunction, please read the note on the back first. Printed by the Central Standards Bureau of the Ministry of Economic Affairs, Shellfish Consumer Cooperative, printed on the prevention or suppression of proliferation of omental blasts. It does not mean that the package is independent and then separated from the positive cases including thyroid leukemia, retinal breast cancer, lung cancer, and pathological effects. The result is no cell colonization. Controlling cancer or cancer are different preventions that cannot be controlled, but do not produce normal cells, but are not limited to cells, cells or germ cells, lung cancer, kidney proliferation cells, cells lacking cells > cells and peptides or 0-cells Contains a great deal of fear of cell type control. Limited to regulatory properties • Whether it is in retinal breast cell lymphoma leiomyomas, bone and flesh, bladder cell carcinoma, lack of proliferative effects, functionally effective opioids, protein in contact with growth or cells in growth or growth in tumors * And its autonomous growth energy mechanism. This category is not related to disease-like cells, forefront, colon cells. Sha means tumor, fibrosarcoma cell carcinoma, sensory neuroma, cells and effective amount. Contact with functional retinal germ cells in vitro or in vivo is acceptable. External body 2 8-law; this paper size is printed in the CNS in China (210X297 mm) A7 B7 printed by the Shellfish Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (26) -, i Patients take the cell sample and mix the retinoblastoma protein or polypeptide with the cells. Alternatively, the RB protein can be microinjected into the cell. Cell cultures of known species can also be used, especially to select proteins that are believed to be equivalent to the function of 1? B protein, such as β B protein equivalents. Alternatively, the peptide or protein can be added to the cell culture medium and then to the cell sample. In vitro methods can be tested with M to identify whether the protein therapy of the present invention can be used to treat cell growth or tumors beyond the control of a patient. If cells are inhibited in vitro, the examples provided below show a direct correlation between the in vitro and in vivo efficacy of the method. Or * The sample of suspicious sister can be injected into human or mouse. Wait for it to form a tumor in the animal. Proteins or peptides can be injected into human animals to identify whether the protein therapy can be used alone or in combination with traditional anticancer therapies. If effective, the same or similar course can be applied to the patient. Therefore, the successful use of the therapy at the cellular or tissue level predicts the medical use of the method in patients with hyperproliferative cells, such as tumors or cancer. After somatic mitosis is completed, at the end of the cell division phase, the cells enter the interphase, which may be a short period or a long period of time depending on various factors. Therefore, 'for example, after the cells of the neural group have differentiated and minted, the nerves can have a long interval. Traditionally, the intercellular phase is considered to have three phases: G1, during which cell growth occurs without DIU replication, S phase, during which DNA replication occurs, and G2, during which DNA replication is completed and the cell is ready to divide. As described in detail under this patent specification, the protein products of certain genes, or fragments thereof, can be terminated reversibly to terminate the G1 phase evolution * and has the ability to control evolution throughout the entire cell cycle. According to this -29- ί · m n--— ^ ϋ ^^ 1 .., ——-. N ί ... cl ·. (Please read the note on the back before filling this page) Order 0.

466117 A7 B7 V. Description of the invention (27) Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs .. Invention-I After entering the cell, it can be reduced to 0. There are discussions within the cell. Termination of interstitial injection. The dose of M treatment or protein cytoma is also related to this oil. It is contained in any oil. The control of the cell cycle of the Gan-Han cell is in its cell cycle, so that the cell cycle evolution is reversed. After a period of time, when the eosinophil was observed in the cell, the cell cycle was observed to return to the first stage below the interphase. The pathological bark in the organism, such as tumorigenicity, showed an accelerated undesired cell cycle. The following are tumor suppressor protein products, such as 1ΪΒ protein or Saos-2 osteosarcoma cells in the G1 phase. In cells, sealed cells do not cause toxic effects, and the method of the present invention can be used to treat diseases with or susceptible to cytoma-related diseases or malignant diseases, which penetrate proteins or peptides into patients . Therefore, it is characterized by the inability of cells to express functional retinal embryo fine lesions, and the treatment is through contact of cells with functional peptides. For animals with 1¾ omental blastoma tumor-associated lesions, the neo-peptide or protein is first mixed with the medicinal substance when the protein therapy described in the instruction manual is used in conjunction with the new internal contact. "Pharmaceutically acceptable crossbones" intends to include standard pharmaceutical carriers, such as sodium phosphate hydrochloride, sucrose, human serum albumin, Tween 80, and some cells in the interphase still maintain a fully degraded cell within the cell In some finer details, its fragments are used for cyanosis, the protein is reversible and the effect of over-injection of the visual network is effective. Including, but not limited to water, water, glycerol, please smell the notice of tit before firing it again, '\ This water page order!

C -30- -r This paper size is applicable to China National Standard (CNS) 7ι4 (210 X 297 mm > A7 B7 4661 1 7 V. Description of the invention (28): carbazide "Sodium carbonate and milk fb Substances, such as oil / water emulsions and various types (read the precautions on the back of the first and fill in this page) moisturizer. The term "in vivo" used in the description of this patent means to provide an effective dose to the patient RB peptide or protein can effectively prevent or inhibit the proliferation of target cells or the growth of tumors. The methods of administering the drug compound are widely known to those skilled in the art, including but not limited to intratumoral injection, Oral, intravenous or parenteral administration. Drug administration can be used continuously or intermittently throughout the course of treatment. The most effective way and dosage method is to be widely known by the artist, and because of the therapeutic · Protein or polypeptide, the purpose of treatment, the cells or tumors to be treated, and the patients to be treated. For example, to sharpen, the appropriate dosage range is from about 0.1 mg / kg / body weight to 10 mg / kg / body weight . / For the function of this patent explanation and clear book The retinoblastoma protein H is intended to include naturally occurring retinoblastoma protein * isolated from a human cell source as described in the examples below or a recombinantly produced retinoblastoma protein, as shown, for example, in FIG. 2 by c DH A Encoded. In a specific embodiment of the present invention, the retinoblastoma protein is designated as the protein province of PRB110. PRB11. The amino acid sequence is shown in Figure 2. In the Central Ministry of Economic Affairs The employee's consumer cooperative printed version also includes functional equivalents of purified retinoblastoma protein or PRB11Q protein produced by the elder sister. Functional equivalents are different from purified retinoblastoma protein or pRB 11 (5 The amino acid sequence of the protein has the ability to produce the same preventive effect. This type of equivalent protein can have additives, deletions or substitutions that do not significantly affect the ability of the protein to inhibit the proliferation of pupae. The method for identifying equivalent proteins is The above-mentioned in vitro test -31- · Paper 逋 逋 逋 Use Chinese national standard (TNSTi ^ specifications (210 X 297 mm),--~~ Economic Printed by the Central Consumers Bureau Consumer Cooperatives 4661 1 7 A? B7__ 5. Description of the Invention ('29). Trial. Phase proteins or peptides include purified retinal germ cell protein or PRB110 segment. Biologically active Examples of this segment include 'but not limited to, P56κβ, and the segments released from amino acids 393-772, 393 to 870, and 871 to 928 as shown in Fig. 2. The operation of the present invention is performed when the cell is an animal cell' For example, mammalian cells such as humans or mice are suitable. The appropriate patients are mammals, such as mice or humans. The above method may not be used to inhibit the proliferation of pathologically proliferating cells. It can also be used to prevent cell perception. This type of proliferation. For example, 'hereditary retinoblastoma's disease has the risk of developing secondary cancer as defined above. As known to those skilled in the art / who knows, the effective amount or dose for prevention can be the same as the therapeutic amount or Dosages are not the same | but can be determined by those skilled in the art. The present invention also provides a method for treating a disease caused by a patient's lack of a functional retinoblastoma or polypeptide, or a lack or mutation of a retinoblastoma gene. As described above, this method requires administering to a patient an effective amount of a functional retinoid blastoma cell protein or polypeptide. As detailed below, the RB gene protein or RB protein is an alternative phosphorylated protein. It has a surface-surface molecular weight of about 110 kDa to n6 kDa on SDS-PAGE. M is normal, i.e. non-transformed cells are present in various vertebrate breeds. In some cases, a pathological or lack of RB protein is the result of a mutation in the chromosomal position of the RB gene. In normal cells * RB protein is the transcription product of a gene located in the 13ql4 region. In the family case of retinoblastoma -32- This paper size is applicable to the Chinese family kneading rate i CNS Μ size (210X297 mm 1 (please read the note on the back first ^ fill this page) Order 1b6l 17 A7 B7 5. In the description of the invention (30),-("Hui ethnic bipedal vision_embryocytoma "] the sudden love of the gene's dual gene on the embryonic vision, eventually leading to retinal embryos in developing children Cell tumors. For one- or two-test versions of the disease without familial inclinations, somatic mutations that affect the dual gene dual genes must occur in the same retinal germ cell. See Proc. Hati · Acad. S ci. USA (1971.) 68: 820-823.®Yes, the present invention provides a method for the treatment and prevention of unilateral and bilateral retinoblastoma cell tumors caused by such mutations. RB protein is an alternative phosphorylated protein, which can be Affinity-purification in cell extracts, due to differences in protein phosphorylation, migrated to osmotic bands of molecules from 110 to 114 kDa on SDS-PAGE. RB. Egg. Phosphorylation occurs in silk Amino acid and threonine. (Ce 丨 1 (1 9 8.9) 56 : 57-65 and Oncogene Res 1: 205-214); This characteristic changes during the cell division cycle. The pattern of low phosphorylation is in the G of the cell cycle. And the pattern of dominating and highly phosphorylated in the G τ phase. Dominated in G 2, M and S phases. For a comprehensive study of the biological properties of RB protein, please refer to Biochemica Biophysica Acta (1993) 1 1 55: 4: 3-61. 1. Off-line consumption at the Central Standards Bureau of the Ministry of Fine Economy Cooperative printed (please read the note on the back f, write this page first) In the cell division cycle, the phosphorylation of RB protein is agitated. As the term "protein" in this patent specification is intended to include all kinds of protein or Alternative phosphorylation patterns. In some embodiments, more than one pattern will be used in the method of the present invention, and alternative patterns can be administered simultaneously. 0 is used for "functional retinal germ cells in this patent specification" Oncoproteins, "RB proteins" or "I? B polypeptides" are also intended to include naturally occurring retinoblastoma tumor proteins isolated from human or vertebrate cell sources as shown in the following examples. Paper ruler Degree Applicable to China National Standard #CNS (A4) (210X297 mm) 4 6 61 1 7 Α7 Β7 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (31) • Retinal embryos produced by quality or M Cell tumor proteins, for example, are encoded by c D N A as shown in Figure 2 in the prokaryotic and eukaryotic expression systems. In a specific example of the invention, the retinoblastoma protein is a protein designated P P R β °. p p R B 11. The amino acid sequences are shown in Figures 2 and 30. The terms "purified" or "'equivalently purified" used in this patent specification refer to a degree of approximation obtained through the experimental procedure described below. Purity can be measured by any method known in the art, such as by imaging or using a densitometer. Also included in this definition are functional equivalents of purified retinoblastoma protein (ppRB110) or pRB110, a protein produced by the sister-in-law. θ The functional equivalents used in this patent specification have egg's white matter with a different amino acid sequence than the purified zygomatic omental blastoma protein p P "" 11 (> egg white protein capable of producing the same preventive effect. Such equivalent proteins Additives, deletions or substitutions that do not significantly affect the ability of the protein to inhibit uncontrollable cell proliferation. The method for identifying equivalent proteins is as described above in vitro testing. The equivalent protein or peptide includes purified retinoblastoma protein -Or PllORB2fi segment. Several examples of this type of fragment are described below. The term "synonymous protein or polypeptide" in this patent " specification " is a product of recombinant expression defined as C & NA (as shown in the figure) (2) or a fragment thereof. The recombinant expression system is the stable transformation of the epidermal epidermal vector into a suitable host cell for the production of the RB protein epidermal body. The vector containing the DNA sequence, and the sequence is other sequences that can affect its gauges. The following are examples of several expression vectors. Although not shown All must understand clearly, but implied that this kind of paper, please read the note on the first page. The paper ruler is used for remote control. China Xingjia Junzhe (CNS jA4% 4M 210X297 mm wide: Λ 嚷 嚷 :: 5 4661 1 7 Α7 Β7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs. 5. Description of the Invention (32). The expression body must be able to replicate in the host organism as a gene accessory body, chromosome ΰ NA integration part or other chromosomes. In other words, the vector is a functional definition given that any D Η A sequence can influence the arrangement of the fixed D N Λ sequence in which it behaves as if it is suitable for a particular sequence to be included in the noun. System, refers to the transformation of infected host cells with a vector constructed using double-body DNA technology. The insertion of the vector or DNA can be microcellular transfer, retrovirus-mediated gene transfer, transformation infection, and cell fusion. The vectors and methods disclosed in this patent specification are applicable to the prokaryotic and eukaryotic and biological host cells of Guangfanyuan. It includes the definitions of basic technology, methods and equipment for molecular biology of the present invention. Physics textbooks are used as references. See, for example, Maniatiseta 1., Molecular CI ο ning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1982.) and Sainbrook et al., Holecualr Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, New York (1989) and various references cited in the document. In addition, the recombinant DNA method currently used by this artist includes polymerase chain reaction (PtR). The combination of oligonucleotide synthesis makes DNA sequence easy to regenerate ... DNA fragments up to about 6000 base pairs in length can be expanded exponentially from a single gene copy by PCR. In the present technology, two oligonucleotide primers of denatured DNA samples are cultured to guide the synthesis of DNA polymerase, each of which generates approximately double the amount of tritium. Splitting each cycle by changing the temperature promotes the denaturation of DNA strands, attaches primers, and synthesizes new DNA strands. Using hot DHA polymerase can eliminate the need to add new enzymes to each -35- I 1 I > Binding (please read the notes on the back first ^ ¢ 1 ^ write this page) S ·

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V n «l I Paper ^ Applicable to Chinese national standards: (3) 4 size T 210 乂 迚 7 male dragon) 4661 17 a? B7 Printed by the Central Laboratories Bureau of the Ministry of Economic Affairs and a separate consumer cooperative. 35) The key of the wedding ring, and thus the promotion of a fully automatic cycle can increase the subject matter of the national patent No. 4,683, 195, 4,800,159, 4, SS3, 202 by about 100 times. Therefore, the nucleic acid molecule (shown in Figure 2) or its use as a heavyweight of the RB protein. Restructuring the method of the invention. Appropriate cells for operation of the present invention are animal cells, animal cells such as human, old flat or mouse cells, milk, such as mice, monkeys, mice or humans. The above method can not only inhibit morbid increase but also prevent cells by M! Subject to this type of proliferation for cell growth. For example, the hereditary vision network defines the risk of secondary cancer as described above. This suffers. As is well known to those skilled in the art, the amount of prophylactic treatment or agent S is different, but it is easy to do so. The present invention also provides a method for treating a disease caused by a patient's lack of functional peptides, or lack of or mutation in the visual network. As mentioned above, this method requires the functional retinoblastoma protein to be sold or more. The following examples are described by M threshold rather than purification of the anti-R Β antibody and RB protein of the present invention. DKA expanded effect. 25 倨 sequence. PIC technology is US 4,754,065, and fragments can be inserted into expression vectors and purified in vivo and used in cells, for example, mammals. Appropriate patients are therefore fed with the proliferation effect of germ cells, and also used to prevent the disease of uncontrollable mesoblastoma. The method is particularly suitable for the effective amount or dose of such diseases. Decide. Capsule retinoblastoma or mesangioblastoma genes cause an effective amount of peptide to be administered to a patient. Clearly set limits. Color body 1 3 mark, in 1 3 q 1 4 -36- present 0 囷 for Α < X ο 21 • J- 々 '

4661 17 V. Description of the invention (34) .: U 岖 ^, I 3 Chromosome omentoblastoma gene and amino acid sequence of the edited sequence were identified. By using the ester-D c D N A selection strain and screening gene and c D M A stocks, the selection strain can be obtained. From this type of selected strains, 16 kb and 0.9 kb were identified in the human c DNA stock, respectively. C D.ff A heavy barrier selective strain RB-1 (as shown in Figure 2 Acid 100 to about nucleotides 1,700) and RB-2 (as shown in Figure 2 about nucleotides to about nucleotides 1,000). Later, another g clone RB-5 was also identified (approximately nucleotides 1,250 to approximately 4,75? As shown in Fig. 2). The fetal retina and placenta hybridized with 4.8 kb raRNA transcripts. In retinoblastoma samples, R β-1 selections detected abnormal mRNA transcripts or were unable to observe πίΝΑ transcripts. Later identified The RB-5Sf. Colony has a 3.5 kb insertion sequence, which gives the same results as RB-1 in ra RNA hybridization. Restriction enzyme analysis suggests that the RB-5 and RB-1 clones are in a 0.4 kb region. Again, the two together define a period of about 4.6 1 ^ 0 ", the size is 1 ^ > ^^^ transcript is close to 0 MProc, Natl. Acad, $ ci. (1977) 74: 546 3-5 46 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (please read the note on the back π # '. Fill out this page) 2 Deoxy-terminator method for breeding strains RB-1 and 0-5 Nucleotide sequence analysis yields complete c DNA sequences that are reconstituted. The M "cell selection strain eye 丨 one" method (Ibid) generates different deletion templates in single M13 phage selection strains, A sequence of more than Q 5% was obtained. The gap between the thorns is based on the extension of the primers in the double strand. The complete sequence identified by this method contains 4,523 nucleotides. The open code appears from the 5 ′ end to the base 2δδ8. There are several others down: Edge, _ This paper is inadequate, using the National Standard for Cattle (CNS) system of the cattle country {ηοχ297 mm) A7 B7 Central Standards Bureau of the Ministry of Economic Affairs, Shellfish Consumer Cooperative Seal 466117 5. Invention Explanation The (55) end-shoulder (¾ stop codon ° translated from the first methionine codon (chaeme 241) produces a hypothetical protein of 816 amino groups (size 940,000 channels). Section The second structure is A. Thiamine is at base 3 4 6. Since the nucleotide sequence surrounding the first ATG is not typical of other known mRNAs (Nucleic Acid Res. (1984) 12: 857-863) The designation of codons is considered to be temporary. The computer searched the protein sequence database of the National Biological Research Fund and found no strong homologues in more than 4,000 published amino acid sequences. However, some nucleic acids -Binding proteins and viral proteins show the highest homology score The weak sequence homology of several yeast-inducible RNA polymerases. The putative protein sequence contains 10 possible glycosylation sites. (CRC Crit. Rev. Biochem (1981) 10: 307-366) but does not No possible sites of membrane penetration were found (at least 20 consecutive water-repellent residues). The amino acid hydrotherapy chart shows a region of mild water repellency in the hydrophilic region near the putative amine terminal and carboxyl terminal. Two pairs of short amino acid sequences have been identified, and the metal-binding functional sites seen in nucleic acid-binding proteins by M are merged by cysteine and histidine (Science (1986) 232: 4 * 8 5-487). 54 amino acid regions from positions 663 to 716 contain 14 amino acids or about 2626 were also observed in the nuclear tumor gene proteins royc and myb (RNA Tumor Viruses (1 9 8 5) Cold Spring Harbor Laboratory Cold Spring Harbor) ° When the early significance of this type of observation has not been established, it has been suggested that the RB gene protein may be a nucleic acid-binding protein. But consider past discoveries and -.38-

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4661 1 7 A7: _____B7 V. Description of the invention (56) Sequence analysis of nucleotides of RB c ~ 0 NA clones confirmed the long-on code encoding hypothetical protein, which has the characteristics of DNA binding function. The original purpose was to identify and characterize RB proteins used as antigens to obtain specific antibodies and to measure their putative DNA binding. Protein antigens can be prepared by expressing fusion proteins in E. coli. In order to achieve this, Wei B. The fusion protein of T. sapiens and TRP E protein 1 should be fused in E. coli. From the 816 amino acids and about 98 kD molecules, although the hypothetical RB protein sequence data can 'build three pATH plastids that express " TRP E fusion proteins. The complete cDNA clones were divided into 3 parts and became 0.7 kb, 0.9 kb, and 1.S kb. These three fragments contain the coding sequence of the RB cDNA. The plastid pATO-0.9 RB is a 5 ′ 0.9 kb segment inserted into the EcoRI-EcoRI position of PATH3. Plasma PATH3-0.7 RB can be constructed by inserting the 0,7 kbH segment of the RB-1 clone to the PATH3 EcolU-EcoRI position, and inserting a 3.1.8 kb fragment into the Bglll-Bglll position of the PATH3 vector can construct the plastid PATH3 -1.8 RB. A detailed location map of the restriction enzyme positions determines its ranking. The Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs printed the recombined physique% ATH3-0.9.9RB, MTH3-0.7RB and pATH3-1 * 8RB and transformed it into E. coli mni294. The concentration in supplement M is preferably about 20 mg / M9 tryptophan is grown in medium with minimum nutritional requirements of M9. The M9 medium was diluted with casein amino acid and ampicillin. The culture mixture was from 1:10 to 1: 150, preferably 1: 100. J. Viro 1. (1984) 49: 132-141 describes the procedure for constructing a reconstituted constitution. .P r 〇c. NatU Acad. Sci. (1986) 83: 4685-4689 describes fusion at the restriction enzyme position (. ≪ < 1: 1., »; National Standards and Regulations ⑼ Love Private 4 Rules Building " r2i〇X 297 mm) * 4 G β 1 t 7 A7 B7 V. Description of Invention (57 Printed by Duan St. P * ATH vector architecture. Only one of the three PATH3 constructs, namely pATH3-0,7RB, represents the fusion protein. The maximum molecular weight of the resulting fusion protein is 57 kD. Since the molecular weight of TRYP E is known to be derived from the RB protein Fusion protein of 37, 2 〇k D of the white pupa part. The other two plastid constructs do not produce protein at all, and gp is TRyp itself. Because the RB selection strain contains many hypothetical protease break locations, it is not possible in E. coli The production of protein is not surprising, and it may be due to the instability of the fusion protein. Using the above procedure to fuse pATH3 and RBH segments, a large fusion protein can be prepared. Then according to Nature (1970) 227: 680-685 Procedure M Preparative SDS Gel Acetamide Gel Electrophoresis Purification Fusion protein was isolated with overnight extract and 'SDS, soluble acrylamide was removed from the dialysis. The protein was then concentrated. The purified fusion protein was used as the antigen in the production of specific anti-RB protein antibodies. According to Proc. Natl, Acad Sc i. (1986) 83: 6790-6794, a rabbit polyclonal antibody specific to 0 protein prepared by the procedure. Μ mixed with 2 micrograms of complete Freund's adjuvant , Preferably 10 micrograms of purified fusion protein (initial injection), preferably repeated injections of rabbits every 14 days, followed by additional injections of a medium amount of fusion protein in incomplete Fredezol auxiliary (repeated injection > complete Fredezo auxiliaries usually include antigenic emulsions in a mixture of saline and emulsifiers, in this case fusion proteins, like. For example, Ariacel A, in a 40-release testimonial from the Chinese version (Jun Zhuan; Huanyang Jiyi has jurisdiction over 21 297 public branches)) Please read the note of the back to the order I * 4 661 1 T A7 B7 V. Description of the invention (58) Mineral oil pops' Dead Mycobacterium Medium. Incomplete Fred The rib adjuvant is the same, except that it does not contain mycobacteria. Repeat the injection until the high-priced anti-melt protein is measured to react with τ RPE and fusion protein, which takes about 2 months. In order to increase only the RB determinant is identified Antibodies, Natl; Acad. Sci (195 1) 37: .5 7 5-578 · and Immunoadsorbents in Protein Purification, Scand, .J. Mmunol. (1976) Suppl. Prepare a .2 or more® affinity column as described in 3. At least one column is packed with TRP E protein, and at least one column is packed with fusion protein. Each column is properly pre-circulated. Antibodies were first isolated through a fusion protein-sepharose column, glycine buffer at M pH 2.3. The neutralized eluate was passed through a TRP E column several times for M removal. The antibody directed against Ti? PE was specifically guided. Purified antibody: "! IgGffCj 豊 is used for immunoprecipitation or immunostaining for the localization of RB protein. It is also useful for the diagnosis and identification of RB protein in human tissue samples. For the identification of RB proteins *, select several human cell sources that are known to have normal or mutated gene performance. L A N-1 Kobe blastoma cell source, normal human fibroblasts, human hepatocellular carcinoma Alexander cell source and osteosarcoma U20S marital cell source containing normal RB mRNA positive control sister. All such cells were obtained from American Type Culture Collection (ATCC) storage. Cell sources with predetermined short or no RB roRNA, such as retinoblastoma cell source Y79 (.ATCC) * RB355 (gift from Robert Philips Toronto; Canada), WERI-l, WEH-24 and WERI-27 (T. Sery Wills' Eye Hospital, Philadelphia) was used as a negative control group. All normal human cell sources mentioned above were labeled with 3 s S -methionine and obtained from -41- Miao Ben Zhi Tian JS: used in the Chinese country «JunSCNS} 210 X 297 public} ^ Han: '々Ψ & 4 6 61 1 7 Α7 Β7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs. 5. Description of the invention (59) All cells of 5 kinds of tadpole omental blastoma, and rabbit antibodies IsG or rabbit anti- RB IgG immunosuppression. According to the procedure described in J.V.i. 〇1. (.1981) 38: 1064- 1076, all human cell sources are labeled with 3 5 S-methionine metabolism ', and K 20 μl, preferably 10 microliters from 50 micrograms / ml to 200 micrograms / ml, preferably 100 microliters of anti-RB antibody UG, using the procedure described in J Virol, (19.81) 38: 1064-1076 to immunize the labeled cells Mixture. Protein pair analogs of Π0-Π4 kD molecular weight were measured in all control cell sources '. Proteins were not measured in retinoblastoma cell sources' or in cells immunoprecipitated with immunized serum in childhood. The RB protein of Μ rabbit anti-RB IpG immunoprecipitation was analyzed by SDS / polyacrylamide gel electrophoresis and self-radiated. The results are shown in Figure 1. In lines 2-5, the RB protein can be seen in an area of about 110 kD.琨, release the normal positive immunosuppressant, that is, the RB protein labeled with 3SS-methionine. Line 1 is the control cell source of retinoblastoma cells that immunoprecipitated by K immunogen in childhood, so it is not resistant -RB protein antibody, rabbit IgG. Lines 6-10 are from immunoprecipitation ~ 5 kinds Omental blastoma is obtained from 3SS-methionine-labeled cell sources. There is no RB protein in any of these cell sources. There is no antigen-detectable RB protein in retinal cells, which supports abrupt type. The tumorigenesis caused by the RB gene is due to the idea that the function of the gene product is completely lost, even in a cell source containing truncated RB mRKA. The hypothetical protein inferred from the nucleotide sequence is considered to have about 98 kD- 42- (Please read the notes on the back before filling this page)

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1 7 A7 B7 — One — 'Description of the invention (40) μ w. -The precipitation-free protein has a Mw ° complete R B amino acid sequence of about 110-114 k D is shown in Figure 2. This complete sequence was obtained from reconstituted clones and contains up to the 5'-end extension sequence not found in the original cDN A clones! Science U 987) 235: 1 394-1 399). The first and second starting methionines are framed, while the alanine and proline groups are underlined. Amino acid sequence (Figure 2 丨 written with a single-letter abbreviation code recognizable by this technique. RB ^ nhiAFg ^ il (Science (1987) 235: 1394-1399) contains nucleotides ΐ to 2 6 8 8 open The codon 'is translated from the first methionine codon, producing 816 amine' thio acids and a hypothetical protein with a molecular weight of 98 kD. Another isolated RB cDNA clone contains an additional 23 4 bases at the 5 'end Yes. The re-modified RB c DNA sequence (Figure 2) still retains the same initiation code in the original selected plant, while another methionine codon is issued at nucleotide 139. When the methionine acid When used as a start codon, the RB protein is speculated to have 928 amino acids and a molecule of 100 kD, though--the same as the surface MW measured by SDS-PAGE. The extra 5 'sequence contains a GC-rich region * The translation is different from the usual alanine and proline residues (Figure 2 >. The difference between the surface molecular weights on the real and SDS-PAGE can be explained by the second protein modification 'the putative amino acid seen in Figure 2 There are several possible N-linkages in the sequence .. 'Glycosylation' positions. However, when 1 ^ 11 cells are in the supplement-galactose When grown in a 3H-glucosamine medium, the labeled RB protein could not be measured even by prolonged autoradiography. In addition, sugar was sugared according to the method described in J. li UC hem '(1975) 250: 8569 -8579. The endonuclease digests the 35S-labeled RB protein without reducing the molecular weight of the surface. — -43- -T —--------- pack --- ο. (Please read the first note on the back first (Write this page) \ __ Η. _ --------- 4 paper rulers for easy use. China's family standard. Ί CNS). A4 size. (-210X297 mm 〇16611 A7 B7

V. Description of the invention (u) Printed tooth-optic epiphyseal germ cell tumor UN-1 of the Central Laboratories of the Ministry of Economic Affairs, Shellfish Consumer Cooperative, is labeled with 32 phosphonium phosphate metabolite and immunoprecipitated * The immunoprecipitated protein runs into a single Band, molecular weight is the same as 3 s S-standard RB protein. The interpretation of Figure 3 shows the 35S-labeled bands of 110-114 kD on the second and third lines, and the u P-labeled bands of 110-140 kD on the fifth line. Lines 丨 (3 5 s) and 4 (3 2 P) are immunoprecipitated with young cross-immunized rabbit IgG. When a whole M-glycosidase digestion of 35S-methionine standard RB samples was digested overnight, there was no measurable molecular weight reduction of 110-114 kD. The above findings prove that the purified retinoblastoma is a phosphoprotein of MW110-140 k0. This phosphoprotein was named ppRB110. The RB gene can also be measured at the DMA level of other vertebrates (also recorded in Science (1987) 235: 1394-1399 and Figure 4). It is suggested that the RB gene exists in many species during the evolution process, and a very important Physiological tasks. Cells from several vertebrate species such as QT6 (gull quail), NI Η / 3 T3 (mouse), Rat-2 (mouse), and c 0 (monkey) M 3 2 P-. The protein was immunoprecipitated with anti-RB IsG using the same procedure used in humans. As shown in Figure 4, antigen-associated proteins can be measured in all cells. There are 108 kD M-like molecules in quail, 120 kD in mice, 128 kD in mice, and 10δ-110 kD in monkeys. Compared with 110-11 4 k D of human cells. Antigen-associated proteins with different molecular weights observed in different vertebrates such as quails, mice, mice and monkeys suggest that the RB protein is preserved during evolution, most likely in proportion to the evolutionary relationship. Because the antigen and molecular weight are stored in this type of vertebrate, the RB gene product may also exist in other breeds and function similarly. 44- The national standard of this paper (CNS} A4 ^ ㈡ X 297mm :) City shortage • HI 1 ^ 1 i Read the notes on the back ^^ Write this page) ---- 、 Order -----

4661 IT A7 B7 Printed by the Shellfish Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (42) p p * R 1 〇 The predicted amino acid sequence of the protein has several similarities to those found in other tumor genes. Therefore, p p R B 1 1 was detected by the fractionation effect of girl cells. Localization of sub-cells. Two methods must be used. To find out the distribution of ppRB110 in the nucleus, cytoplasm and membrane parts. Consistent with chemical properties, p p R B ^ ^. The sequence suggested a possible D N A binding interaction 'to identify 8 5 S; p p 〇 11. Appears in the nuclear part, the proportion is smaller, although P P R B 1 1. (Less than 丨 〇u is located in the membrane. No measurable presence in the cytoplasm. / To further prove that PP〇u〇 is mainly located in the nucleus, use a source of osteosarcoma cells known to have beneficial cell morphology for immunohistochemical staining. U 2 0 S. As an experimental group. Υ 2 0 S cells were anti-PR PR Β 11 0 I g G immunoprecipitation. As a control sister, [J 2 0 S cells were immunoprecipitated with UG immunized in childhood. Both sisters were K commercially purchased rhodamine conjugated sheep anti-rabbit IgG culture from Sigma. Immunofluorescence was observed in cells that reacted with anti-pρig igG, that is, in the nucleus (Figure 5). The cells of the control response of the immunized children did not show fluorescence (Figure 5Bii). The subcellular localization of ρρίΒ110 in the nuclear part suggests that the protein is in regulation + other genes. It has a very important regulatory function and has a DNA-binding activity. -Sex. Certain cell sources, especially those from tumors other than retinoblastoma, such as neuroblastoma LAN-1 cells, are radiolabeled with K 32P-phosphate. Cell lysates of such labeled cell mixtures are based on Mol. Cell. Biol. (1986) 6: 4450-44 57, single or double-stranded bovine thymus-4 5 ~ (Please read the precautions on the back before filling this tile) „--.---, 0: 尺度 Zhang scale quick-use wood country countries knead Fengye 2ΪΟΧ297 公 *) The jealousy is better: Rabbit age?: ⑼ ifei 0 4661 1 7 A7 B7 V. Description of the invention (u) 0 NA ~ ini element column separation. The obtained results suggest that PpRB ^ o only binds to the saturable, DM A position. In the past, other proto-tumor genes η-myc, c-myb, and c-f 〇s are With .DNA binding activity (Mol. Cell. Biol. (19δ6) 6: 4450-4457; U982) 296: 262- 266). This type of proto-tumor group. Activation due to gene expression loss of modulation or structural modification The product is necessary for tumorigenesis. The absence of ppRB110, but not its existence, appears to be a tumor that partially or completely inactivates the RB gene. Therefore, the inhibition of tumorigenic activity of other genes by ppRBii0 does not make ppRB11 evil. It is an important regulatory protein that prevents and inhibits, and causes malignant growth due to its absence. The essentiality lies in regulating other genes. Pp RBU 〇 Deficiency or production . Problem, the RB protein with several first ρρκβΛι utility of such a limited number of memory c -. M y c, the core protein dish Nature consequent tumor gene, a tumor gene, due to the presence of some cell growth. Predicted by its existence, the loss of pp RB mediator tumors in the absence or absence of membranous tumors that can act as tumours or cells in fine embryos ao * iH 0 0 Vision or children become sick new or non-embryonic ' Guan Erxiang fetal tumor cell movement-fine and germ membrane human network scrutiny judged it to be the first to listen to it ri strike order · Central Ministry of Economic Affairs, Central Bureau of quasi bureau shellfish consumer cooperatives printed fine embryo membrane network . Treatment and treatment of tumors and tumors associated with swelling of the tumors and early-onset Μ results can prevent pre-diagnosis and early Μ. Tumor IV -iE I. It diagnoses and diagnoses the tumor's embryonic membrane defense net to be used as a sexual pass. The picture of the figure can also be selected by the method of screening. law. The history of the Fang family's home diagnosis reveals the atrial papilloma of the tumor cell carcinoma: The pre-thinning of the tumor cell cytometer K master gel can be used by the method of the god, the Fang tumor is broken / meat diagnosis, the fibrous tumor and the flesh. Bone selection, after the sieve is said to be precedent >

-T 'This paper size applies to China National Standards (CNS) (210X 297 mm): .w, dB6l 17 A7B7 V. Description of the invention (u :) Cancer, etc., indicating whether it has occurred in the past with retinal embryo Patients with cell tumors 0 2. Preparation and description of the RB fusion protein. The BB fusion protein is used as an antigen for immunization. 5, 0.9 kb, 0.7 kb and 3, 1.8 kb regions of the retained RB cDNA et; a 1. Molecular Γ ΐ π ntng * ___ L · Laboratory Manual (1982) Cold Spring Harbor Laboratory, Co 丨 d Sping Harbor, NY Among the vectors described in the standard procedure for subpopulation selection to induce high TRP E expression, pATH-3 (University of California San Diego). Contains three RB cDNA subfragments of the codon sequence, ie 5 *

EcoRi-EcoRI), 0.7 kb (EcoRI-EcoRI of RB-1) and 3 'Please read the note above f Book binding 1.8 kb (Bgin-Bglll) printed by the Central Consumers Bureau of the Ministry of Economic Affairs (Bgin-Bglll) PATH3-0 7 The TRP of the plastid 19δ4) 49: 132-141 The PATH3-0. 7 RB is the position map of the pATH-3 plastid which can be inserted and confirmed according to the figure. Medium, and then supplemented with 20 mg of MG growth. The culture was diluted to 1: 100 in M9. The expression of 10 mg / ml indole (I) gene in photoalcohol. Granulated bacterial cells and fused human PATΗ3 vector in a separate framework. The E-R β gene product uses the method described in J, Virol. · Constructed in E. coli " 7A. CDNA RB fragment frame EcoRI endonuclease site. Detailed restrictions on the transformation of recombinant somasomes into E. coli mm294 / ml tryptophan M9 minimum nutritional requirements plus casein Density of protein amino acid and ampicillin (600 nm of K2, added to the 1: 1000 dilution of 100 ¾ acetic acid M induced TRP Laensmli gel sample buffer -47- Standards: National Standards for Difficulties (CNS) A4 (210X297 mm) 中央 Central Standards of the Ministry of Economic Affairs 印 Printed by employee consumer cooperatives 4 6 61 t «A7 • _ B7 V. Description of the invention (45) Boil God 4 5; Bell Then, the β-gel was stained with Coomassie blue by polyacrylamide gel electrophoresis analysis. A large number of fusion proteins were prepared, purified by preparative polyacrylamide gel electrophoresis, and separated by liquid extraction. s D s and soluble propylamine M were removed by dialysis. Protein was concentrated again It is mixed with a rib adjuvant to immunize rabbits. Approximately 6 mg of protein * is recovered and then rabbits are immunized with K. Only one of the three PATΗ3 constructs, namely PATΗ3-0.? RB gauge fusion protein. The molecular weight of the resulting fusion protein is 57kD Since the molecular weight of TRPE is known to be the 37 kD and 20 kD protein portion of the protein-derived fusion protein. The other two plastid constructs do not produce protein at all * even TRP E itself. Since the RB clone contains many assumptions The proteolytic cleavage position, the inability to produce proteins in E. coli is not surprising, and may be due to the instability of the fusion protein. 'After induction, the surface of E. coli produces a 57 kD fusion containing 20¾ of all E. coli proteins. Protein. PATH-0.7 represents a fusion protein of MW 57 kD, of which 37 kD is derived from TRP E and 20 kD is derived from RB. In Figure 7A can be seen the production of TRP E-RB fusion protein deficiency, 7B is P Results of AG E / gel electrophoresis. 58 kD protein appeared in the induced culture (line 2), but not in the control culture (line 2). Using the above procedure for fusing PATH3 and RB fragments, a large number of fusion proteins can be prepared and passivated by preparative SDS polyacrylamide gel electrophoresis to K in accordance with the procedure described in Nature (1970) 227: 680-685. The extracts were isolated overnight. Fusion protein; SDS and soluble acrylamide are removed by dialysis. Then -48- ft right; Zhang Chi Bianxiao uses Chinese national standards: (CNS) A4M ^: (210 X 297 mm :) 乂; ----- ----- Ί Feng-·. V (please read the notice on the back page first) -5 Printed by the Shellfish Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 4661 1 7__b7 _ V. Description of the invention (shirt) -Egg a quality. The purified fusion protein is used as an antigen for the production of a specific anti-R β protein antibody. Anti-RB-specific antibody The fixed rabbit polyclonal antibody against the RB protein was prepared according to the procedure described in Proc. Natl. Acad. Sci. U 9 8 6) S 3: 6 7 Q 0-¢ 3 7 9 4. As described above, New Zealand red rabbits were immunized with the obtained TR p E-RB fusion protein according to standard experiments. 0 Rabbit M was mixed with complete Freund's adjuvant_ 2 μg, preferably 10 Micrograms of purified fusion protein (most injections) is preferably repeated injections of rabbits at 14-day intervals, followed by additional injections of the fusion protein in incomplete Fredezo auxiliary (repeated injections). Completely. The Fredezo auxiliaries usually include antigen emulsions in a mixture of saline and emulsifiers, in this case fusion proteins, as, for example, Arlacel in mineral oil and killed mycobacteria. Incomplete Fredezol is the same ’except that it does not contain mycobacteria. Two months later, rabbits produced high-value antibodies that reacted with TRP E and fusion proteins. Collect oil from rabbits, collect blood into plastic containers and coagulate. Y, iooo s centrifugation ο min minutes to obtain serum. Rabbit anti-ρρ 〖βΐι. Immunoglobulin (IgG >) was purified by antiserum via two affinity columns. To increase antibodies that recognize only the R B. determinant, two affinity columns were prepared, one containing the trp ε protein and the other containing the fusion Protein. The antiserum was isolated through a fusion protein-polysaccharose column and M 0,1 Mole concentration glycine HC1 緵 wash solution (pH 2 · 3). Using the same buffer, the eluate was passed through the TRp E column several times to remove antibodies directed against TRP E. Repeat the segregation several times. -49- (Please read the note on the back before filling this page) -------------- 1 i --- οι- κ. V Η II an · m ί i ¾. 4661 1 / __—---- V. Description of the invention (47) A7 B7 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs: owing to the fibrous lake 1 5 x after the entanglement contains precipitated RB eggs to resist -RB reference h test plan contains somatic fiber a (ϋ 2 〇s) bundled nutrient cell source such as WERI-24 obtained above for M lid culture dish approximately 1.5 XC i / ml) continued operation in methane- The antibody prepared by the above steps under the salt Non i det Aproti η ΐ AV is sufficient to immunize the RB protein in 103 cells. The purified anti-RB antibody was used for immunoprecipitation or immunostaining in all assays, and white matter was immunized according to the procedure described above among several cell sources of RB mRNA as demonstrated above. Identification of RB protein by antibody immunoprecipitation—Urol .. (1981) 38: 1064-1076 standard LAN-1 neuroblastoma cell source of normal normal RB raRKA, normal human cells * human liver cancer Alexandria cell source and osteosarcoma The cell source was used as a positive control sister. All such cells were obtained from a US collection depot. Retina ' blastoma cell sources Y79, RB355, WERI-1, and WER 1-27 with pre-stretched truncated or lacking RB ibRNA were used as shade control controls. This type of cell source is based on the protein of 0 3BS-methionine-labeled cells. It is cultured in a medium without methionine for 30 minutes at a temperature of 60 mm, at 37 ° C, and the cells are starved for 10 seconds. Incubate with 35S-methionine (150 micron _. 3 ml of methionine-free medium for 3 hours. All after 4T: at a concentration of 25 millimolar trimethylolamine (pH? .4), cell extracts were prepared in a lysis buffer of 50 mmol NaC !, 0.2¾ P-40, 0.5¾ deoxycholic acid and 200 units / ml of η deactivator. At 20,000 M g Centrifuge for 15 minutes to clarify the lysate. -50- You & this paper dazzle / 9 steps sleepy country (CNS) A4 regulations (21〇) < 297 public dust) Please read the note above first Cover page> ll. Order _ 4 661 A7 B7 V. Description of the invention (48) f Please read the precautions on the back before filling out this page. J Free-Epidemic disease is performed by immunizing rabbit antiserum with 5 microliters at an early age. 'Jiefan was absorbed into Folin Forest-Fixed Staphylococcus aureus from Enzyme Center, Inc. 10 μl of 100 μg / ml of anti-ppRBtl0 IsG was added to the supernatant of each test sample, and 10 μl of juvenile immune serum was added to the supernatant of each control sample as a control. Then added to the protein Λ 添加 refers to sugar beads (Sisraa). The immune reagents were sequentially cleaned with the following reagents: 1) lysing buffer, 21 [Mole concentration of NaC1, 3) in lysing buffer, 0.115 Hac 1 in lysing buffer, And 4) Lysing buffer M removes non-specifically bound egg white matter. The immunoprecipitated protein was analyzed by 7. 5¾ SDS-polyacrylamide gel electrophoresis and autoradiography. K-acetic acid-salt-based 2.5-diphenyoxalic acid was saturated, and the protein gel of Crab. Spectrum was measured at -70¾. The results are shown in Figure 110-114 kD. The protein was found to be immunoprecipitated with anti-P P RB 11 ° I g G. The description of the gene product is to further describe the protein purified from cell extracts. Cells are labeled with 32P-gallic acid or 14C or 3H-glycosamine, and then digested with endoglycosidase. Cooperatives are printed to test for protein phosphorylation * UN-1 cells are labeled with 3 2 P-gallic acid metabolism. For M 32P-labeled LAN-1 cells, in a 60 mm covered petri dish, 37 ° C, culture in phosphate-free medium for 80 minutes to starve about 1.5 X 106 cells, and then supplemented with 3P 〇43- (1 milliCi / ml.) Of 2 liters of phosphate-free culture medium for 1 to 2 hours. Contains 2 5 millimoles: ^ turn ::: This paper scale soil is used by the Laos country to fly to the CNS) (210 X297 male K '4 6 61 1 7 A7 B7 Shellfish Consumer Cooperative, Central Bureau of Standards, Ministry of Economic Affairs Printed V. Description of the invention (49). Degree of Jg-synylaminomethane-hydrochloride (P 丨! 7.4). 50 millimolar concentration, hC 1, 0. 2¾ Mon ideb P-4 0, 0.5 ¾ Deoxycholic acid and lysate from Ca! Biochera 200 units / ml kallikrein deactivator: Prepare cell extracts in the wash solution. At 4 t: under 2 0, 0 0 0 Centrifuge at xs for 20 minutes to clarify the lysate. Immunoprecipitation was performed with M anti-ppRB11. IgG. Radical standard procedures were performed. Immunoprecipitation and killing were absorbed into formalin-fixed Staphylococcus aureus from Enzyme Center, Inc. The following reagents were sequentially washed 丨) lysis buffer * 2) 1 Molar concentration of Na.Cl, 10 Molar concentration of trimethylolamine methylamine-hydrochloride (p 丨 17. 4 > And 0.15: Ν ο nidet P-40; 3) 0.15 HaCl, 10 millitorles concentration trimethylolaminomethane-hydrochloride (P 丨 丨 7 · 4), 0. 1 Ν ο η idet P- 40; and 4) lysis buffer. Eucalyptus J. V 丨 rn 1.. (19δ0) 36: 6 1 7 -62 1 Prepare the immunoprecipitated protein for electrophoresis. Immunoprecipitation / killing protein ran out of a single band, its molecular weight was 3SS-labeled ppRB11. The molecules of the protein are the most identical. • Shows that the RB protein is a phosphoprotein. Secondary cell localization of full-length RB protein Human neuroblast B cell tumor cells L A N-1 were labeled with 3 2 S-methionine as described above, and then fractionated into cell membrane, cytoplasm, and nucleus. Labeled protein-and then immunoprecipitated with anti-RB UG. The experimental plan for cell fractionation is basically the one described in ^ J. Cell ·. Βίοι. ^ (1983) 97: 1601-1611. Prior to use, 2 to 5 100 mm 'culture plates contain a total of 2.0 X 107 to 7.5 X 107 cells κ 3 sS-methionine metabolism markers for 2-3 hours. All the follow-up procedures are on _ 5 2 · The standard of the next step, the standard of the courageous business) 8 4 specifications $ 210, 297 mm, shame 4661 1 7 Α7 Β7 Printed by the Shell Consumer Cooperative of the Central Government Bureau of the Ministry of Economic Affairs Explanation (50) 4 t:-the next i line. The cells were buffered with phosphate buffered saline (PBS) i. The supernatant was twice, scraped to PBS, and pelleted in a table centrifuge at 375 × g for 5 minutes. Half of the cells were resuspended in the lysate solution as complete cell lysis production%. The remaining cells were washed in hypotonic RSB buffer (10 millimoles concentration HEPES pH 6.2, 10 millimoles NaCl, 1.5 millimoles concentration MsC ", 200 units / ml Aprotinin), and then washed again Suspension in RSB. 懋 Float in a tight Dounce homogenizer and mix 20 times to make it uniform. Adjust the volume of KRSB buffer to exactly 3 ml. On a Sorvall HB4 rotor that rotates 375 X g, K 1 The homogenate was centrifuged at 50 ° C per minute, and the cell pellet was resuspended in the lysing buffer to produce the nucleus. The supernatant was placed on a Beckman SW50.1 rotor and the thick-walled polya-llomer test tube was placed in a test tube. M3 $, 000 was rotated (150,000 X s) per minute and centrifuged for 90 minutes. Cell pellets were resuspended in the lysate solution to produce a portion of the membrane, and the supernatant was adjusted to a concentration of 1 X lysate solution to generate the cytoplasm. Section. All 4 sections tested the RB protein inclusions as described above. Immunoprecipitation with 7.5 »: SDS polyacrylamide gel electrophoresis and autoradiography analysis. The results are summarized and explained in Figure 5A The following classification methods can also be used q All procedures are performed at 0 to 4 t 2 to 5 tilted 100 mm culture plates containing a total of 2,0 X 107 to 7,5 x 107, LAN-1 cells, M phosphate-Yuanchong saline solution (p B s), rinsed twice, scraped to PBS The particles were pelleted for 1 minute at 1,000 × s in a clinical centrifuge. Cells were pelleted in low-tension TKM buffer (20 mmol / mL trimethylaminomethane pH?. 1,5 mmol) KC 1,1 millimolar concentration MsCl2, '1¾ Aprotinin) After washing once, the cells are dispersed in ΤίίΗ and pupil size is 15 (please read the precautions on the back first and fill in this page)

C attack ------- ir ----- V susceptible -------- I. 0Τ 民. Jj · ^ · Cheng Weilian and Shu Guoer family rubbing accurate " (: 'C Fine >): Α4 ^ ΐ ^ ..: 拉 1 (ί > < 297 Gongqing A7 B7 4661 1 7 V. Description of the invention (51) minutes-. Cell suspension in a homogenizer Stir 20 times to make it uniform, (please read the precautions on the back to fill in this page) > Loading · Adjust the volume to TK buffer to exactly 3 ml, and take out the sample for immunoprecipitation analysis. Μ Low-speed centrifugation produces nuclear particles The supernatant obtained from the initial embryonic heart was subjected to high-speed centrifugation to produce a precipitate and a soluble fraction. In order to obtain nuclear particles, the homogenate was homogenized on a sorvaU ΗΒ4 rotor (375 X s) Ot, MU 500 Centrifuge for 10 minutes, and suspend the coarse nucleus particles in 1 ml. The nucleus particles are homogenized 5 times in a Dounce homogenizer. Aspirate the liquid 3 times through a tube equipped with a 25 gauge needle. And suspended in Tkh buffer solution and aspirated 5 times through the same tube. After the final centrifugation, the nucleosomes were suspended in TKM buffer and analyzed for "protein content and secondary cell markers. The original nuclear supernatant (p NS 丨 .. and The supernatants of the nuclear washings were combined, and were turned on a "ckroan SW50.1 spinner" in a thick-walled polyheterogeneous test tube at a rotation rate of 3,000,000 per minute (150,000 x, centrifugation at 90 ° for 90 minutes to Particles (Pls〇) and soluble (S15〇) fractions were generated. Each fraction was adjusted to equal capacity with TKK buffer and tested for ϋΒ protein and secondary cell markers directly. Printed by the Central Standards Bureau of the Ministry of Economic Affairs The substance was identified by M measuring 51 nucleotides. The sample was dissolved in TKM buffer, and the culture strategy contained 10 millimoles MsCU, 0.1 millimoles concentration AMP, 100 millimoles concentration glycine ( In the test mixture of pH 9.0), 2 microliters of C i 3H-adenosine in the test mixture was measured by liquid flash count (I bid). The soluble protein of each fraction was according to Proc. Natl. Acad. Sc i. (1962) 48: 2123-2130 Tested for lactate dehydrogenase activity. 'Identified * The content of the succulent web is. According to Biochem. Biophys.

Acta. U971) 233-334-347 described in the procedure to test NADH myocardial yellow-5 4-pull: the paper size is suitable for the Chinese national standard, (CNS, A4 grid: (¥ 2 to / 297 mm. :) Pi :: _ Weaving round 4661 1 7 A7 B7 Printed by the Shellfish Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (52) .. The results are illustrated in Figure 5B. Using biochemical fractionation and immunofluorescence (as described in the previous example), the RB protein was identified primarily in the nucleus. (Fluorescence is mainly released in the nucleus * when the immune control sister is negative in childhood). Most (approximately 8 5: «) 3 5 S-labeled proteins are located in the nucleus and a small part (less than 10 2 > is connected to the membrane. There is no measurable RB protein in the cytoplasmic part, or secretion The secondary subcellular localization of the RB protein detected by the rabbit epidemic fluorescent light was obtained from the U.S. human osteosarcoma cell source U20S of the American-style culture collection collection store for immunofluorescence staining. Approximately U20S cells were seeded to 12 On a glass cover slip * after 18 hours for immunofluorescence staining. Cells were washed once from PBS buffer and fixed at room temperature with cold acetone for 10 minutes. The fixed and permeabilized cells were hydrated in PBS for 1 to 2 minutes. At room temperature, in a humid chamber, 200 μl of rabbit anti-RB IgG (1:20 dilution) or childhood immune serum were cultured for 45 minutes. After washing three times in PBS, cover 200 μl of erythramine-conjugated goat anti-rabbit ISG (25 μg / ml) from Sigma was incubated at room temperature for 45 minutes. Coverslips were then 'cleared' in PBS and cleared with a Zeiss light microscope I Observation. Alternatively, the immunofluorescence staining of L〗 N-1 neuroblastoma can be performed as follows OK. Cells of about 104 were seeded on 12 mm glass lids and used for immunofluorescence staining after 18 hours. Cells were made from 0.06 Molar concentration Pipes, 0.025 Molar concentration HEPES, 0.01 Molar concentration EGTA, 0.002 Molar concentration MsC U, pH 6.9. Clean the PHEM buffer once, then fix it in PHEM buffer with 2¾ polyformaldehyde for 20 minutes at room temperature. After fixing and permeabilizing pi --- (please (Read the notes on the back first and write this page), ίτ

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Printed by the Central Bureau of Standards of the Ministry of Economic Affairs, Beiya Consumer Cooperative 4 6 61 17 at • _ Β7 V. Description of the Invention (56) .. Feng Bing name # Appropriate post-transcriptional modification, as in proteins in higher eukaryotes. In order to test the feasibility of expressing a functional r B protein by the baculovirus system, a human r b c D K A containing the complete sequence of the R B gene was included in the AcNPV expression vector, and the re-elder virus was propagated in insect cells. Achieved by the host-vector system a large number of successful epitopes of human P 11 〇 β. The resulting protein is phosphorylated by SS and properly gnawed to the nucleus of an infected female cell. In addition, methods for purifying RB proteins have been developed. The purified protein can bind DNA in the same way as the original human 'pp110rr' and form a specific complex with SV40 antigen. The rapid nuclear translocation of proteins after microinjection further suggests the activity of purified RB proteins. In order to achieve the maximum production of RB protein / plasma in the baculovirus epitope, the 'most 5' non-codon sequence of the RB gene was deleted to construct a heavy body transfer vector. Through position-specific mutation generation, two Ba in Η I positions incorporate nucleotides 116 and 2935 of the RB cDNA to accelerate

Framing. The resulting vector encodes an mRHA, which contains 23 base pairs fused to the 5 'untranslated region of the RB cDNA followed by the complete (60 痼 base pair) polyhedral gene V non-coded sequence of the full codon sequence, The recombinant gene does not contain the ATG codon downstream of the nucleotide position of the original nucleotide 139 *. Therefore, the recombinant gene encodes a non-fusion * full-length RB protein. With reference to FIG. 31, it is described that the transfer vector P Ac YM1 'has all the upstream sequences of the polyhedral gene, including the A' of the original codon, followed by the unique BamH1 position. This transfer vector is designated as all upstream of pAcYMI ’with a multifaceted gene -59-

4661 1 7 A7 B7 Printed by the Consumers' Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs. 5. Description of the invention (57) Sequence-indicates the A of the initial ATG codon. The connection is a unique Bam 丨 丨 1 position. This transfer vector has been described in Hauwa et al. J, Gen.! 1 r 0 1 · (I987) 68: 1233-1250 apRB44 ~ 2. It contains the complete RB cDHA codon sequence from nucleotides Π6 to 2935, followed by It was bred to the position B am Η Γ of the blood prize PG EM 1 (Pr ome ga). The recombinant baculovirus vector, pAcYM1 / RB2.8, was constructed in an appropriate arrangement, inserting a 2.8 kb BamHI fragment from PRB44-2 into the BamHI position of P / \ CYM1. Therefore, the transcription of the RB gene is in Polyhedral genes are under the direct control of genes. -For constructing a baculovirus epitope vector for RB synthesis, consider the following facts. pRB44-2 contains the complete RB cDNA codon sequence from nucleotides 116 to 2935, sub-selected into the BamHI site of pGEM1. pAcYMl contains about 7 kb EcoRI 倒 segment of the viral DNA sequence of the polyhedron gene, in which the leader sequence remains intact, except for the first A of ATG, all polyhedral gene code sequences are replaced by BamHI concatemers. The recombinant baculovirus vector containing the polyhedron gene gene-RB cDN A fusion, pAcYM1 / RB2.8, is constructed by inserting the RB2.8 BamHI segment into the BamiH position of pAcYM1. Therefore, the transcription effect of the RB gene is Under the direct control of the polyhedral gene promoter, the sequence at the fusion junction is shown below in Figure 31. The lowercase font represents the polyhedral gene promoter, the uppercase font represents the RB cDNA sequence, and the BamHI linker is underlined. The translation effect K arrow of the ATG (nucleotide 139) fusion gene using RB is shown, and FIG. 312 3 ^ = (+ 1) represents the first A 0 -60- of the translation start codon ATG of the polyhedral gene (Please (Read the notes on the back first, write this page). 1.

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V. Description of the invention (58) Transfer of RB cDNA from viral plastids to viral genes is achieved through DNA and wild-type Aut. Ngrapha californica nuclear polyhedrosis virus D K A co-transformation infection with lipid infection (B R L). Polypeptide virus, in which the polyhedral gene is inactivated by the replacement of the homologous gene with the R B gene pair, because polyhedral gene absorbers are not shown in infected cells, and can be identified based on their different plaque morphology. The diseased larvae were purified three times to obtain a pure RB-containing baculovirus, designated as AcNPV-Y4 RB. Within the infected insect cells and the surface of the exogenous RB protein, determine whether the AcHPV multifaceted promoter gene can be used. Before the expression of the human RB gene in xenogeneic non-sacral animal cells, 蔺 S f 9 cells were first produced. S f 9, Spodoptera frugjpftrdn IPLB-Sf2 AE selection colony in vitro, 13: 213-217 (1977) at 27Ϊ: supplemented with 3.33 g / liter yeast lysate, whey protein hydrolysate t GIBC 0), and 10% heat-inactivated fetal bovine serum (GI M INI > Bull. 1 5 5 5 Π 9 8 7), (Texas Agricultural Inspection Station, University Station, Dezhou 丨 Grace Insect Medium) Into a monolayer or maggot floating culture. In the large-scale preparation of cell lysates, the spider culture of Sf9 cells is produced in EX-CELL 400 serum-free limited medium (J, printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs R. Scientific). Molt-4 cells, a human skeletal T cell leukemia cell source, were cultured in suspension in RPMI 1460 supplemented with 20¾ bovine serum. Saos-2 cells, a source of osteosarcoma cells, were grown in. Beige-modified Igloo medium (D u 1 becc 〇's in 〇dif ed ed E ag 1 e s nt edium > supplemented with M 7. 5¾ fetal bovine serum) in deciding whether Ac NPV multi-faceted promoter gene Can drive in heterogeneous non-vertebral motion -61- ii, i »· * A7 B7 Printed by the Central Bureau of Standards of the Ministry of Economic Affairs and the Industrial Cooperatives Cooperative Association 466117 V. Description of the Invention (59) Before the performance of the human RB gene is revealed, Sf9 cells are infected with K plaque-purified AcKPlY4 RB. After 40 hours of infection, collect The lysates of the infected cells were immunized with M anti-R BO. 47 antibody. The samples were subjected to SDS-PAGE and then analyzed by westward smear. As shown in Figure 32A, the M PMG 3-245 single colony antibody was immunosmeared. The appearance of the full-length RB protein is similar to that of mammalian cells in cell extracts infected with AcNPV-Y4 (line 3) > but different from cells infected with pseudo or wild-type AcNPV (lines 2 and 4) Regarding Figure 32B, cell extracts from / \ c'H PV-Y4 RB infected cells were prepared at different times after infection to determine the appropriate timing of RB protein production. Lysate M anti-RB0.47 antibody immunoprecipitation KpMG 3 -245 single colony antibody immunostaining. In Fig. 3 B, pp 1 10 RB represents unphosphorylated and phosphorylated RB protein, respectively. During the period after the infection, monitor the RB protein. Production situation Time. As shown, four hours after infection in 2 R Β detection of protein production 32Β, increased significantly in the 12 hours following. The amount of protein produced was maintained during approximately 72 hours of infection, during which time significant viral cytolysis began. In order to reduce the degradation of proteins associated with cell lysis to a minimum, infected cells were routinely harvested approximately 40 hours after infection. In detecting the expression of RB. Protein, Sf9 cells were infected with AcNPV-Π RB at 0.5 MOI at 24, 36, 48, 60 and 72 hours after infection, and 5 X 104 cells were lysed in 1 ml of buffer (50 mmol) Ear concentration Trimethylolaminomethane-HCI, pH 7.4; 0.2¾ Nonidet P-40; 1 mmole EDTA; 100 mmole NaCl; 50 mmole NaF and 1 mmole ---- ------ „--------------- ir ----- line -----, --------- (Please read the Note P fill in this page) d A7 B7 Printed by the Central Standards Bureau of the Ministry of Economic Affairs and Consumer Cooperatives 4 6 611 V. Description of the invention (60) Ear concentration search PMSF), the lysate was centrifuged (4 t :, 2 0, 0 0 0 X g) 5 min clarification. The lysate was then cultured with anti-KB 0.4 7 antibody * 7.5: ϊ SDS-PAG EI separated the immunoprecipitate. The protein was then transferred to nitrocellulose Paper * Followed the traditional method. After overnight blocking, nitrocellulose paper was cultured with PMG3-245 anti-fRB single colony antibody for 3 hours, followed by alkaline phosphatase · 'conjugated goat anti-mouse IgG and Color substrate, as described in Cell (1 988) 54: 275-283. Verification of foreign RB protein Effect and post-translational phosphorylation The RB gene encodes a nuclear phosphoprotein of about Hr 1_1 0,000. In order to identify the nucleus of the RB protein produced by baculovirus in insect cells, the nucleus of the RB protein was infected with AcNPV-Y4 RB-infected Sf9 cells Immunostaining with Μ anti-RBP.47 antibody for 40 hours. This condition / specificity is the pathological effect of baculovirus-infected cells. When performing immunostaining, color analysis, and the following steps. In false, wild-M AcNPV, or 40 hours after AcNPV infection * Sf9 cells were seeded on poly-L-lysine (Sigma) coated trough glass plates (Miles Scientific) and cultured overnight. The glass plates were treated with Saline washing: The cells were first fixed with U formaldehyde for 20 minutes in 0.4 Molar phosphate buffer solution (pΗ7.4) or 20 minutes in M propionium (-20 it). 1-2020 minutes in yeast. Fixed cells in M23 in PBS; normal sheep serum pre-cultured for 10 minutes, and then diluted in M. 0.02 $ Tee X-100 rabbit antibody -RB0.47 is baked overnight. After cleaning, 'Biotinylated goat anti-rabbit IsG (TAGO, Burlingame, CA) & 1 Hours later * Cell M was cultured with Vector Ubs, Burlingame Ca, conjugated AB complex for 45 minutes, and then subjected to quality support. The subject matter is included in the 0.05 mol -63- 隹 ^ This paper is used in the paper and used in the country quasi TCNS ') A4 specifications # 210X297 public and)

Printed by a member of the Central Bureau of Standards of the Ministry of Economic Affairs and a Consumer Cooperative. V. Description of the invention (61) Concentration-Trimethylolaminomethane-丨 丨 C 丨, 0. 0 5 «3,3 ' -Diamino-diamine dibenzyl tetrahydrochloride and 0.01¾ H2O2 (SiSma >. After 3-5 minutes the cells were washed with p B s to stop the reaction. Then, the cells were treated with Nikon double images Microscopy. Photographs # See Figure 3 3 and Oncogene. ≪ 1989) 1: 205-214 and Ceil (1989) 56:57 -65, the phosphorylation of RB protein occurs in multiple serine and threonine Residues cause differences in the molecular weight of the RB protein on SDS-PAGE. In order to identify whether the RB protein produced in insect cells undergoes ortho-acidification after translation, 40 hours after infection, 3 SS-methionine or 32P-orthophosphate SS salt metabolism marker AcNPV ~ Y4 RB-infected S'f9 sheaf ^ 3 hours. The extract of H. pumila was immunoprecipitated and analyzed by SDS-P AGE, followed by autoradiography. At the same time, immunoprecipitable RB proteins from the same extract were treated with lipase diphosphate phosphatase (PAP) to test the effect of dephosphorylation on RB protein mobility in SDS-PAGE. After dephosphorylation, 3t5S-labeled RB protein was reduced from a pair to a single band of Mr 110,000, and radioactivity was almost completely released from 3 2 P-labeled RB protein. Westward smear of lysates of unlabeled cells infected with AcNPV-Y4 RB. The dephosphorization analysis performed showed the same pattern of band reduction after PAP treatment. This type of observation showed that the RB protein produced in insect cells was phosphorylated-acidified, and that the modification also caused the molecular weight difference of the βB protein observed on SDS-PAGE. When performing radiolabeling and dephosphorylation analysis of Sf9 insect cells, perform the following steps. 40 hours after infection, in a 60 mm culture plate, Sf 9 cells (3x10β) K lacked methionine or phosphoric acid but supplemented with fetal bovine serum -64- Mm > ^^^ 1 ·---I — ^ 1 fl ^ i tj— 1 ^ 1 (Please read the notes on the back first to write this page) -I. Order

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Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs. 5. Description of the Invention (62) ~ DME-Pei Tzuji training 30 minutes. Radiolabeled by metabolic supplementation with 0.25 raCI / ml asS-methionine (1134 Ci / milline, NEN) or 0'25 milliCI / ml of 3: 2P-n-sulfonium 8¾ salt (without carrier, ICH) Cells for 3 hours. Then in lysis buffer (50 millimolar concentration trimethylolaminomethane-HC i; (^ 7.4; 0.2 $ ^ 〇 to < ^ 1; 卩 -40; 1 millimolar concentration £ 0 Cell extracts were prepared in 4; 100 mM _aCl; 50 mM NaF and 1 mM PMSF), and immunoprecipitation was performed with anti-RB 0.47 antibody. Two-thirds immunization Precipitated RB proteins from 3SS- or 32P-labeled and unlabeled cytolysates were analyzed for diphosphate phosphatase (PAP, Boehringer) dephosphorylation (Onc.QKe..ne.Res. (1 989 ) 1: 205-214. Immune complex containing RB protein in 37 ΐ :, reaction buffer (20 mmoles MES, f > H 5.5; 100 mmoles NaCl; 1 mmoles MCU .; 50 micromolar concentration of PAP incubation of Leutopin) M1.5 units for 60 minutes. After the reaction, the RB protein was analyzed by 7.5¾ SDS-PAGE, followed by autoradiography or westward beading. According to the following Programmatic purification of proteins from infected insect cells. S f 9 cells were infected with MoI 1.0, Ac ΡPV-Y 4 R, and cultured in suspension (1 X 16β cells / ml, 1,000 ml) , 40 hours after staining, the cells were pelleted by low-speed centrifugation to prepare cell lysates. In this case, the total RB protein expressed in the baculovirus system is liters of infected insect cell culture (about 10 β cells per liter). ) About 17-1δ mg. For this point, see the table below. -65- —-------- 'V Pack-(谙 Please read the note on the back # ^^ write this page) __ Order

Second-line®); A4 ^ Τ: 21〇 × 297 ^ '): -ΐ'-'-_ Tin "6U7-" 6U7--Purification of heavy carcass R Β protein from diseased and infected insect cells Printed by the Order of the Ministry of Labor of the People's Republic of China Naked Associate Bureau · 11 Attacks &

5. Description of the invention (M) Step 缌 Protein RB protein yield Purity purity (mg) (rag) (%) multiple U) Cell extract 670β 16b 90c 1.0 2.3 PMG3 -245 Immunophile 13. 5b 1 2. G1 " 79c · d 41.3 95 and column a. Protein quantification by Bradford (B io-RAD). b. Micro BCA (PIERCE) and spectrophotometry for protein quantification. c. Westward smear and optical density determination of legal protein. d. Coomassie blue protein staining and densitometry are legally the most protein. As mentioned above, after cell rupture • 90! «(16 mg) of the protein appeared in the supernatant, and '10% was in the insoluble part. Since all cellular proteins representing 2.33¾ * RB protein are easily detected in cell lysates. After a single step of purification by immunoaffinity affinity chromatography, approximately 13.5 milliliters of protein can be recovered from the column's alkaline eluate. To estimate the purity of the isolated protein, the eluate corresponding to 2.5 X 105 cells was fractionated by SDS-PAGE and Coomassie blue staining Iff. We provide more detailed purification procedures * Wash the cell lysate and resuspend in trimethylolaminomethane-HC1 with a concentration of 50 millitorles-Η 7.4; 0.2 2 4 4; 1 millimolar EDTA concentration; 100 mM NaCl; 10¾ (V / v) glycerol; 1 mM DTT; 1 mM PMSF; 25 μg / ml leucopeptin and 50 units / ml aprotinin

V. Description of Invention (64) -67- After incubation on ice for 15 minutes, the samples were clarified by centrifugation (1 0, 0 0 0 0 X g, 4 1 C, 10 minutes), and the supernatant containing RB was collected. Immunoaffinity analysis of RB protein was performed in a 2 ml volumetric column containing an anti-fRB single colony antibody (pHG 3-2 45) linked to albumin G-lolipose. After the supernatant passed through the column 4 times, the column was washed with 200 beds of the following reagents, and the column was cleaned: lysis buffer, lysis buffer containing 500,000 mmol NaC1; and a cleaning solution ( 200 millimolar concentration NaCi; l millimolar concentration EDTA; 1 millimolar port pMSfr; 103! Glycerol >. The bound protein was reconstituted from the column containing 20 milliliter triethylamine, pH 10.8 200 millimolar concentration "C1; 丨 millimolar concentration EDTA; 1 millimolar concentration DTT; 1 millimolar concentration PMSF and 10% glycerol in alkaline separation 鑀 eluate decantation. Collect milliliter portion, stand g三 Trimethylolaminomethane-substituted 1 (? Only 7.5) with a molar concentration of 1 / 10th of a mole is neutralized and stored in -7 ^ :, 1035 glycerol. Purified from When the RB protein of the infected insect cells is measured, the total protein mass is measured, and then the southwest DNA-binding test and the SV40 T-antigen binding test are performed as follows. The total protein mass in the isolated portion of the immunoaffinity column is micro BCA (PIERCE ) Measure __. The total isolated protein samples were analyzed by MSDS-PAGE. The amount of RB protein in the eluate was K. Coomassie blue staining, followed by M. Densitometry estimates. Total protein mass in cell extracts was measured by Bradford (Bio-Rad) Anal. Biochem. (1976) 72: 248-254. RB protein, purified by serial dilution

4 0 61 t β Α7 Β7 Printed by the Central Bureau of Standards of the Ministry of Economic Affairs and Consumer Cooperatives. V. Description of the invention (65) RB egg θ side is used as a standard for westward smearing ', followed by a band density optical density measurement comparison ° Protein smearing uses traditional techniques As described in ftcid Res. (1980 丨 8: 1-21), the radioactively labeled DNA culture smears were performed. The procedure was performed at room temperature. The smears were washed with water and then washed for 6 months. Ear concentration of urea and NP-40 was washed three times (20 minutes each), followed by KDNA-binding buffer (10 mmoles of trimethylolaminomethane-HCl, pH? · 〇; 1 mmoles of EDTA; 40 millimoles NaCl; 0.2¾ BSA; 0.2¾ Ficoll 400 and 0,2¾ polyethylene-nitrogen five-copper copper) Wash four times (30 minutes each time smear on 3HA-DHA-binding Incubate for 30 minutes in buffer. EcoRl linearized pGEMI ONA is randomly labeled with ot 32, P deoxynucleotides (Araersham. ≫ 3000 Ci / mmol) and used as a probe. After hybridization, the DNA-binding buffer wash the smear three times (10 minutes each) 'air dry Shuo 'was analyzed from autoradiography. TRP E-RB fusion proteins were included in the control group. Each TRP E-RB fusion protein was named according to the expression sequence of the RB gene contained in the protein. Therefore, RB19-22, RB23-27 and RB19 -27 crossed the regions of the recombinant proteins from expression sequences 19 to 221 amino acids 612-775), expression sequences 23 to 27 (amino acids 776-928 "and expression sequences 19 to 27 (amino acids 612-928), respectively The SV40 T-antigen is purified from Ad-SV XI-infected 293 cells by M immunoaffinity chromatography, see J. V. [Rot (1985) 53: 1003 1004 and "Eucaryotic Viral Vectors " Cold Spring Harbor Press. (1 982) Cold Spring Harbor, NY PP. 1 87- 1 92--68 This paper was once again used in China National Rolling List (CNS) A4 (210X297mm) Please read the note on the back first Written on this page, I order _ line A7 B7 4 6 611 V. Description of the invention (66,) The anti-T drug selection strain antibody (PAB419 antibody) was obtained from Oncogene [nc · Known complex formation test ce 1 1 (1 988) 5 4: 2 7 5-2 8 3 slightly modified. In short, 800 micrograms of baculovirus expressed ββ protein and 1 milliliter of EBC buffer (50 millimoles of trimethylolamine) containing 1 millimolar concentration of PMSF 25 micrograms of white wins 50 units / aprotinin Methane-HC1, pH δ.0; 120 mM NaCl; 0.5% Non-idet P-40). 800 micrograms of purified T antigen was added to the mixture and incubated on ice for 90 minutes. The aliquots of the mixture were immunoprecipitated with anti-47 or PAB419 single colony antibody. Killed and subjected to a westward smear analysis " The smear was continuously reacted with pMG3-245, followed by PAB419. After culturing with alkaline phosphatase-conjugated goat anti-mouse IgG, the smear developed from chromogenic receptors to DN / \ and protein knots. 1-combination test Figures 10 A and 100B illustrate south-west orientation DNA-binding test. 6 micrograms of purified TRP fusion protein (truncated RB protein> and full-length doublet RB protein M 10¾ produced from the baculovirus system described above. SDS-PAGE test. In the test illustrated in Figure 10A, Coomassie blue staining was used. In the test of FIG. 10B, the parallel gelatin was transferred to nitrocellulose paper; it was cultured with 32P-labeled J) NA glutamate and analyzed by autoradiography. Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs. In Figure 10B, DHA is combined with protein and analyzed by K-radioradiography. A fusion protein was identified. RB19-27 contains the main functional site that interacts with DNA. It has 20 times higher DHA affinity than two subregions, R Β 1 9-2 2 and R Β 2 3-27. In this regard, the third row of Figure 10B can be compared with the first-...-and the second row, and the purified full-length RB protein is similar to 019-27. This paper size is applicable to Chinese countries (CNS > Α4 Specifications (210X297 mm) 466117 A7 B7 V. Description of the invention (67) Printed by the Shellfish Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs " DiTA binding activity (Figure 10, line 4 >). Isolated from the virus 糸The DNA-binding activity of the RB protein of the system can also be proved by the analysis of the DNA- pheromone retention protein and the NaCl from the column M after about 400 mM NaCl. As mentioned above, Sf9 Niu cell KM0I 1.0, AcNPV-Y4 infection, cultured with maggot floating solution (1 X 16β cells / liter, 1000 ml). 40 hours after infection * Centrifuged granulated cells at low speed, washed * resuspended in trimethylol with 50 milliliters concentration Aminomethane-HC1, pH 7.4; 0.2¾ HP-40; 1 奄 Ear degree EDTA; 100 奄 Molar concentration NaCl; 10¾ (v / v) glycerol; 1 奄 萁 Maer DTT; 1 奄 Mole Concentration PMSF; 25 μg / ml leucopeptin and 50 units / ml aprotinin in the extract tincture. After 15 minutes of incubation on ice • Centrifuge 10,000 X 4t, 10 minutes) clarified sample * Collect the mash containing RB above. In a 2 ml volume tube containing the protein G-sepharose-linked anti-fRB single colony anti-horse (pMG 3-2 45) Immunoaffinity chromatography of the RB protein was performed in the column as described in this patent specification. After the supernatant was passed through the column 4 times, the column was continuously washed with the following reagents of M 200 bed-capacity: lysis buffer, containing 500 mmol Ear perfusion degree KaCl lysis buffer; and Lu washing solution (200 mmol of NaCl; 1 mmol of EDTA; 1 mg of DTT; 1 PMSF; 10¾ glycerol). Combined The protein was further extracted from the column with triethylamine containing 20 mM Molar, pH 10. δ; HaCI at 200 mol Molar; EDTA at 1 mol Molar; DTT at 1 mol Molar; 1 mM Separation of PMSF and 10¾ glycerol in alkaline isolation buffer. Collect 1-pen liter fraction • Immediately one-twentieth volume of 1 mole of trimethylol limb methane-HC1 (pH 7.5) And stored in -701 :, 10 »glycerin. -R-70-

L I --------- install-(Please read the note on the back 1 for writing this page)

Line P 0 · Heart-size paper This paper uses Chinese bacteria ® Home Accuracy (CNS) A4 ^ fM 210 X 297 mm) 166117 V. Description of Invention (68) A7 B7 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs · Pure pupa obtained from Qi-stained insect female cells in step pp 1 10 14 B, the total protein mass was measured, and then the southwest DNA-binding test and SV40 T-antigen binding test were performed. The total protein mass in the isolated portion of the immunoaffinity column was determined using -micro B C A (PI E R C E). The isolated protein samples were analyzed by M s D S-P A G E. Although the R β protein in the isolated solution was stained with Coomassie blue, it was then estimated by densitometry. The limulus protein in cell extracts is most commonly measured by the Bradford method (Bra-Rad). Anal. Biochem (1976) 72: 248-254. In order to determine the RB protein in the cell lysate, the RB protein diluted in the column was used as a standard to perform the westward coating * followed by a band density optical density measurement comparison. For this, see the table above. Protein smearing is using traditional techniques. According to the experimental plan described in Nucleic Acid Res ^ U 980) 8:: 2-1, radioactively labeled DN A Pei-Sai was applied. The procedure is performed at room temperature. The smear was slightly washed with water, and then washed three times (20 minutes each) with 6 mol urea and 0.2¾ NP-40, followed by KDNA-conjugated base solution (10 mM trimethylol limb methane -HC 丨, pH? .0; 1 耷 Mole concentration EDTA; 50 millimolar concentration HaCl; 0.2 BSA; 0.2¾ Ficoll 400 and 0.2¾ polyethylene monoazapentanone) Wash four times Γ 30 minutes each time) . The smears were incubated in DNA-binding buffer containing wp_labeled DNA for 30 minutes. EcoRl pG E M 1 D N A M α-3 2 P deoxynucleotide HPA (A hi e r s h a m, > 3 0 0 0 C [/ millimolar) randomly guides the label and uses it as a probe. After hybridization, the smears were washed three times with -binding buffer (10 minutes each time, air-dried, and analyzed by K-radioradiography. T i? PE-RB fusion protein was included in the control sister. Each TRPE-RB fusion protein Named according to the expression sequence contained in the protein. -71-Please make a note on the front page to order a thread. Paper size: China National Broadcasting Standard (CNS) A4 specification (210X297 mm > 466117 A7 B7 Central Standard of the Ministry of Economic Affairs) Printed by the Bureau ’s Consumer Cooperatives V. Description of the Invention (Sand) So ~, 1 9-2 2, RB 2 3-2 7 and R β 1 < J-2 7 respectively pass from Tables 19 to 22 (amino group) Acid 612-775), ppllORa region of Table VII sequence 23 to 27 (amino acid 776-92S) and expression sequence 19 to 27 (amino acid 612-Q2S). SV40 T-antigen is M immunoaffinity purification by decantation From Ad-SV Π-infected 293 cells, J. Vir. (1985) 53: 1001-1004; " Eucaryotic Viral Vectors " Cold Spring Harbor Press. (1982) Cold Spring Harbor, NY pp. 1 87-1 92 j. The anti-T single colony antibody PAB419 antibody was obtained from OnCOgene Inc., with a few modifications to make known duplicates. Containment formation test. Among them:> 800 micrograms of RB protein expressed by baculovirus and containing 1 millimolar concentration of P MS F 25 micrograms of leukopeptin 50 0. units / ml aprotinin 1 milliliter of EBC buffer (50 milliliters Molar concentration Trimethylolaminomethane-HC 1, pH 8, 0; 120 mM NaCl; 0.5% Non-idet P-40) mixed. 800 μg purified T was added to the mixture on ice Incubate for 90 minutes. An aliquot of the mixture of M anti-RB0.47 or PAB419 antibodies is immunoprecipitated and subjected to a westward smear test. The smears are reacted with PMG3-245, followed by PAB419. At M alkaline phosphatase-conjugation After the goat anti-mouse UG was cultured, the smear M produced chromogenic hair .. In order to test the ability of pure lib protein and SV40 T-antigen to form a specific complex, equal amounts of full-length RB protein and SV T- Antigen, mixture fractions K-anti-RB0.47 antibody or anti-T antibody PAB419 immunoprecipitation. Figure 11 illustrates the formation of a complex of RB protein (pllOSB) and SV40 T-antigen expressed by baculovirus. In a test tube, that is, in vitro mixed purification -72 Please read it on the back of Sr.

-T Chinese paper _ rate (CNS (210X297)). Ν '4 6 β 1 1 Α7 __ Β7 V. Description of the invention (7〇) II —-Ρ / (装 I __.. 5 ^ # ·. 线 (Please read the notes on the back to fill in this page first) | _ Full length white matter and purified τ_ Antigen The same mixture is divided from PAB419 (line 2) or anti-Rb〇.47 (line 3) Immunoprecipitation and killing are then applied in a westward direction. Lines 1 and 4 show _ and P Α β 4 1 9 purified SV40 T-antigen from immunosinking hall, and purified baculovirus immunoprecipitation with anti-RB0 · 47 antibody_ -Table RB protein. Mixing RB protein and SV 4 0 T antigen in vitro results in co-immunoprecipitation of RB protein and PAB419 (line 2>), and co-immunoprecipitation with T and anti-RB0.47 antibody Kill (line 3). This type of resource proves that the full-length protein isolated from the purification of the baculovirus system can form a specific complex with the SV40 T antigen. The nuclear translocation of the purified RB protein peptone identified the purified RB protein in vitro After two known biochemical activities of the preserved wild-type RB. ', The in vivo behavior of the purified protein was detected. Purified RB protein. P 11 0 R B. Injected into the cytoplasm of Saos-2 cells derived from osteosarcoma cells, which had a defective RB gene with deleted gauge sequences 21-27, encoding a short C-terminal RB protein (p95), Central Ministry of Economic Affairs Printed by Shell Bureau Consumer Cooperative

Proc. Kat I. Acad. Sci. USA U990) 87: 6-10 〇 p9 5 1 white matter located in the cytoplasm is extremely M and cannot be identified by the anti-RB0 .. 47 antibody used in this patent specification, even if The antibody is directed against the C-terminus of the RB protein. Cells were fixed immediately after injection and immunostained. Regarding microinjection, the purified protein was dialyzed to an injection containing 20 mmoles of trimethylolaminomethane-HC1, pH 7.4; 10 mmoles of KC1; 0.1 mmoles of DTT and 2 丨 glycerin The buffer was brought to a final concentration of 0.5 mg / ml. According to traditional technology, glass capillary needles (for paper and paper used in China) are used in A4 format (210X297 mm > ”): 1. Consumption cooperation printed by employees of the Central Standards Bureau of the Ministry of Economic Affairs

4661 W V. Description of the invention (71) Eppend “f) Microinjection of S a 0s-2 cells grown on glass trough plates. Use a vacuum and pressure device M and inverted inverted microscope (N 〖K ο η) micromanipulator for micromanipulation of the capillary and observation of the microinjection process. Immediately after the microinjection, the cells were fixed in a phosphate buffer solution (pH 7.4) at a concentration of 0.4 mole. > An immunostaining assay was performed. Figure 12 shows the nuclear translocation of the RB protein p 110 R β after microinjection into the cytoplasm of Saos-2 cells. The purified RB protein was injected into the cells and performed Immunostaining analysis. The arrow on circle 12 indicates the deep staining of the nucleus after microinjection, compared with the uninfected. Compared with the infected control sister, the deep staining of the injected cell nucleus showed rapid delivery of the injected protein into the nucleus. Since the RB protein is known as a nuclear protein, the rapidly and correctly shuttle-shifting effect of the purified protein after microinjection crosses the nuclear membrane of cells, and it is further suggested that the heavy body RB protein is active in vivo. The above results prove that Human retinoblastoma Because the product can be effectively expressed in the baculovirus expression system. It has been considered difficult to try to express a large number of proteins for a long time, because it is suspected that RB protein may hinder cell growth or even be "toxic" to cell growth. Multifaceted genes transcribe foreign genes The amount occurs in the late 7th stage of infection. After the production of in vitro virus particles and blocks the cells and most viruses ... genes. The baculovirus-insect cell system is therefore conducive to protein synthesis, such as RB protein. The growth of harmful cells. Another silly aspect of this system is the similarity of protein processing pathways between insects and mammals. The RB protein produced in the baculovirus expression system has been shown to target the nucleus of insect cells correctly, meaning mammals. Animal nuclear shift messages can also be " 74-

A7 B7

A 6 61 1 T V. Description of the invention (72 Recognized by insect cells. Although the glutamation of heavy protein in the baculovirus epithelium appears to be limited to the high-mannose-type 0-link or N -Linked_ * But it has been reported that proper phosphorylation of foreign proteins can be used for the performance of ciyc and HTLV-I p'4 011. RB proteins have been shown to be phosphorylated rather than glycosylated in the past | make rod-shaped The virus system is suitable for the production of functional proteins. The RB pliORB protein produced in infected worm cells is phosphorylated after translation | Western smear analysis can differentiate into multiple bands, just as in the case of the original mammalian RB protein. However, depending on the band density Judgment, compared with highly phosphorylated RB proteins> Un or lowly phosphorylated forms accounted for the majority. It is not known whether this phenomenon reflects the cell cycle status of the population during viral lysosomal infection, or simply because of a large amount of external R source Beta-protein peptone appears in cells and insect kinases are not sufficiently sulfonated. · The precise map of ortho-acidification on RB proteins can identify whether the phosphorylation pattern is really the same as that of mammalian proteins. The amount of RB protein expressed in the baculovirus system is about 17-18 mg per liter of infected insect cell culture (~ 100 cells). This gauge is equivalent to the other produced by the system Mammal Proteins, such as 10-20 mM of Leukoglobin 2 / L The Banbury Report. Fields, B., et al ... (ed.) (1985) 22: 319-328 Cold Spring

Harbor Laboratory Press, Cold Spring Harbor, and P210 BCR-ABL 4-5 mg / L, flncogenp (1 9 8 9) 4: 759 -766. The use of a recombinant transfer vector containing a 5 'untranslated region of a complete polyhedral gene that is fused to an RB cDNA that removes most of its 5' non-code region | promotes the large expression of RB protein. R B «1 R N A's the -75- 广 L Please read the notes on the back ^: Fill in this page) -pack-

Mr. D. Printed by the Consumers' Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs 4S6117 A7 B7 V. Description of the Invention

The 'sequence is very rich in G + C, except that it is favorable for the formation of a stable second structure. When this type of structure appears before the start codon, it is believed that the translation efficiency corresponding to m 0 A will be reduced. Replacing RB 5 with alfalfa cyanovirus (A MV) R Μ A4, or callin-hemoglobin mRNA, untranslated sequences have been shown to promote the in vitro translation of RB raRNA by 5 to 10 times. It is further suggested that RB 5 Translation of non-cryptographic sequences, EHBQ J. (1990) 9: 1815-1822, possible negative reactions. The length of foreign genes5 and the presence of untranslated sequences have been shown to affect the performance of recombinant proteins in the baculovirus system. Since the polyhedral promoter gene is very rich in A + T ', it is concluded that for the optimal expression of the rb DNA sequence, before inserting into the transfer vector, the long, G-C-rich 5, non-codon sequence is excised from the RB cDHA. In an attempt to reduce the denaturation of proteins during purification, several self-affinity column isolation tests have been tested. Since there are not many biological and biochemical properties of the R B protein > there are only two variables that can be used to measure the complete properties of the purified protein, DNA-binding activity and activity with the SV40 T antigen complex. It was found that the current isolation conditions using triethylamine at a concentration of 20 millimoles at pH 10,8 can effectively preserve the biochemical properties of proteins. Purified eggs after micro-injection are rapidly transferred from the cytoplasmic nucleus to further prove that the protein is active under the J1I analysis conditions ° At the hibiscus pH (200 millimolar concentration of glycine 'pH 2.3 or 100 millimolar concentration of triethylamine 'pH 11.5) isolated proteins' tends to denature proteins, and the aforementioned two activities are also significantly weakened. The stomach & stomach stomach aggregates obtained evidence. .......... Although past reports indicate that only non-disc acidified * ^ protein is in D2C2 cells -76- (Please read the note on the back first, write this page on seven pages)-Pack · P: , ¾ Line

In 6 6 ”7 V. The description of the invention can bind to the SV40 T antigen, and the D2C2 cell line was stably transformed by monkey S-cell-derived CV protein P (Cell (1989) 56: 5? -65). The low-phosphorylated form of RB protein can form a complex with SV40 T antigen. This phenomenon can be achieved by mixing T-resistant tadpoles in vitro with purified RB protein or Mo It-4 lysate from AcHPV-Y4 RB-infected insect cells. Repeated proof was obtained. This phenomenon can also be observed when monkey cells are used for complex formation in vivo. Due to the phosphorylation of RB proteins in the cell cycle in a specific divisional manner *, between RB and virus tumor proteins The complex formation involves the conversion activity between the low-phosphorylated RBylated protein and the SV40 T antigen, which needs to be further explained. Using the baculovirus-insect cell system to provide a large amount of soluble | Complete and presumed active RB protein indicates in the future Major advances in studying the biochemical and biophysical properties of RB gene products. Possible applications include analysis of cellular proteins in the phased phase, isolation of specific DHA sequences that interact with them, and X-ray crystal map study of the three-dimensional structure of RB protein can also accelerate the interpretation of the biological function of retinoblastoma genes in cancer suppression. Currently, direct K-microinjection tests are being used to study the role of RB and cell growth and differentiation. Probably not. "85.li, 06f, printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs. A7 B7. 4. Preparation of RB protein-terminated growth cells produced by the elder sister. Osteosarcoma cell source, Saos-2. In this study, it was obtained from the American Culture Collection. This cell source lacks the expression of wild-type RB protein, but contains a 95 kDa carboxyl-truncated protein that cannot bind SVO T to cytoplasm that binds to I. Saos-2 cells can add genes __- 77 -_ which encode foreign genes to RB. This paper scale is applicable to China National Standard (CNS) A4 (210 * X297 mm)

JL___JL V. Description of the Invention 1 35.11.08 Please read the notes before you read it | This page shows the response. The gene is a gene that expands the cell size and loses the tumorigenic ability to metastasize to nude mouse cells. Incorporated by vector gene transfer. It has been determined that such cells may be particularly sensitive to the injection of RB proteins. Cell SR-40 can be spiked. Exogenous RB gene becomes RB protein * This cell is after retrovirus infection with RB gene as described in Science (1988) 242: 1563-1568. • Single cell selection It is derived from 5303-2. Sources of African green monkey medical cells (^ -1 and (: 05-7) are also available from the American Type Culture Collection. COS-7 is transmitted through the K-defective SV- as described in Cell (1981) 23: 175-182. 40 transformation effect was derived from CV-1. All cells were cultured as recommended in the culture medium of Beibei modified with 103 {fetal bovine serum. The Central Laboratories of the Ministry of Economic Affairs and the Consumer Cooperatives printed and used Proc. Hatl. Acad · Sci. USA · (1982) 79: 4083-4087, starvation through isoleucine for 36 hours · Incubate in complete medium supplemented with aphidocolin or hydroxyurea (both from Sigma) for an additional 12 or 16 hours * Synchronize the cells at the G1 / S boundary. Wash three times with phosphate buffered saline (PBS) to release cells from the G1 / S blocker and resupply for complete culture. According to Exp. Cell Res. (1 980) 126: 397-40 5 After 8 to 12 hours of 0.04 μg / ml nocoda-zo e) (SUaia) treatment, collect cells that are stagnated in the middle stage of cell division. To increase the number of mitotic cells Yield, cells were first aphidicolin treated for 12 hours to stop the cells at the G1 / S boundary. Aphids were removed after 6 hours Lin (aphidocolin), add nicozole to the culture medium. Mitotic cells M are cleaned and shaken gently to be collected, and then smeared on this paper scale. Applicable to China National Standard (CNS) A4 (21 × 297). V. Invention Description (Xishi)

Printed by the Consumer Standards Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs

35 mm petri dish. After δ hours of seeding, unattached cells were washed away. Cells were then injected at different times. Preparation of RB protein Two forms of RB protein were prepared for microinjection experiments. The full-length recombinant protein (p110rb, a low-acid and unphosphorylated form) was purified from baculovirus-infected insect cells to be nearly homogeneous. However, when the degree of Mae is close to 1 mg / ml, the protein aggregates into a form that cannot be injected. In order to partially solve the problem * RB protein is purified, stored and injected in a tincture containing 10¾ glycerol. Unphosphorylated, amine group ~ truncated 56 kDa RB protein (456〃 or truncated R8 protein) contains a complete T-antigen binding functional site, expressed in E. coli and purified to near homogeneity. Since the truncated protein can be concentrated to 1 mg / ml and injected in a standard 嫒 solution containing 2¾ glycerol *, it is used in most of the following experiments. p56rb (" RB truncated protein) is the 0-terminal half of the RB protein and contains two regions necessary for SV40 T-antigen binding. It is produced in E. coli from Nature (Γ991) 350: 160-162 The revealed T7 UNA polymerase behaves systematically. ρΠ〇μ was produced in the insect cell line and revealed in the Cell Growth and Dijff. (1990) 1: 429-437. Mouth 110 ^ and P56RB proteins were purified to homogeneity by conventional decantation. Sister Weaver H1 and rabbit anti-sheep IgG were purchased from Boehringer Mannheim and Vector Laboratories, respectively. Antibodies, 495 (against the TRPE-RB fusion protein formed by the sequence of self-expressing sequences 19-22), .47 (against the TRP 79 of sequences containing the self-expressing sequences of RB 23-27-this paper is for Chinese countries Standard (CNS) A4 size (210X297 mm) (Please read the precautions on the back * .. Fill out this page) Installation · M. -5 ^

Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (: ^) E-RB fusion protein) and R2 are concentrated in a microinjection warm rinse solution to a concentration of about 1 mg / ml. T-peptide and p5 3-peptide were given by Nicholas Lin. Such antibodies are described in EMBO J, (1990) 9: 1815-1822. The T-peptide includes the amine group 101-118, and is dissolved in a microinjection tincture of 1 mmol or 5 mmol as described in Cell (1939) 56: 57-65. Mutant T peptide contains a substitution of aspartic acid with lysine and K5 mol concentration. Using Cell (1989) supr ?, p5 3 is dissolved in microinjection buffer at 5 mTorr. in. Protein preparations, in addition to the full-length RB protein, containing trimethylolamine methane at a concentration of 20 milliliters * pH 7.4; 0.1 millimoles degree EDT A; 10 millimoles degree KC1; 1 millimoles Degree of 2-Hydroxyacetic Acid and 2 Glycerol Injection Yuan Concentration Solution "was concentrated using a Centri con 30 micro-constrictor (Eb. 1 <: 〇11). ? 1101 ^ Food exists in a buffer containing 10: »; glycerol to reduce agglutination." For the experiments described in Benwei (3), when the retinoblastoma gene is referenced * intends to have the nucleus described in 臛 2 The cause of the nucleotide sequence. When referring to the RB protein (pRB110), it also means that the protein having the amino acid sequence as shown in FIG. 2 is derived from the protein 2 and the truncated protein fragment, p56rb. In addition, p56RBk, the fragment is illustrated on plaque 2, starting with arrow A and ending with arrow B. In the second circle, the C-terminal peptide is explained, starting from amino acid 917 (arrow C) and finally amino acid 928 (tendon B). Protein preparations were analyzed using standard techniques KSDS-P AGE. Figure 12 shows the titer and purity of a protein preparation used to discuss the microinjection technique below. Protein preparation for microinjection is KSDS-PAGE analysis, line 1 (Please read the precautions on the back before filling this page) '' pack_

I Order the paper standard General Chinese National Standard (CNS) M Specification (210X 297 mm) 661 1 7 A7 U7 V. Description of the Invention (planning) Printed by the Consumers ’Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs: P110RB was obtained from M Reorganization Insect cells infected with baculovirus baculovirus; line 2; biotinylated anti-sheep antibody; line 3; anti-RB. 49 5 anti-crow; line 4: anti-RB R2 antibody; line 5: Anti-RB.47 antibody; Line 6: Sister Weaver 1; Line 7: p56rb from E. coli. 1 liter of each sample was placed on a 15 acrylamide gel. Mass fresh M staining. The position of the molecular weight standard is shown in thousands of ear ridges. Cells grown on 35 mm petri dishes were directly injected using Eppendorf micromanipulators and micro syringes with a micro-tip capillary dropper. The injection pressure is typical Set between 50-100 hPa, injection time. 3-.5 seconds. It is estimated that the protein preparation is diluted about 20 to 50 times at the time of injection. Assume that the typical cell volume is 50-100 picoliters and the RB segment irrigation is 5-1 mg per milliliter, about 5-50 million molecules are injected per cell. After injection, the growth culture is based on the plant. Supplement MBrdU (AmershaB). After appropriate labeling period, remove the culture medium and wash 3 times with KPBS. M ice-cold • Absolute formazan culture 30-minute furnace M fixed cells. After washing with PBS and rehydration, at room temperature, sample K guide Anti-BrdU (ABershaB) mice were colonized for 1 hour. After washing for 3 times in PBS / dihydrofluorescein-conjugated anti-rat anti-rat (Amershaia), it can be cultured for 1 hour at room temperature. Washed 3 times with MPBS to remove anti-mouse antibodies. Sample M Texas Red Conjugated Key Biotin (ABershaiB) was cultured. Chains of anti-biotins were combined with biotinylated RctG co-injected with all protein preparations. Therefore, it is regarded as the cytoplasm of injected cells. After the last washing, the solution of the most equal volume of glycerol and PBS is added, and the sample is covered with a glass coverslip. Under the fluorescence microscope, K Texas red and dihydrofluorescence Samples for the inspection of elementary organs. — .. 81- The paper size is applicable to the Chinese National Standard (CNS) A4 specification (210 × 297 mm) -------- •, -'...---- Please install first κ Read me back Φ ^-^ '-while writing this page) Order

A Qian_ V. Description of Invention

A66117 The following examples demonstrate the experimental results obtained by incorporating RB protein into tumor cell sources. In particular, the examples show the evolutionary capacity of RB proteins and fragments thereof through the cell cycle ' In addition, examples show that the SV40 T antigen can unblock this blocking effect #. In certain specific examples * the RB gene product is a sarcoma cell source treated with MM full-length RB protein or a fragment thereof, and the cell cycle evolution of Saos-2 *. It was found that cell cycle evolution is at G1, and that evolution is reversible. In Figs. 14A to 14P, the results of microinjection and immunostaining of MBosU-labeled Saos2-2 cells are explained. Saos-2 cells are injected as described in this patent specification. Figure 14A shows RB protein injected immediately with ρ56 * β, immobilized immediately and indirectly stained with Texas Red. Figure 14B contains uninfected cells labeled with MBrdU, re-fixed and immunostained with K-dihydrofluorescein conjugated anti-BrdU antibody. Figures 14C and 14D are the single sites of synchronized cells that were fixed and stained in the early G1JWp56rb * RctG co-injection * M BrdU culture for 24 hours. K Texas Red stained cells marked the injected cells, while M dihydrofluorescein stained cells showed that the cells had incorporated BrdU. Figures 14E and 14F show the single location of cells injected by RotG alone in early G1K. Printed by the Consumers' Cooperative Standards of the Central Bureau of Standards of the Ministry of Economic Affairs in order to identify whether the protein can cross the cell membrane and be transferred to the nucleus. 110 ~ cytoplasmic microinjection, and then fixed for 5 to 15 minutes. Cells were immunostained with K-rabbit anti-RB anti-SI. 47 and Texas Red conjugated anti-rabbit. Antibodies. Staining was observed primarily in the nucleus (Figure 14), although some staining was observed in the cytoplasm of some injected cells. The p11〇rbWp56hb protein can be transported to the nucleus within 15 minutes. 82 This paper size applies to China National Standard (CNS) A4 specification U 丨 0X297 mm) 5. Description of the invention (γ

Printed by the Consumers' Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs. The experiment of Houyi was marked by co-injection of rabbit anti-goat antibody (Ra G), which is a typical KRB protein. It is estimated that each cell is injected with 5-50 million parts. The number of exogenous RB protein molecules per cell is estimated to be about 1. A protein represents at least 5 to 50 times the endogenous amount. Cells that have undergone S-phase cultures will incorporate BrdU into their DNA. Dihydrofluorescein-conjugated antibody immunostained BrdU, and avidin immunostained R «G (Figure 14). The percentage of ejected cells can be identified under a fluorescent microscope, and the sex cells are also difluorescein-positive. During the 4-hour labeling period, the incorporated amount of BrdU in the hydrocarbon RB-injected cells was only slightly less than that of the cells injected without uninfected R ct G alone (Table 1). This reaction shows that the quaternary substance may not be toxic to cells in general, and can be determined by its DK A incorporation and cell attachment. -83-This paper size is in accordance with Chinese National Standard (CNS) A4 specification (2! OX 297 mm). Biotinylated * is the RB protein with injected protons. million. So injected. * Cells after injection. After the fixation period, cells were conjugated with K Texas Red and incorporated into the BrdU via injection. Because of Texas Red-Yang, cells that grow asynchronously or the RB protein used by K have sustained viability | Table 1 Asynchronous Microinjected protein from growing cells Yang BinU stained cells Total number of hydrocarbon injections entering S phase β Total number of cells b Cells pp110rb 1094 416 38 p56rb 1315 408 31 R ct G 1725 707 41 Sister Weaver 106 42 40 Uninfected 41 ]: -----, installed ------- ordered -----, 'line (please read the notes on the back of the world first. Written copy f). 46611 f V. Description of the invention (笱 A7 B7 The total number of injection cells printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs (Texas red positive)) White at least five (except for tissues that represent an experiment) except for injections 0b, injections that are incorporated into BrdU -· Cell number 1 can be confirmed by the staining of all anti-Br dU antibodies linked to dihydrofluorescein. The cells were cultured in growth medium > 1 B r dU for 4 hours before staining. RB protein shot in early G 1 blocks the incorporation of Gr dU. Since only a small effect on BrdU incorporation can be observed during the injection of RB protein, cells may only be in the cell cycle m-僩 specific point Sensitive to RB protein 0 To test this hypothesis, RB was injected intra-synchronously to treat synchrony cells * Stop cells at mitosis 9 Release and culture for another 6 hours »At this point, remove non-adherent cells (adher en t) and inject Remaining cells. After BrdU was cultured for 24 hours, the cells were fixed and stained for BrdU and RaG. At least 80-90¾ of uninfected cells were stained with BrdU. However, cells injected with the 56 kD truncated protein almost completely inhibited the evolution from G1 to S during the labeling period. At this point, please refer to Table 2 and Figure 14C. The same inhibitory effect was observed when 14D 0 was injected with K full-length RB protein. 0 Opposite-injection of K-spinweed and R ct G or cells with only 60 -70X of Ra G * can enter the S phase during the labeling phase ( (Draw 14E and 14F) 〇Although the full-length RB protein inhibits the incorporation of Br dU 9 under these conditions, the results are slightly different. During the hydrocarbon transition period, the RB protein preparation may agglutinate and cause injection difficulties. Compared to the full-length preparation The truncated RB egg activity is very consistent. In order to extend the observation to cells that have exhibited wild-type full length, for truncation, please listen to the back of ik and fill out this page-Ri-This paper applies the Chinese national standard (CNS) A4 specifications (210X297 Mm) 466117 £ 1

Amendment 4 M A7 B7 Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention RB protein was injected into the cell source SR-40 derived from Saos-2 * Its predetermined expression is full-length RB protein. The effect of RB protein injected in early G1 on synchronous SR-40 cells is the same as that on Saos-2 cells (Table 2); k 24 hours of labeling, very few cells injected with RB truncated protein enter the S phase. Therefore, the presence of endogenous wild-type full-length RB proteins does not interfere with the effect of RB truncated proteins on cell cycle evolution. In order to determine how long the ϋβ truncated protein can block the entry into the S phase, the labeling period in the presence of dU was extended from 1 day to 2 or 4 夭. B 15 shows the hydrocarbon injection of BrdU during the labeling period Number of Soas-2 cells. 6-7 hours after the release of Nicozole treatment, Saos-2 cells were co-injected with M56 kD RB protein and RaG. After injection, cells were cultured in BrdU supplemented medium before fixation and staining. Number of days shown. The percentage value shows the number of injected cells that were incorporated into BrdU during the labeling period. This value represents at least 100 injected cells. After 2 days of KBrd II culture, cell circulation can still be observed before fixation and staining Inhibition of evolution. 4 days after injection · 80X KRB truncated protein-injected cells can incorporate BrdU. This type of observation shows that the blocking of cell circulation by the 0 protein is reversible and extends to 0 and 2 truncations between 2 and 4 days The dose-dependent effect of the RB protein effect was also measured. Diluted protein fractions were injected early into G1 into synchronously growing cells. Figure 16 illustrates the dependence of cell cycle arrest on p56rb dose. Saos-2 cells Ny 6-8 hours after the azole treatment release, truncated protein at the concentration indicated by K and 1 g / ml RaG were co-injected. After cultured in BrdU for 24 hours in the growth medium, the cells were fixed and stained for BrdU and RcxG. Merge into a 8 5_-This paper size applies to China National Standard (CNS) A4 specifications (210X297 mm >

In i --- 二 ί —II 1 \ — / ^ I ^ 1 (Read the precautions on the back-%-fill this page) J *-丁

The percentage of BrdU injected cells. Each value represents at least 150 injected girls. As shown in Figure 14 = A truncated RB protein diluted 5 times from its original concentration weakens its inhibitory activity *, but an integer fraction diluted to 2 times still retains the inhibitory effect. Therefore, the blocking effect of p56r & s entering S phase depends on the amount of protein injected. The lower limit of blocking effect incorporated by BrdtJ is 0.2 and 0.5 mg / ml of injected protein. In order to prove that the inhibitory effect of entering the S phase is caused by the RB protein, the blocking effect can be weakened with a reagent that specifically binds to the truncated protein. ϋΒ protein (P56RB) was mixed with 1 mg / ml rabbit polyclonal antibody .495, .47 * or R2 solution. These antibodies are cultured against unique RB fusion proteins and recognize RB truncated proteins on westward smears (31, 49, 56). The injection of a mixture of RB truncated protein and .495 resulted in approximately 30¾ of BrdU in the injected cells, compared to the almost complete lack of incorporation in the cells injected with the lutein alone (Table 2). The cells were co-injected with the protein and antibody .47 and humans were approximately 16X BrdU at that time. Co-injection of truncated RB protein and antibody R2 also showed reduced inhibitory activity, although its effect was not as strong as that of antibody .47 or .495. Since the antibody bound to the RB protein weakens the inhibitory effect of the protein, the blocking effect on BrdU incorporation is specifically caused by the RB protein. (Please read the precautions on the back first, fill in this page}-Printed by the Central Standards Bureau of the Ministry of Economic Affairs, Consumer Work Cooperative, this paper is printed in accordance with the Chinese National Standard (CNS) A4 specification (210 > < 297 mm) 4661 1 " 7 V. Description of the invention (济 Α7 Β7 Printed by the staff of the Central Standards Bureau of the Ministry of Economic Affairs and Cooperatives. Table 2: Microinjection of cells grown in the same area in the early stage of G1, _. __. Teach s **! I 「dU [[i welcomes the siblings. The number b 讹 人 S 叩 妁 turbidity is the sage worker Saos-2 pll0 '^ c 255 8 3 RaGc 270 173 & 4 ρ5δ? 3 388 12 3. RaG. 295 202; S3 sisters know 59S 333 '' 5S without dyeing 84 dSS ^ 3 d 247 10 4 p56? 3 d +. / 4 9 5 80 24 30 d5S? Jd -f: 47 596 95 16 pSS ^ d + R2 712 43 S SR-4 0 p56 ^ 89 3 3-20X 105 52 RaG 134 74 55 (Please read the notes on the back first and fill in this page) __ ~ η 7 I_ This paper size Applicable to China National Standard (CNS) A4 specification (210X297 mm) Amendment date, _ supplement first A7 B7 V. Description of the invention (pulling). Number of cells injected (Dezhou Hongyang The number of injected cells incorporated into BrdU can be confirmed by the uniform staining of the anti-BrdU antibody linked to dihydroluciferin. After injection, the cells were stained before staining. The growth medium M BrdU was cultured for 24 hours. The injection was performed in a buffer containing 10¾ glycerol. The concentration of the truncated RB protein injected in this type of experiment was about .3-.5 mg / ml instead of 1 mg / ml. If the truncated protein printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs is injected in the S phase and is not injected after the early injection of RB protein in G1, it can be explained as the inhibition of DNA synthesis. Processing causes Saos-2 cells to stagnate in the early S. Such as Figure 17 | If the protein does not inhibit the protein. Saos-2 cells are stagnated at G1 / S, and after K injection, the cells are released from the stasis and hydrazide) or 4-6 hours ( Aphids) * This coxazole treatment also stagnated the cells at the fibroblasts and injected after about 6 hours. After the injection * K was fixed and stained. It is shown that after the preparation of the protein used for injection, the cells were stained with dU staining. Released in the Becklin treatment and K BrdU culture 4- for BrdU and RctG immunostaining. Insufficient effects of BrdU and G1 in combination with G1. Absence of BrdU incorporation * To test this explanation, K aphidolin | Re-injection of truncated RB protein in S phase injection "treatment of BrdU with hydroxyurea or aphid * Injection. Cells were cultured with BrdU for 6-δ hours (cells were fixed and stained at hydroxyl time. Epithelium. After release, the cells were collected and cultured with BrdU for 24 hours, and reproduced. The head shows the proportions during the relevant labeling period. After injection * cells from aphid 6 Hours, fixed, and reversed the injection of cells as described in the period. • About L, -------- installed ------- ordered -----, Xiao (Read the notes on the back and read # '4!' Write this page) This paper size is applicable to China National Standard (CNS) A4 specification (210X297 mm)

"The party" Α7 Β7 Printed by the Consumer Standards Cooperative of the Central Bureau of Standards of the Ministry of Economy After injection of truncated RB protein, the cells arrested by hydrocarbon aphids can be positively stained due to the incorporation of BrdU. The percentage of cells entering the S phase and the density of BrdU staining and M. weave and RctG, or RaG injection alone The cells are similar. In order to control any possible delay in the function of RB protein after injection, K RB protein was injected into the cells arrested by aphidolin and cultured for an additional 6 hours before release and KBrdU labeling. Although the density of BrdU staining was slightly lower · May be due to K aphid collin cultured for too long, the percentage is equivalent to the cells injected with MRctG alone. Similar experiments were performed with hydroxyurea instead of aphid collin arrested cells. Hydroxyurea is different from aphids near the G1 / S boundary Kolin stagnated the cells and stagnated the cells. Saos-2 cells were stagnated near G1 / S with hydroxyurea treatment, and then injected with truncated RB protein. After injection, the cells were released from the hydroxy gland treatment and KBrdU was selected for 6 to 8 hours. Again, the truncated protein injected was incorporated into BrdU No measurable inhibitory effect. The percentage of DNA synthesized by cells was the same whether KRB protein and RaG * tissue spleen and RaG, or RaG injection alone. This result is consistent with the observation of asynchronous growth cells injected with MRB protein | In terms of DNA synthesis itself, RB truncated proteins cannot significantly block this effect. Therefore, the inhibitory effect of DNA synthesis cannot explain the complete lack of BrdU pooling effect seen in the early injection of protein into synchronized cells in G1. RB protein Blocking the evolution through the G1 phase at a specific point 'according to the results of the above experiments • The inhibitory effect of truncated proteins must be due to blocking the evolution through the G1 phase. To define the location of this stagnation during the G1 period, first determine In the cell cycle of 2 cells, the precision of this paper at the beginning of the S phase applies the Chinese National Standard (CNS) Α4 specification (2Ι〇Χ2ί > 7 mm) ----------- packing ---- --Order ----- 1) 0 ---------------- "One (please read and read the note of memorabilia first continued # ,. Fill in this page) Year B5- ， Amend and add A7 B7 V. Inventive (^?) Scale. Circles 18A to 18C illustrate cells Dependence of circulatory arrest on the duration of truncated protein injection. As shown in Figure 13A, Saos-2 cells are arrested by mitosis by K Nicozole treatment. After release from arrest, cells are cultured in the presence of Br * dU as shown Hours, re-fixed and stained with anti-Brdu antibody. The head of the bar graph shows the calculated percentage of cells stained with BrdU. Each value represents at least 200 cells. As shown in Figure 20A, uninfected cells are approximately 20 hours stained with BrdU. By 30 hours * cells of 80-90¾ are actually human BrdU. Based on this *, a time course experiment was performed in which cells treated with nicozole were injected with} > 56RB protein at different times after stagnant release. Figure 18B. The time in K hours describes the time since the onset of S phase in Saos-2 cells after release from stagnant mitosis. The G1 period is about 22 to 24 hours long. The S phase is about 7 to δ hours long. The length of G2 phase has not been precisely determined. The arrows indicate the injection time points for the experiments described in the description of Figure 18C. Printed by the Central Bureau of Standards, Industrial and Consumer Cooperatives, Ministry of Economic Affairs About Figure 18C, after Saos-2 cells were released from Nicozole treatment, M 1 mg / ml truncated RB protein and RcxG at each time shown (hours) Total injection. After the injection, BrdU was continuously cultured in the medium for 30 hours from the original release of Nicozole. After BrdU labeling, the cells were fixed and stained for BrdU and RctG. The head of the bar graph shows the percentage of cells injected with BrdU protein. Each value represents at least 200 injected cells. Very few cells enter S phase 30 hours after release, if the protein is injected 5-10 hours after nicozole release (circle 20C). If the truncated RB protein is injected 14-16 hours after the stagnation, the number of cells in the pardant is incorporated into BrdU. In this paper size, the Chinese National Standard (CNS) A4 specification (210X297 mm) is applied, 661 t

A7 B7 was injected 17-19 hours after release. The truncated protein had no measurable effect on entering the s phase. Assuming that the S phase begins 22 to 24 hours after the release of nicozole, the results suggest that about 6-10 hours before the G1 / S transition, the cells become difficult to control the inhibitory effect of the injected RB protein. This result confirms that the inhibitory effect of RB protein on cell circulation is due to the evolution of gall bladder through the G1 phase, not the DNA synthesis of the sister. Furthermore, this type of observation implies that RB may be acting at a specific * fairly early point in time during the G1 period. Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Light of the People's Republic of China —---- install—I (please read the precautions on the back, then 4: write this page) The blocked cell cycle is used as a test for functionally inactivating RB protein when SV40 T-antigen is bound, in the presence or absence of SV40 T antigen. • The truncated RB protein was injected into G1 evolution. influences. A solution of 1 millimolar T-peptide capable of binding to p56rb and an equal volume of truncated RB protein was mixed and injected into the early synchronized cells of G1. At this concentration * peptide is M100 to 200 times more molecular weight than RB protein. Synchronized cells injected with K-truncated RB protein / T-peptide mixture during the labeling period * approximately 28X entered S phase (Table 3). If the concentration of T-peptide is 増 to 5 mmol, or the molecular weight is 500 to 1,000 times, the injected cells of about 70¾ are incorporated into BrdU. In contrast, after injection of the K-truncated RB protein and the carboxy-terminal P53 peptide that did not bind to the truncated RB protein after 500 to 1000 times the molecular weight *, almost no cells progressed to the S phase. The effect of truncated RB protein injection on CV-1 and COS-7 derived from kidney cells of African green monkeys was also compared. The COS-7 cells are selected for release because they are derived from CV-1 cells by transformation with the original-deficient SV-40 gene. The T-resistant of the big fi 91. The paper size applies the Chinese National Standard (CNS) A4. Specifications (210X297 mm)

Printed by H Consumer Cooperative, Central Standards Bureau of the Ministry of Economic Affairs. 5. Description of Invention (sp Therefore, it can be used as a good test system for the effect of the presence of endogenous T-antigens on the activity of injected RB proteins. K truncated RB proteins in early G1百分比 The percentage of injected synchronized CV-1 cells incorporating BrdU * is at least 5 times lower than that of those injected with RG alone (Table 3). However, the synchronized COS-7 cells did not inhibit evolution to S phase at all. The results show endogenous T-antibodies and show a large amount of T-antibodies. Therefore, they can be used as a good test system for the effect of the presence of T-antigens on the RB protein activity injected. In the early G1 period, K is truncated. The percentage of synchronized CV-1 cells that were injected with RB protein and incorporated into BrdU was at least 5 times lower than those injected with RctG alone (Table 3). However, the synchronized COS-7 cells did not inhibit evolution to S phase at all The results show that the presence of endogenous T-antigen or co-injection with T-antigen peptides can neutralize the inhibitory activity of RB protein. Therefore, those skilled in this art can use this system to screen RB protein fragments and Comparative entry in the presence of RB protein The percentages of CV-1 and COS-7 cells in the period are identified as fragments that can cycle the evolution of the sister cell. The foregoing results provide functional proof that RB protein can inhibit evolution through cell cycle. Injection of truncated p56rb protein does not inhibit DHA synthesis itself * Because the cells that are stopped by aphids or hydroxyurea are not affected, it is the evolution of G1 through G1. The inhibitory effect of this cycle of evolution is dose-dependent, not because of the general effect on cells. Toxicity is specific to RB protein • Because antibody-specific and truncated RB protein binding can reduce its activity. Circulating cells are very sensitive to the inhibitory effect of RB protein, until about 6-10 hours before opening DNA replication; thereafter * Exogenous addition of truncated RB protein no longer inhibits evolution to S. It is assumed that injection of truncated RB protein -92-This paper size applies to China National Standard (CNS) A4 (210X297 mm) --- Γ- ------)-install ------, order ---- (谙 first read the precautions on the back to write this page) 1,

! I I J t. * I 661 1 T 4

5. Description of the invention There is a short delay between the quality of the product and its active form in the nucleus. It can be concluded that the RB protein inhibits evolution to the S phase at a key point in the G1 phase. The 6-10 hour time window before DNA synthesis may be similar to the period after the G1 limit point, which is the irreversible point at which mammalian cells enter the S phase. The defined time window does not necessarily accurately reflect the point in time when the 1ΪΒ protein acts under normal physiological conditions. From this type of experiment, it is not clear how long the injected full-length RB protein or truncated RB protein box can appear in its active configuration and / or location. However, RB protein was buried in the nucleus within 15 minutes after cytoplasm injection. * Kaphidicolin cultured K-truncated RB protein-injected cells prior to release and labeling did not reduce the percentage of cells incorporating BrdU. The observation shows that the time for RB protein to become functional is very short after injection. Because the protein is densely phosphorylated at this point. But * because an earlier period has been defined as described above * it seems to be bondable to the activity of the truncated RB protein. Therefore, RB may involve regulatory decisions made by the cell about 6-10 hours before the G1 / S transition. RB protein can be acidified at multiple positions in vivo and in vitro, but it is not clear that the position of phosphorylation is most important for the function of RB. A slight increase in scalylation during G1 may be related to the cell's transformation from RB-sensing to non-sensing. ° Some phosphorylation of full-length RB proteins has been observed in G1, although it may also be due to incompleteness. Caused by synchronized cells. The basal-terminal 56-kilophones of RB is sufficient to inhibit evolution through the G1 phase. Shows that the carboxyl-terminal half of the full-length protein produced by her sister is used for her cell cycle 93-This paper size applies the Chinese National Standard (CNS) A4 specification (2 丨 0X297 mm) years

A B7 Fifth, the effect of the invention (f /) ring is the functional part. The two biochemical activities of the carboxy-terminal half of the protein, DNA binding and protein binding. According to the current findings, the specificity of the RB sequence that binds to DH A is very low. Although M high G / C contains a slightly higher affinity for SK and DNA binding, but does not particularly prefer a particular sequence. On the other hand, Specificity was observed in the combination of RB and protein. The transforming proteins K and cell proteins of several DNA-transformed viruses bind to the same functional site of the full-length RB protein produced by the older sister, so they can compete with each other for binding to the RB protein. This region is where most naturally occurring inactive RB mutants are located. It appears that blocking the evolution of G1 by RB proteins depends on specific protein-protein interactions. Printed by the Central Bureau of Standards, Ministry of Economic Affairs, Consumer Cooperatives—r -------- ri—I (please read the notes on the back first and fill out this page) Order truncated RB protein and derived from SV 40 Τ- Antigen and can be mixed with peptides that bind to truncated RB proteins * to avoid their inhibitory consequences. Co-injection of truncated RB protein and peptide containing K with a single amino acid substitution more than 500 to 1000 times the molecular weight can also weaken * to a certain degree * the blocking effect on cell cycle evolution. Although amino acid substitution reduces its affinity for RB protein * The mutant T-peptide can still bind to truncated RB protein in vivo when K disparity is exceeded. In contrast, the injection of P53 peptide, which was not effective after injection of gold, had no effect on the inhibitory activity of the RB protein *, thus enhancing the specificity of the interactions observed between the ΙβΒ protein and the T-antigen peptide. CV-1 cells injected with truncated RB protein early in G1 stagnated before the S phase, while COS-7 cells exhibiting a large number of T-antigens did not stagnate. These observations together show that T-antigen binding is indeed functional for RB proteins. Since the truncated RB protein for injection is not phosphorylated, the results of the test and the inhibitory effect on cell differentiation have been shown to be on paper. The standard standard is 2I / 1 «centimeter 97 2 46611 July 7 η Corrected the hypothesis that A7 B7 was printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs. 5. Description of invention (f >) The active form of KB protein is an unphosphorylated form-due. Therefore * some of the DNA tumor virus transforming proteins, including SV40 T-antigen and adenovirus E1A, can promote cell growth, at least in part, by binding and deactivating low-level, full-length RB proteins produced by the older sister. The immortality of the cell is consistent with the phenotype of the inability to rest induced by the expression of such transformed proteins and the loss of regulation of cell cycle. Assuming that the carboxyl-terminal half of the RB protein is biologically active, the question is what is the function of the amine-terminal half of the RB. The sequence in this region may be required for proper protein acidification. In mouse cells, peptides similar to the truncated RB protein were not overphosphorylated. Moreover, several consistent positions of cdc2 / MPF kinase appear in this region. It is consistent with the statement that the amine-terminal half of the RB protein may contain regulatory functional sites. The unblocking effect of the truncated 0 protein on the evolution of G1 does not occur until 3-4 days after injection; this lengthy stagnation period may be due to the inability of cells to regulate RB protein in a normal manner * or because of the considerable amount of RB injected protein. In this test * various RB proteins with mutations in the phosphonium acid position, comparing their activities can help answer this question. If the truncated RB protein is structurally active • Multiple wins due to mutations can inhibit cell differentiation and thus place the cell at a disadvantage. This phenomenon may explain the lack of naturally occurring mutants in the amine terminal half of RB. The results described here show that RB participates in the control of the cell cycle by acting on a control point in the G1 phase. This point is significantly earlier than the hyperphosphorylation of RB protein produced by the heavy body when the cells enter the S phase. Therefore, it is reasonable to propose a combined test result and other RB proteins. ___________- QS -_ This paper size is applicable to the Chinese National Standard (CMS) A4 specification (210X297 mm) ll · --------- } .Installation-< Please read the precautions on the back-sample page) Order / m 466117 Month: δ5 · 1ΐ · 0 6 / /: f -yr- j A7 B7 V. Description of the invention (strict; >) The Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs printed a model with known characteristics. Since RB has been shown to be related to the protein of several cells, and the functional part of RB's protein binding is sufficient to block the evolution of cell cycle * The binding effect of fine SSS white matter seems to be very relevant to the function of RB observed _ Sex. This model suggests that rb encounters cell proteins that are important for DNA synthesis and / or cyclic evolution at a special time in the G1 phase, and participates in the regulation of cell cycle. Inactivation of PHORB by mutation • Phosphorylation at key locations • or binding to transformed proteins, prompting them to provide the functions necessary to continue cell circulation. Once the cell releases this type of protein, it will continue the cell cycle itself. The full-length RB protein can no longer effectively inhibit its function, thus removing the blocking effect of cell cycle evolution. The secondary nuclear localization of RB may be important for its ability to bind other proteins in inactive form. The RB protein K is inactivated to isolate other proteins. When RB protein is infected, the replication protein of K cells is isolated to the replication site of herpesvirus DNA, and its normal regulation of cell cycle is broken. In (II period), the low-phosphorylated form of full-length RB is tightly bound to the special nuclear position. The purified protein also tends to polymerize, which can explain the difficulty in maintaining its solubility at high maize degrees, and the Fibrillar protein intermediates have limited homogeneity. This type of observation suggests that the full-length protein of the heavy body may provide certain nuclear structural components, and the combination of the protein and the composition is necessary to inhibit cell evolution. It is speculated that high phosphorylation The protein can be released from a second nuclear position to provide some additional functions to K, or to restore its ability to bind other proteins in the posterior cell cycle. This model predicts that the regulatory effect of RB activity can be achieved by specifying its nuclear position and / or The binding of the cell protein is achieved. This paper is compliant with the Chinese national standard (CNS) A4 (210X297 mm) (please read the precautions on the back before filling out this page) .—— r. • 装 _

.1T,.: 4 46611 7 Printed by X Consumer Cooperative, Member of the Central Standards Bureau of the Ministry of Economic Affairs " H5. Ii. 'OF Afternoon £ 3 V. Invention ^ Full retina participation in enhancement until phosphorylation of tumors in RB function. Contains truncated G1 early S phase. The synthesis injected into Brd! J was recirculated and modified A7 B7 lost the RB gene and blastoma tumors to explain the phenomenon that the cells continued to receive may be due to the permissive effect. The aforementioned changes in the acidification status of the RB gene. T-antibody binding. • Injection of any protein into fine K. Co-injection guide pair. Stagnation on the G1 / S side. Incorporation without shadow. This result provides that BM regulates the function of cells and / or proteins and other progressive tumors. Method. RB can act as an appropriate signal for M cell cycle. Irregular cell differentiation The physical consequence is that RB in the cell cycle stops passing through G1. Therefore, the loss of the product is a nuclear change. In order to test the purified cells in the test area and then test the anti-RB antibody boundary or G1 at the end of the injection, it is suggested that the RB protein R Β can penetrate through the phosphoprotein, which is the apricot RB modulates the RB protein into S cells, which can antagonize the first 6 -10 small white matter is not directly evidenced in one of G1 and ganglion cells * regardless of the period of the shadow can inhibit the effect of "the specific point in the S phase" is also cyclically synchronized and the progress is full-length or loud. After its evolution to the RB protein cell, it broke the DNA restriction cells and established the RB egg shooter. It was able to co-operate with the peptide cells. The peptide cell had a finer quality. Anti-egg-JU, Β T i R Inner whites make RB fine knots. The test of the original test resistance T T emerges the strength. The nature of the test is true or false. Note that the quality of the paper or the size of the white paper is applicable to the Chinese national standard (CNS > A4 specification (210 × 297 mm)) {Please read the precautions on the back first. Fill out this page )

6 6 4 Printed by the Central Bureau of Standards of the Ministry of Economy—Industrial and Consumer Cooperatives, Invention Description (Knowledge)

A7 B7

Table 3. SV40 T-antigen releases P56RB for the evolution of 01 for the sister-in-law —… ～ ~ cell-derived protein β the number of injected cells b BrdU-stained cells the number of injected cells entering the S phase ”S a 〇s-2 p56RS 247 10 4 P56RB + T P56RB + 724 203 28 p53 727 7 1 CV-1 p56rb 459 46 10 Histone 471 259 55 R a G 332 176 53 COS-7 p56rb 257 224 87 Tissue 脒 191 159 83 All proteins at about 1 mg / Injection at a concentration of milliliter, except that p56rb in Saos-2 cells was microinjected at approximately .3-, 5 mg / mL of injected cell source 6-8 hours after release from nicozole treatment. The total number of injected cells (Texas red positive) is shown. K BrdU cells were cultured in growth medium after injection and before staining. -98-: The dimensions are applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm). Please read the notes on the back ^ and then 4 'write this page) ... ·' installation ----- ----) d .--- 1-^-..

I A7 B7 4 8 6117 V. Description of the invention (fg) 5. Exogenous RB protein inhibits tumor growth. Passivation of RB protein. (DE3 > / pLyS / pETRBc passivation. In short | 120g of moist cell suspension was resuspended in a lysate warming solution treated at 13,000 Psi with a micro-liquefaction processor, (10 millimolar Metridotrimethylolamine- Cl, 10¾ glycerol, 1 millimolar EDTA, 1 millimolar concentration DTT, 1 millimolar concentration benzyl fat · 1 μg / ml leucopeptin, pH δ. 5) * 50¾ ammonium sulfate is added to the soluble portion The precipitated protein K1 10,000 X s was recovered by centrifugation and re-dissolved in the lysate solution. • The solution was clarified by centrifugation and the lysis buffer was dialyzed. The dialysate was added to the cellulose androgen acetate P-11. (Wa tun) tube. Column 10 奄 mole concentration of trimethylolaminomethane-C1, printed by 10¾ glycerol, 1 奄 mole concentration EDTA, 1 millimolar DTT, pH 8.5, and K0-0.7 Mol NaCl gradient washing and isolation. The part containing p56RB * pllORB (determined by SDS-gel mold swimming) was combined, and the protein K 70% ammonium sulfate precipitation. K centrifugal recovery of precipitates and dialysis to 20 millimoles concentration of sodium sulphate * 0.2 mole of HaCl, 10¾. Glycerol * 1 millimoles concentration £ 0 Ding,?} { Refill in 7.5 * into 36? 3 (; 1 ^ 1-200 (Pharmacia) distribution column. In this purification step * t > 56RB was split from p110rb. The combined portion of pllOB was reloaded into DEAE (Column 11131>) and 10 millimoles of trimethylolamine methane-(: 1,105 (! Glycerol, 1 millimoles concentration of £ 0), 卩} 18.8, washed with 0- 〇_25 Molar concentration NaCI gradient analysis. The pllORm p56rb purity M Coomassie blue stained SDS-polyacrylamide gel used for this type of research> 95 ×. Hydrocarbon purification _-99-_ Paper size Applicable to China National Standard (CNTS) Α4 specification (210 × 297 mm) 466117

A7 B7 Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs. 5. Description of invention (%) pllOR ”Dp56Re is divided into wholes and stored at -80t3 at a concentration of 20 mils of sodium acid. 0.2 Molar degree HaCl, 10¾ glycerol * pH 7.5. MNC1-H596 NSCLC cells, each part of the RB protein • The 3H-thymidine absorption method described below was used to test its biological activity. The inactivation (control) part of PiiOB was prepared using the above procedure, except for lysis and The column column flush solution does not contain glycerol. The inactivated part of P110RB does not cause the growth inhibition of ΝΠ-Η596 cells at a concentration of less than 50 μg / ml in vitro. 125I-p110rb and 125I-P56RB use the amine-T method ( Nature (1962 194: 495) was prepared from purified p110rbSp56rb protein and 125 I (ICH BioBedica 1, Irvine, CA). Cell Source The cell source used in this study was obtained from the American Culture Collection. Cell Source NC1 -H596 (lung adenocarcinoma), A5 49 (squamous cell lung cancer) * 5637 (human bet bladder cancer), ΝΠ-Η69 (small cell lung cancer) | MDA-MB468 (breast cancer) * Saos (precious sarcoma), FHs 738B1 (Normal human bladder) · MRC-9 and VI-38 (normal human (Lung) and CCD 976Sk (normal human foreskin fortinoblastic mother cells) across Duback-modified Yage medium: at 37t: under humidified air / 7S; in a C02 incubator, 2 millimoles F-12 (v / v) of glutamine and 10¾ (v / v) fetal bovine serum (heat deactivation). Cell-derived phenotype: RBPC in this study) S indicates wild-type p110rb2 expression Cell-derived. RBne * means the cell-derived source of mutant, non-functional RB or the expression of RB protein in it Kot-RB MAb 3C3 (J.

IfflBuno 丨. Meth. (1994) 1δ9 ·· 231-240). Immunostaining or westward smearing of cell sources that cannot be measured.一 100 _ This paper ruler is used in Chinese National Standard (CNS) A4 size (2! 0X 297 mm) L ---------- tPlease read the notes on the back first. Moth )

• 1T 4 6 61 1 Yin Sang 85, the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs, 85.IUS revised year Ft α Α7 Β7 V. Description of the invention ------- In vitro growth inhibition test pllORB-Dose-induction curve of growth inhibition of media 2x 104 cells were seeded in a 96-well culture plate, allowed to stand overnight and allowed to attach and cultured at 0-25 μg / ml fesiB in RPMI-1640, 10X fetal bovine blood maggot (heat deactivation). Sai Gongzi 輋 抒 __ Xi Ying Lian 3 ΐ ΐ 咿 咿 捶 捶 捶 每-thymidine per C1 to each question. Collect cells and follow Proc. Hatl. A cad. S ci. -153) to test the absorption of 3H-thymic nucleus spines. For the time course of P110RB-mediated growth inhibition, 2 X 104 HCl-H596 (RBne *, KSCLC) cells were seeded in 96-well culture plates, and RPMI 1 640, 1 0¾ fetal bovine serum (heat deactivation) 50 μg / ml of p110rb. Cells were collected at the indicated times and subjected to a 3H-thymidine spatula. The nuclear-localization effect of P110RB The time-dependent nuclear localization effect of p110rb was determined as follows: NC1-H596 tumor cells MlOe cells / slots (triple troughs at each time point) were seeded in a 6-slot culture dish of RTMI-1640 (103! FBS) , Let stand overnight to allow it to adhere. 0.75wCi of 12M-pllORB was added to the medium at 8-hour intervals for a total of 72 hours. The medium was changed every 24 hours. At 0, 8, 24, 48, and 72 hours, the cells were cooled to 4¾ and rinsed 3 times with 1 ml of PBS to remove the remaining 12 s I-P 11 0 R B. Trypsin (Irvine Scientific) of cells K 0 5 ml 0.2 5 S! Was cultured in normal saline for 5 minutes, and then RPMI-1640 containing 10 ¾ fetal bovine serum (heat deactivation) was added. Collecting detached cells -101-This paper size applies Chinese National Standard (CMS) Α4 specification (2) 〇 × 297 mm) (Please read the precautions on the back before filling this page) 'Pack.

, 1T, Yu Η, 466117 -ψ ~~ η Α7 Β7 Five instructions, please fill in the l t t-item and then-fill out this page and centrifuge at 4lCM5 00 x g for 5 minutes to granulate cells. The supernatant was removed * and the cell pellet was resuspended in 1 ml of ice-cold hypotonic solution (0.01 moles of trimethylolaminomethane-HC1; 1 millimolar degree of MsCU, pH 7.6). K homogenized cells were sown for 15 times in a 15 ml dounce (yheaton). Centrifuge the homogenate at 4 t for 5 min * at 500 sec *. The particles (core) are resuspended in 1 ml of ice-cold hypotonic buffer. Contain the membrane and cytoplasm above the mash; below, centrifuge at 10,400 X g for 45 minutes. The remaining cytoplasmic fraction and granules (membrane fraction) of the supernatant were removed and resuspended in 1 ml of ice-cold low Zhangyuan solution. Liquid fraction scintillation calculation (Cytoscint liquid) was performed for 1/10 of each part (0.1 ml). Immunoprecipitation and identification of 125i-pllORB in the nuclear part of HC1-H596 3 X 10e NC1-H596 non-small cell lung (HSCLC) cells were seeded in 100 mm tissue culture dishes (Costar) 10% RPMI-16 40 medium (10X fetal calf serum (heat deactivation) * Allow to stand overnight to allow it to adhere. Remove the culture medium and add 10 liters of 1036 fetal calf serum (heat deactivation) and 0.8wCi 125I-p110rb (specific activity 0.008 wCi / μg) of fresh RPMI-1640. At the indicated time point * Μ 5 ml of HBSS-washed cells were printed by the Central Standards Bureau of the Ministry of Economic Affairs, Shellfish Consumer Cooperative, and collected and classified according to the above classification. Cells were collected within 48 hours Cells were replaced with fresh RPMI-1640 containing 0 × fetal bovine serum (heat deactivation) and 0.8 / iCi 125Ι-pllORB at 24 hours. 125I-p110rs Μ α-RB MAb 3C8 (Revealed in J, Immunol. Meth (1 994) 1 69: 23b 240) Immunoprecipitation, the complexes were subjected to fractionation on an 8-16¾trimethylolaminomethane-glycine SDS-polyacrylamide gel by immunoprecipitation, and transferred To nitropyroxin and phosphate imager model SF (Molecular Bio- 糸 -109 paper size) According to the Chinese National Standard (CNS) A4 specification (2 丨 0X297 mm) 4661 1 7 correction date δ5. II. Oli -fit! A 7 B7 5. Analysis of the invention ((0) system) analysis. Other lungs Cell source M is tested in the same way except that the culture medium is removed at 24 hours and replaced with fresh KPMI-1640 containing fetal bovine serum (heat deactivation) and 0.8iuCi 125I-p110rb. The purity of the nucleus and cytoplasm is determined by _Measure the NADH oxidase and NADH oxidase of each part according to the methods described in & 丨 £;} ^ 111. Biophys. Acta, (1971) 233: 334-347 and Biochen. J. (1982) 203: 245-251 5 'Nuclease enzyme activity measurement. The comparison of pllOR ”np56RB SHCI-H596 NSCLC cell core localization was performed as follows: 5 X 10s NCI-H596 cells were seeded in 1 ml RPMI-1640 medium (10 ¾ fetal calf Serum (heat deactivation) in a 6-slot hybrid culture plate (Costar) and let stand overnight. Remove the nutrient base * Add 1 ml of 10¾ fetal bovine serum (heat deactivation) to each tank And 230 pci 12BI-pllORB (specific activity 9.23 pCi / μg) or 991 pCi 12eI-p56RB (specific activity 39.6 pCi / micro ) Of fresh RPMI-1640. At the indicated time points • Ml ml of HBSS cells from 3 wells were washed twice • collected • pooled, and the nuclear fraction was isolated as described above. Nuclear-specific radioactivity is quantified in a scintillation counter. Treatment of subcutaneous tumors around the tumor The Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs printed a group of three nude mice (Ba lb / C NCR nu / nu female, SiBonsen, Gilroy, Ca) each M107 Ν (Π-Η596 (RBn ··· ) Or 107 A549 (RBPCJS) NSCLC cells are inoculated subcutaneously. Mice with tumors are allocated to different cages with the urn. When the tumor size is about 9 square meters, RB protein is injected subcutaneously around the tumor. Each tumor is divided into 4 portions every 6 hours. Alternately inject 25 micrograms of RB protein subcutaneously into each quarter for a total of 10 days. Each animal receives a total of -103 -_ This paper size applies to China National Standard (CNS) A4 (210X297 gong) 4 6 611 Employees of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 printed by the consumer cooperative V. Description of the invention (id) 100 micrograms of RB protein, a total of 1 milligram during the 10-day eaves. Use a vernier caliper twice a week to measure the tumor in two dimensions. Intravenous pllO 30 Nine (39) female Ba and b / C naked M (Sonnonsen Labora-iUWfeS, 81 Πδ) were swollen, stunned, and stunned for 1 day, and then measured by a K caliper for 81 days. Mice are randomized according to tumor size Each time it is divided into four sisters, each sister contains 9-10 mice. During the three-week period, the mice received one dose (5 doses / week) of active P110RB 50 micrograms per dose per dose; the active P110RB 200 micrograms per dose Or inactivated p110rb 200 micrograms / dose. The biological activity of p110rb is determined by the ability of K to inhibit HC1-H596 tumor cells in the 3H-thymidine absorption test described in Figure 22. Tumor size (1, w, h ) And body weight were measured twice a week by two individual investigators and observed the pathological signs of mice every day. Assuming that the radius of the spherical geometry is equal to half of the measured tumor length, the tumor volume of each animal was estimated. Using Abe (Abe us Software Berkeley, CA) completed the Mann-Whitney U-test to compare the average tumor size between sisters. The effect of RB protein on tumor cell differentiation The RB protein selectively inhibits RBnee non-small cell lung cancer cell source NC1- The ability of H596 to grow is shown in Figure 19A. The results demonstrate that 110 ^ is a M-dependent way to inhibit the growth of NC cells 596 cells. In this study, pllORStIC50 was about 5 μg / ml [about 50 μmole concentration]. Interesting Yes, it contains growth inhibitory P110r6 C-terminal derivative of the "pocket" region, p56RB (amino acid 379-928) (Hoi, Cell -104-This paper size applies to China National Standard (CNS) A4 specification (210 × 297 mm)) (Please Please read the notes on the back *, and fill in this page) Order I. 6117 Half ag correction heart 1.0. Move 'Punch A7 B7 Printed by the Staff Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of Invention (1993) 13: 3384 -3391; Genes and Develop. (1992) 6: 953-964), which only slightly inhibited growth in N (Π-H596 tumor cells) (Figure 19A). More importantly, what's more important is that pii〇rb is more resistant to picking RB " e * NSCLC cell source NC1-H596, because whatever? Neither 110 ^ nor p56rb inhibited the growth of rbpos HSCL {: cell-derived A549 (Figure 19A). The time dependence of p110rb-mediated NU-H596 tumor cell growth inhibition was also investigated by M. When tumor cells were treated with 25 μg / ml P110RB, a decrease in 3H-thymidine uptake was observed after 24 hours (drawing 20B) and a maximal inhibitory effect at 48 hours was observed. This delay may be due to a combination of several factors * including the internalization dynamics and the fact that the cell population is non-synchronized. In order to prove that the observed growth inhibitory effect can occur in various tumor cell types, 4 types of tumor cell sources, 1 type of ΙμΒμ »tumor cells and 4 types of normal epithelial cells in equal amounts? 110 ^ subcutaneous treatment (Table 4). All RBne tumor cell sources tested showed a significant decrease in 3Η-thymidine uptake at 72 hours (60-80¾ inhibition of DNA synthesis). In contrast * under these conditions * RBPCS HSCLC cell source A549, normal lung epithelial cell source HRC-9 and VI-38, normal bladder epithelial cell source FHs 739B1, and foreskin Panveum cell source CCD 976S1C did not inhibit growth. This data is consistent with the modest effect of RB gene expression on cell growth in large numbers in normal cells, and a surprisingly large number of p110rb manifestations may be growth-inhibitory.

Please read the precautions before reading it, and then fill in J and I. 0_5_ This paper size applies to China National Standard (CNS) A4 (210X297 mm) 4661 1 7 Β5.Π.〇δ month a A7 B7 5 Description of the invention: Cell-derived cell form RB status Relative growth rate Normal cell-derived FHs739Bl Bladder epithelial cells + 84 ± 1 CCD976SK foreskin fibroblasts + 89 ± 1.5 HRC-9 lung epithelial cells + 86 ± 3 VI-38 76 ± 3 tumor cell source A549 NSCLC + 99 ± 1, 5 H596 NSCLC 11 ± 1.5 HC1-H69 SCLC-30 ± 8 MDA-HB-468 Breast adenocarcinoma-24 ± 4 5637 Bladder cancer-37 ± 1.5 Table 4 in 72 The effect of pllOB on human 3H-thymidine in cells at hour. 5 X 103 cells were seeded in each well of a 96-well culture plate and allowed to stand overnight for attachment. Add p110rbM medium to a final concentration of 25 μg / ml. Including pi 10RB medium printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs is replaced every 12 hours for a total of 72 hours. Cells are collected and the 3Η-thymidine absorption is tested as described above. The cell division rates shown are the average of 12 individual data points and the standard deviation of the percentage ± mean of the M Yuan wash control. "+" RB status indicates wild-type RB status indicates mutant RB or lack of measurable performance. -1 0 fi-This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) 4661 17 Amendment ^ 1 ... Supplement 4 A7 B7 V. Invention? Cell absorption and nuclear localization of P110RB Central Bureau of Standards, Ministry of Economic Affairs P110rb printed by Shelley Consumer Cooperative is a kind of nuclear phosphoprotein, and its interaction with the transcript elements of the bond such as E2F underestimates its regulatory role in the cell cycle (see TIBS (1992) 17: 312-315 and Science ( 1992) 25δ: 424-429 for research). Therefore * If P110RB enters the cell and its normal pathway affects its growth, it must be located in the nucleus of the target cell. Accordingly • Add 12SI-? 110118 to ^ 1 (: 1-11596 tumor cell culture. Collect the cells and tiller to the membrane according to this patent specification. • Enriched sister fraction of cytoplasm and nucleus. Figure The time-dependent internalization shown by 21A becomes various subcellular parts. The acidic-sinking source of radioactive accumulation occurs earliest in the nucleus * although the measurable amount appears between 8-14 hours after treatment. Part of the membrane. The time course (peak appears at 48 hours) and nuclear localization (internal radioactivity within 70X in the nucleus) are consistent with the time previously described for p110rb in plutonium 21A-growth-inhibitory effect by particle. To prove The complete 12βΙ-Ρΐ10κβώ is now processed in the nucleus of the cell * to isolate the nucleus. The immunoprecipitate prepared by Mc ct-110RB antibody was fractionated on an SDS-polyacrylamide gel and analyzed by autoradiography (Figure 21B) ). A 110 kD radiolabeled band appears in a time-dependent manner * shows that the complete 126I-p110rb appears in the nucleus. The acid-irrigable 12 * 1 cpm was tested in RBPQS lung tumor cell sources MRC-9 and A549. Time-dependent nuclear accumulation The results are similar to those of 12eI-i> 110RB which can be immunoprecipitated, and the results are similar to Π (Π-Η596 (drawing 21C). The results show that the relative insensitivity of this type of RBP ° S cell source to RB protein is not because it cannot be internalized or Locate p11〇rb in the nucleus. The RB " e «cell source's weak growth inhibition of p56rb is reversed. The paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) 466117

A7 B7 V. Description of the invention Ui) 懕 can be interpreted as too little P561 21D) in the cell. The diagnosis of ppRB11 <? was obtained from biopsies of patients or labeled with 32P-phosphate and precipitated. Or * extracted from active radioactive or non-radioactive direct diagnosis to smear analysis. As a diagnostic tool for judging other diseases of retinal germ cells. Tumor suppressor gene of lung cancer. Tumor suppressor gene plays a role in mutating tumors. Tumor Suppression • The first to appear is the retina in retinoid meningoblastoma. The retinal specimen is rejected and the second indactyl is inactive. Description of the RB gene (pi 10Re). Pi 10RB appears to be factor E2F binding and regulates the amount of ΙΒ stored in the nucleus of the target cell (graphical identification of isolated tumor cells M 355 methionine above procedures M anti-ppRBUo Is (3 immunostaining protein) The lysate can be controlled by the presence or absence of proteins labeled with the labeled anti-RB specific antibody Western immunoprecipitated protein or the absence of tumor or retinoblastoma gene control. Deactivation of white matter therapy. A study of the importance of its genes for human cancers in human germ cell tumors. The deactivation of a hereditary reticuloblastoma (RB) gene has been shown to have an effect on the growth and differentiation of progeny in the offspring. Encodes a nuclear phosphoprotein through several times the target protein * including the evolution of the transcriptional cell cycle. RB gene structure and table-· 1 I II I-1 I ---. 1 I n (Please read the precautions on the back first ^; Write this page)

* 1T. • tl

I Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs. Leiden line of a type II cancer RB. The presynaptic change of necrosis factor is basic- RB-based cancer support M standard lung. The orthodox disease type includes malicious control, sexual bed complexes, fluids, sexually-prone blood, can-eating diseases, and essential malignant cancers, which can cause severe cancer. The main causes are polymorphism of humans into RB. Cell Xu Shengsheng often thin and urinary tumors are heterogeneous edema and swelling into the tumor-cancer in the present-spot Teng table first special paper size common Chinese National Standard (CNS) A4 size (210X297 mm) 46611 7 Printed by the Central Standards Bureau of the Ministry of Economic Affairs, Shellfish Consumer Cooperative, S5.U.㈣ Amendment. ¾ A7 B7 V. Description of the invention ((Ά) The recent decrease in p110rb has been shown to refer to patients with acute bone-associated leukemia and early cystic carcinoma Early warning of disease deterioration (Researched in Annual Rev. Biochea · (1993) 62: 623-651). Eyelash fixation therapy enamel characterized by mutation or mutation of RB gene during the expression of P110RB The paralysis researcher's research on pre-suspension protein replacement therapy "* wherein p11〇rb • or its functional derivative is incorporated into tumor cells characterized by the loss of K-type wild-type tumor suppressor protein. It The results showed that exogenously added pllORBM entered the cells in the culture. It is located in the nuclear region and inhibits the DN Α synthesis of tumor cells. In addition, pH〇rb # can be used to intestine tumor growth on clinically effective animals in human lung cancer. PllORBt truncated body is called p56rb. It also has limited activity in vivo. This result further supports the application of tumour suppressor gene protein therapy in human lung cancer characterized by K-deficient RB. P110rb inhibits tumor formation in vivo. The in vitro growth inhibitory effect extends to nude mice xenograft tumor bodies. Based on this, subcutaneous NC] [-H59S tumors were established in nude mice, and RB protein therapy was started and maintained for 10 days. Subcutaneous * peripheral tumor administration of RB protein sold resulted in Significant inhibition of tumor growth (Pu 24A). In p110rb-treated mice, tumor size did not increase significantly at about 8 weeks *, but in mice treated with M p56RB or control buffer, the progression of the tumor was relatively Fast "The effect of RB protein treatment is long-lasting, because at day 75-90, the tumor rate in tumors in pll0RB-treated mice is increased by -109.-This paper applies Chinese national standards CNS) A4 specification (210X297 mm) (Please read the precautions on the back before writing this page) H. Thread 466117 i5.il. June 2006 £) Amend A7 B7 Staff Consumption of Central Bureau of Standards, Ministry of Economic Affairs Printed by the cooperative V. Description of the invention (t ~ ------------- (about 3 square millimeters / day) is significantly lower than that of buffer-treated mice (about 20 square millimeters / day). A549 NSCLC tumors were established in nude mice and treated in the same manner as described in Figure 22A. The data (Figure 22B) demonstrates that pll0R "Bp5 6Ra protein therapy has no effect on the growth of A549 tumors, showing that only RBne« swelling and swollen growth are reduced. When localized, subcutaneous therapy can be used to demonstrate that RB protein reduces lung cancer in vivo For the ability of cell-derived tumorigenesis, systemic therapy is a more rigorous test to test the possible medical value of ρΠ0κβ. Based on this * intravenous inspection is used to deliver multiple doses of active or inactive p110rb to humans with the above-mentioned subcutaneous NC1-H596 Balb / C nude mice with lung tumors. Data (Figure 23) demonstrate that pU0rbK transported by radon effectively blocked tumor growth in a dose-dependent manner. Day 49 • Mean tumors in mice treated with 200 μg / dose of active p110rb Dimensions significantly smaller than inactive p110rb (p < 0.01) or lower doses (50 micrograms / dose) of pU0rb (p < 0.05) treated mice at the same dose as K. The highest tumor growth rate observed was when receiving RB protein inactivation Preparations (determined by lack of activity in in vitro 3H-thymidine incorporation test) or sham control group-treated mice. K-active p110rb-treated mice No signs of poisoning or morbidity in the study. Previous studies based on measurements of soft agar and subendothelial tumor formation in nude mice have been shown to reincorporate the RB gene into the RBne * background to reverse tumorigenesis in many cell types The phenotype of RB. Including functional RB gene into RBne tumor bundle cells, resulting in the production of RB protein in cells (see -110- This paper size applies to the Chinese family standard (CMS > A4 size (210X297 mm) lr -------- = ') Outfit-(Please read the note on the back first to buy in H copy) Order 4 6 61 17

Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs.

Cancer Res. (1 992) 52: 1968-1 973). If p 11 0RB is included in tumor cells, the growth inhibitory effect of RB gene replacement can also be observed. «This application is shown in the G1 early base? 110 »^ or 956» * 8 microinjection synchronization Saos2 cells stopped growing and were blocked from entering S phase. Because microinjection is very time-consuming and impractical for the treatment of traditional diseases, it was decided to determine whether the cell source can respond to externally added RB proteins. Two NSCLC cells were originally selected for this study. NC-H596 cannot express a measurable amount of pllORe * based on immunosmearing and A549 produces a normal amount of p110rb (see I. Immunol. Heth. (1994) 169: 23 bu 240). The data reported in this patent specification clearly demonstrate the effective dose-sensing effect of P11OκB on HC and H549 NSCLC tumor cells in vitro. The source of I? BPes a549 NSCLC cancer cells was not inhibited by p1 orb, showing the preference effect of P110RB on cells with defective RB expression. This result also suggests that the growth inhibition effect observed with Ntn-H596 NSCLC is not due to the non-specific toxic component of the applied RB preparation.

PllORB inhibits the growth of three other RBne * cell sources. MDA-MB-463 derived from breast cancer cells contains a partial deletion of the RB gene and site-directed mutations in its p53 gene (see Mol. Cell Biol. (1 990) 9: 1628-1634 and Oncogene (1993) S : 279-28S). The single-copy copy of the human RB gene was incorporated into the reverse transcriptase vector into MDA-MB-468, resulting in a reduction of tumorigenesis in nude mice and a reduction of growth ability in soft agar soil, but a growth rate in culture Not affected. In the experiments shown in this patent specification, exogenous pUORb inhibited the growth of MDA-MB-468. The degree of MDA-MB-468's in vitro growth inhibition effect may be similar to that achieved by such experiments. ________ " 1 1 1 ~ This paper is in accordance with China National Standard (CNS) A4 (210X297 mm).

4 6 6117 Printed S5.1L (16. Shaving A7 B7 5 'invention by Central Laboratories of the Central Bureau of Standards, Ministry of Economic Affairs, PillORSfi has been found to be higher in cells. When M SDS-polyvinylamine gel electrophoresis analysis Although 5637 and MDA-MB-46S cell sources cannot produce measurable ϋΒ protein, SCLC cell source NCI Η-69 produces a disordered migration species of RB (Proc. Hatl. Acad. Sci. USA (1990) 87: 2775 -2779). On the NCI H-69, the abnormal conjugation of the raRHA precursor was caused by the sister's site-specific burst fee. • The expression sequence 21 in the nRNA was structurally fused to the expression sequence 23, and 33 amines in the expression sequence 22 dense brick sequence were removed. RB protein produced in this type of cells. K has less surface molecular weight migration than PllORB by ~ 4 kD * and is defective for E1A and SV-40 T-antigen binding. Reintegrated into wild-type? 110 ~ To "(: 1 H-69, the resulting condition is the coexistence of functional wild-type and non-functional mutant KB proteins. The appearance of mutant RB in NCI H-69 does not appear to be in this test. Dry search of growth inhibition caused by wild-type PllORB. Growth of RB media in this type of experiment The inhibitory effect is specific to the RBn · »cell source. PΠ0 does not inhibit the growth of normal bladder, foreskin-like androgenic cells, lung epithelial cell source, and RB NSCLC cell source. Under normal culture conditions * excessive expression of pU0rb & does not affect HIH 3T3纩 The growth rate of male mother cells (Exp. Cell Res. (1 9 9 3) 2 0 7: 99-1 0 6). This data is contrary to another report, which overexpresses wild-type pnoRB, etc. Growth arrest (Oncogene (1993) 8: 2659-2672). In this study, growth arrest of lung epithelial cell sources such as WI-38 was achieved by plastids that showed RB cDHA metastasis infection and then selected stable metastatic infections. The inability to see the growth inhibitory effect in normal cell sources may be limited by the amount reached in this method -112- This paper size is applicable to China National Standard (CNS) A4 (210X297 mm)-] / ------ -:. > equipment-(please read the notes on the back #, write this page) I. > ar. 诛 4 6 6117

Α7 Β7 Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs 5. Description of the invention (, 〆) The concentration of piiorb in the cells. Absorption of radiolabeled pn〇RB shows that only a small portion (0.5-13 added; cpm) of 1.25I-pllORB is absorbed by cells. Studies of microinjection of RB protein in synchronized cells have shown that the time and duration of protein injection A significant delay between the times of growth inhibition observed (Cell (1991) 67: 293-302). The time course of P110RB-mediated growth inhibition (Figure 21B) also reflects this delay. This type of data is consistent with the time-dependent verification of 12SI-p110rb seen in circle 21B. The nuclear localization effect of exogenously added p110rb2 is consistent with the report that p110rb should be localized to the nucleus in order to have a growth-inhibitory active box (Mol. Cel I. Biol. (1 993) 13: 4588-4599) ° RB protein is a cell proliferative effect An important regulator. The research presented in this patent specification provides RB function that does not require genetic modification to return to normal in RBne «. It is significant * because the loss of RB function occurs in a wide variety of tumor types. The growth inhibition of various tumor cells in vitro proved that the exogenous RB protein can functionally replace the endogenous wild-type RB. And normal cells can absorb p110rb * and its growth is not inhibited, which proves that pU〇rb has selective anti-proliferative activity against RBnee tumor cells. Because reduced tumorigenesis in vivo is a more sensitive indicator of restoring tumor suppressive function * experiments include nude mice xenograft models. Recent observations have shown that reintroduction of the RB gene into RB SCLC cells with other gene love can inhibit tumorigenesis in nude mice (Oncogene (1993) 8: 2175-2181). '' Various RBn * · Tumor Cell Growth Inhibition Prove Exogenous 0 Egg_: ----_ 1 1 3-This paper size is applicable to Chinese National Standard (CNS) Α4 size (210X297 mm) /..Package- -(Please read the precautions on the back before you write this page) Order No. 831 1 1748 Patent Application 4 6 β 1 1 7 Chinese Manual Correction Page (August 1990) ^-Description of the invention ( )

White matter can work pllORB > Cells have a choice of complex tumor suppression horizontal. Figure 1 is a rabbit epidemic nursery 2 is a basic acid sequence nursery 3 is a figure 4 is a stubborn figure 5 A is a figure 5B is a dumping garden 6A Ii) Capable of replacing endogenous wild-type RB. While normal cells can absorb the growth of their lines and are not inhibited, it is suggested that pllORB has a fine anti-proliferative activity against RBne * tumors. As the reduced tumorigenesis in wax is a more sensitive indicator of recovery function * Each experiment includes a chromatogram of nude mice xenograft, which describes the identification of RB protein by M rabbit anti-RB IsG precipitation method in various cell sources. Complete RB cDNA nucleotide sequence and putative amine of RB protein. Chromatogram * Reveals modifications to RB protein. Chromatograms * show RB gene production in different vertebrate species. Chromatograms * show the localization of RB proteins. The chromatogram * shows the results of immunofluorescence studies of RB protein in U20S from noble sarcoma cells. Column chromatography photo of RB gene 瞵 protein in single strand DNA cellulose Figure 6B is a column chromatography picture of RB gene phosphoprotein in double strand DNA cellulose The figure shows the production of TRPE-RB fusion protein. Fig. 7B is a photograph of a polyacrylamide gel electrophoresis of the TRP E-RB fusion protein. FIG. 8A is a chromatogram showing immunoprecipitates of RB protein / anti-ILB protein IsG from various human cell sources. 114-This paper size applies the Chinese national standard (CNS > A4 size (210X297 mm) No. 83111748 patent application Chinese manual revision (A7 B7 in August 1990 (Amendment 丨 ^ Supplement 1 Figure 8B is a chromatography garden • The RB protein / anti-RB protein 圄 9 is a chromatogram and shows the effect. Figure 10 A shows the performance of white matter and baculovirus added to 10 × Figure 10B is the autoradiography spectrum from a parallel gel. Figure 11 is a chromatogram, p110rb and SV40 T-anticircle 12 are photographs. • Nucleus shift of the described protein is described. Figure 13 is used for microinjection. SDS-PAGE analysis. Figures 14 to 14F are micrographs. According to injection and immunostaining. Draw 15M to show the immunoprecipitation of IgG from several retinal blastoma cells. Biochemical separation is used to prove the protein SDS-PAGE, Coomassie blue-stained fusion egg. P110RB's south-westward DNA binding test. The complex formation of RB protein protease expressed by culm virus by the coagulated smear shown in Figure 10A with K. Injected into the cytoplasm of Soas-2 female cells and passed through the blunt position. The concentration and purity of the protein preparation Percentages of micro-reported cells labeled with released hydrocarbon-labeled Saos-2 cells up to 4 days after injection (please read the note on the back before filling this page) Figure 16 illustrates the percentage of meridian cells and P56 of the meridian system 0, and Figure 17 illustrates the effects of the 56? Injection on cells in phase 5. Figure 18 A to 18CK chart illustrates the cell cycle response to injection Dependence on truncation of protein. -114i This paper size is applicable to China National Standard (CNS) A4 (210X297 gong). Printed by the Central Bureau of Standards of the Ministry of Economic Affairs, No. 83111748, patent application case 4 * 6611 * 7 Chinese manual correction page (Aug. 90) ^-B7年月 # 死. .. —_ Figure 19 shows that pllORB inhibits the growth of small cell and non-small cell lung cancer cell sources that are defective in the Rb protein epithelium. Figure 20 is the result of the time course study of RB-medium growth inhibitory effect. 圜 21A shows the time-dependent localization effect of 125I-pll0aB on each cell part. Figure 21B shows the 125I- Precipitation and identification of rabbit epidemic of p110rs. Figure 21C shows Immunoprecipitation and identification of i2SI-pll0RB in the nucleus portion of lung cancer cells. Figure 21D shows the quantification of nuclear-localized la5I in N (Π-Η596 NSCLC cells. Circle 22A shows the NC1-H596 (RB " * «) Tumor size represents the mean ± SD of the three animals tested. Figure 22B shows the A549 (RB ~ tumor size represents the mean ± SD of 3 animals of each experimental sister. B 23 shows the effect of intravenous injection of p110rb on the growth of human lung tumor cells in nude mice, and 蹰 24 shows that pllORB can be used in vitro. Inhibits the growth of non-small cell lung cancer cells, but has no effect on the proliferation of normal cell sources "Figure 25 shows that pi 10RB inhibits the growth of small cells and non-small oil cell lung cancer cell sources that are deficient in the expression of Rb protein. Figure 26 shows pi 10RBq enters the cell and localizes to the nucleus. Plant 27 shows that intact p110rb can be measured in the nucleus of K-labeled Pii〇w-treated H5 96 tumor cells. -114b-This paper size applies Chinese national standards (CNS > A4 Specifications (210X297mm) (Please read the notes on the back before filling this page)

Revised page of Patent Application No. 83111748 (90 months in 1990) ^-Invention theory! g () Read correction _ supplement Η 2δ display? 110! ^ And? 5611 & is biologically active for subcutaneous treatment of non-small cell lung cancer in animals. Figure 29 shows non-hydrocarbon enteric therapy for lung cancer using piloRB in an appropriate phantom. Figure 30 shows the amino acid sequence of the retinoblastoma protein produced in E. coli. Figure 31 illustrates the construction of a baculovirus expression vector for pp110rb synthesis. Figures 32A and 32B show the smear of shangxi spores (蹰 32A) and identification of cell extracts from ppRB-infected insect cells. The cell extracts are from infected cells 72 hours after infection. Figure 33 is the result of phosphorylation and dephosphorylation analysis of RB protein released in insect cells radiated from temporal spectrum. (Please read the note on the back before filling in this page) Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs -114c-This paper size is in accordance with Chinese standards (CNS > Α4 size (210X297 mm)

Claims (1)

苐 83111748 patent application 4 6 6, the applicant asked the amendment of the patent scope (August 90) A8 C8 D8 patent application The Central Bureau of Standards, Ministry of Economic Affairs, and the Industrial Cooperative Cooperative has printed ^ Jie Caijie compound, which produces the proliferation of diseased proliferating cells. Among them, the morbidity of the cells is the lack of cells or the amount of cells. The following Result of functional retinal germ cells with polypeptide or protein 2. The drug compound according to item 1 of the patent application scope, which lacks or has-· ·. Functional retinal germ cell replacement polypeptide or protein at the required amount of K is a pathologically mutated retinoblastoma gene Results "3. According to the first patent application scope of the road medicine sister compound * which inhibits the pathologically proliferating cells M no community proliferation (apoptosis) * cell death * differentiation or benign phenotype is characterized. 4. According to the first patent application scope of the road drug sister compound, the pathologically proliferating female cells are retinal cells, prostate cells, colon cells, lung cells, • 'sarcoma cells * leukemia cells * or lymphoma cells. .5. According to the peony compound of the first patent application scope, the morbidly proliferated female cells are mammalian cells. 1; ', V * 6. According to the medical contraindication compound in item 1 of the scope of the application, the cell line, /. ·' Is in contact with P56RB or 'external contact with p56RB p / *'; '' ... 7 . A kind of Lu Cai Cai composition I for treating patients with primary (primary) or epidemiological L. retinal germ cell tumor-related rods. It includes: teaching patients to administer 'Effect.! 1 of p5 6RB. • \ _. 8. According to the 7th patent application scope of the peony medicine omentum. The relevant buried cancer is visual = omental blastoma. 9. According to the scope of patent application No. 7-Road medicine composition. Dairy animals. Among them, the primary view is that the patient is lactating 10. According to the application, please apply for the S-sister compound in item 7 of the patent scope *, where the paper size of this paper is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) ) (Please read the precautions on the back before filling this page) _ j AS 4 6 6117 as D8 VI. Application for Patent Scope Compounds are administered to patients via intravenous administration, oral administration, intratumoral injection or 0 pickled endometrial abdomen (Please read the precautions on the back before filling out this page) Set the paper size of the printed papers of the Central Standards Bureau of the Ministry of Economic Affairs and the Consumer Cooperatives, using the Chinese National Standard (CNS) A4 specification (2 〖〇 × 2 dozen mm)