KR100285292B1 - Human cyclin b2 gene cloning, method for producing anti-human cyclin b2 antibody, and use thereof - Google Patents

Abstract

PURPOSE: A human cyclin B2 gene, a method for producing an anti-human cyclin B2 antibody, and its use as a diagnosis reagent of cancer are provided, thereby easily distinguishing the benign cancer and malignant cancer. CONSTITUTION: The human cyclin B2 gene, which is a regulating a subunit of M-phase/maturation promoting factor(MPF) and isolated from cDNA of human fetal liver, has the nucleotide sequence and amino acid sequence of figure 1a and comprises 5' untranslated region (5' UTR) and 3' untranslated region (3' UTR). An expression vector pMALC-p(KCTC 0458 BP) comprises the human cyclin B2 gene. The method for producing an anti-human cyclin B2 antibody comprises the steps of: transforming E. coli with pMALC-p and pRESET-A(KCTC 0459 BP); cultivating transformed E. coli to produce human cyclin B2 protein; administering the human cyclin B2 protein into an animal such as a mouse to produce an anti-human cyclin B2 antibody. The cancer diagnosis reagent contains the anti-human cyclin B2 antibody as an active ingredient.

Description

Human Cycline Ｂ2 Gene Cloning and Anti-human Cycline Ｂ2 Antibody Preparation and Use thereof

The present invention isolates and clones human cyclin B2 gene to produce protein expression vector (Accession No. KCTC 0458 BP: pMALC-p and KCTC 0459 BP: pRSETC-A), isolates and purifies human cyclin B2 protein, and anti-human cyclin B2. The present invention relates to the diagnosis and treatment of proliferation, division and cancer differentiation of human tissues and cells.

More specifically, a new human cyclin B2 gene is isolated, cloned, and purified by the expression vector (pMALC-p and pRSETC-A) expressed in Escherichia coli (DH5α and DL21) to separate and purify the inoculated in rabbits or mouse high. A novel antibody for the diagnosis and treatment of proliferation and division of human tissue cells and cancer differentiation by the production of polyclonal and monoclonal antibodies using bridoma.

Cyclin genes that regulate the human cell cycle include Hunt T, J Cell Sci Suppl 1989; 12: 77-97, Nature 1991; 350: 462-63, et al., Pines J, Cell Growth & Differ 1991; 2: 462-63) are classified into three groups, G1, A, and B, based on amino acid sequence homology and timing in the cell cycle. Each of these groups has at least two members, including type B cyclins, and type B cyclins have been identified from a variety of species, with human cyclins B1 and B2 being similar to Xenophus amphibian cyclins B1 and B2. (Minshull J et al, Cell 1989; 56: 947-56) and Jackman M et al, EMBO J 1995; 14: 1646-54.

Cell division by Evans T et al, Cell 1983; 33: 389-96, et al. Is due to the activity of protein kinase, an ^MF promoting factor (MPF) consisting of subunits of p34 ^cdc2 and cyclin B. Cyclin B is known as a regulatory subunit that varies with the cell cycle. The end of division in the mid-to-late phase during cell division is induced by the destruction of cyclin B, which in turn causes inactivation of cell division promoters. Cyclin B accumulates as cells enter the interphase, whereby cell division promoters are reactivated and cell division occurs. Cyclin B is the primary cell division cyclin, and the specific role of different types of cyclin B in animal cells is not known yet (Epstein CB and Cross FR, Genes Dev 1992; 6: 1695-706). Recently, we prepared a human fetal liver liver CD (NAD) library (Choi SS et al, Mamm. Genome 1995; 6: 653-57) and expressed by DNA sequence expression (EST). The nucleotide sequence of clone (F1-104D) could demonstrate homology with cyclin B2 in mice.

Currently, human cyclin B2 is not registered in the database and published in the paper, but the base sequence is cited in papers such as Chapman et al, Development 1993; 118: 229-40. have. However, the F1-104D clone cloned by the present inventors was found to have a different nucleotide sequence from the previously cited human cyclin B2 as shown in FIGS. 1 and 2.

In order to study whether cyclin B2 is present and functioning according to various cell types, we cloned the cyclin B2 gene fragment by reverse transcriptase chain reaction (RT-PCR) in several tissues and cells. As determined, the nucleotide sequence was identical to that of the clone of the present inventors. Therefore, the existing human cyclin B2 gene was found to be an error in the determination of clones or sequences.

In general, the method for measuring cell proliferation was dependent on laboratory culture with tritium thymidine or bromodioxyuridine (BrdU) (Hall PA, and Lesion DA, J Clin Path 1990; 43: 184-). 92) This method is uncertain because cell proliferation in culture does not reflect the biotic state. On the other hand, direct immunohistochemistry using Ki-67 antigens or proliferating cell nuclear antigens (PCNA), which show cell cycle-related antigens, is also possible. These methods also have many limitations as markers for proliferating cells (Gerdes J). et al, Int J Cancer 1983; 31: 13-20, McComic D et al, Histopathology 1992; 21: 591-94). In practice, the method of directly calculating cell division is most preferable, but it is time-consuming and difficult to apply in paraffin-embedded tissues (Goodal RA et a.l, Gastroenterology 1991; 101: 1235-41). Therefore, immunohistochemical staining using an anti-human cyclin B2 antibody, which is a novel antibody of the present invention, is a test that can be easily examined on a large scale at a general pathological task level. It will be a marker. In particular, it is effective in distinguishing malignant cancer from benign cancer. In addition, it is very useful as an antibody used for Western blotting, ELISA test, and phagocytosis.

FIG. 1 (a) shows the base and amino acid sequences of human cyclin B2 isolated and identified from an expressed sequence tag (EST) produced by human fetal liver cDNA.

Figure 1 (b) is an amino acid sequence showing the destruction box (X), cytoplasmic storage signal box (Y) and cyclin box (Z) in the human cyclin B2 gene.

Figure 1 (c) shows the difference between the amino acid sequence of a part of the amino acid start region of cyclin B2 and the present human cyclin B2 in several species.

Figure 2 (a) shows the starter (primer) position of the reverse transcriptase polymerase chain reaction to confirm the identity of the first half gene sequence in various tissues and cell lines.

Figure 2 (b) is a picture of the electrophoresis of the reverse transcriptase polymerase chain reaction product on 2% agarose gel to confirm the identity of the first half gene sequence in various tissues and cell lines.

3 is a gene map of the human cyclin B2 expression vector pMALC-p in which the human cyclin B2 gene was inserted into the polylinker region of the E. coli expression vector pMAL-p.

4 is a genetic map of the human cyclin B2 expression vector pRSETC-A in which the human cyclin B2 gene is inserted into the multi-linker site of E. coli expression vector pRSET-A.

5 (a) shows maltose binding protein / human cyclin B2 and metal binding protein / human cyclin B2 in vector expressed in E. coli (DH5α, DL21) using human cyclin B2 gene expression vectors pMALC-p and pRSET-A. The complex protein is an SDS-PAGE electrophoresis picture of the protein.

FIG. 5 (b) shows the maltose binding protein / human cyclin B2 and metal binding protein / human cyclin B2 complex protein expressed in E. coli (DHα5 and DL21) using human cyclin B2 gene expression vectors pMALC-p and pRSET-A. Human cyclin B2 complex protein was electrophoresed by SD page after eluting an amylose column or nickel enantio (Ni ²⁺ -NTA) agarose column.

Fig. 6 is a lysate containing (a) maltose-binding macrophages / human cyclin B2 expressed in Escherichia coli (DH5α) by an anti-human cyclin B2 antibody obtained by inoculating rabbits with human cyclin B2 protein (b) In the gastric cancer tissue, a single band was shown by Weston immunoblot.

7 shows normal gastric (A), benign gastric tumor (gastroadenoma) (B, C) and malignant gastric cancer tissues (D, E) and normal colonic mucosal epithelium (F, G) using anti-human cyclin B2 antibody, Microscopic picture by immunohistochemical staining showing the expression level in benign colorectal cancer (colon adenoma) (H) and malignant colorectal cancer tissue (I, J).

The present invention provides cloning, sequencing of human cyclin B2 gene, protein expression vector of human cyclin B2 (Accession No. KCTC 0458 BP: pMALC-p, KCTC 0459 BP: pRSETC-A), preparation and purification of human cyclin B2 protein. , Antihuman cyclin B2 antibody production step.

<Example 1>: Human fetal liver mRNA isolation and cDNA library construction

Total RNA was isolated from fetal liver tissue at 22 weeks by GTC (guanidine thiocyanate), and poly (A) + RNA was isolated from oligoti cellulose chromatography (oligo (dT) -cellulose. Separated by chromatography to prepare a directional cDNA library (cDNA library) using a cDNA synthesis kit. First, we synthesize primary helix DNA from 4μg poly (A) + RNA using a starter having Xhol restriction enzyme cleavage site using MMO (mLV: moloney murine leukemia virus) transcriptase. After synthesizing the secondary helix cDNA, EcoRI adapters are attached to both ends of the cDNA, cleaved with Xhol restriction enzyme, and then removed with small fragments of base or initiator using a sephacryl spin column and cleaved with EcoRI-xhol restriction enzyme. One plasmid piBluescript SK- (II) vector.

<Example 2> cDNA clone production and nucleotide sequence determination

The cDNA was transferred to XLI-blue MRF 'Diech 5 alpha Escherichia coli (DH5αE. Coli), and a double-stranded plasmid template was obtained by alkali dissolution to obtain 4μg of which was deoxy-chain termination; F et al, Proc Natl Acad Sci USA 1977; 74: 5463-67) determine the nucleotide sequence of the 5 prime end region. Intermittently, the template is alkali denatured and the T3 primer is initiated to label and terminate with the isotope S35diettye ( ³⁵ S-dATP). After the reaction, it was developed on 6% polyacrylamide gel, soaked in 15% methanol and 5% acetic acid for 20 minutes, dried and exposed to X-ray film for 16 hours.

The determined base sequence and GenBank database were compared using the Blast program (http://www.ncbi.nlm.gov/BLAST). In order to find the mouse cyclin B2 homologous DNA and determine the total nucleotide sequence, successive subclones were prepared by a series of nested deletion methods (17), and the base sequences were determined and finally linked. Figure 1 (a) is the entire base sequence and amino acid sequence, consisting of a total of 1401 nucleotide sequences, of which 398 amino acid sequence of which encodes a 44.5 kilodalton (kDa) size protein. Compared with 3 primes, the 3'-UTR site has 185 nucleobases. There are multiple adenine signals and multiple adenine tails attached. Figure 1 (b) shows a characteristic cyclin box and breakdown box in the protein and the cyclin box consists of 150 amino acids and is common to all cyclins. Destruction boxes are common to type B cyclins and trigger ubiquitin-mediated degradation pathways. The clones of the F1-104D expression sequencing (EST) were closer to B2 cyclin than B1 cyclin and showed 89% consensus with the previously cited human cyclin B2. This was lower than the 91% homology of hamster cyclins and the difference with human cyclin B2 previously cited is the nucleotide sequence where the transition of the 10 amino acids (AAQVAKKAQN) has been transferred to the front of the destruction box (Figure 1 (c)). The existing cited cyclin B2 is also structurally different from cyclin B2 in different species, ie hamsters, mice, humans.

<Example 3>: Base sequence analysis of the reverse transcriptase polymerase chain reaction product

In order to confirm the error of the transferred amino acid sequence of Example 2 was to determine and examine the base sequence of the reverse transcriptase polymerase chain reaction product in various tissues and cells. First, synthesize the first helix cDNA by reverse transcriptase, then terminate and amplify the second helix cDNA synthesis, and then use Tfl DNA polymerase and Access RT-PCR system kit (Progma, Madison, WI). The denature was extended at 94 ° C for 30 seconds, the annealing was 62 ° C for 1 minute, the extension was 68 ° C for 2 minutes, and the last cycle was extended for 68 ° C for 7 minutes, followed by electrophoresis on 1.5% agarose gel. It was. The polymerase reaction product was digested with restriction enzymes and purified and inserted into EcoRI and Bam HI sites of pBluescript KS II (+) Phagemid; Stratagene, La Jolla, CA. The position of the primer used (primer) is shown in Figure 2 (a) and the base sequence is as follows.

Figure 2 (b) is a sequence of amplification by the reverse transcriptase polymerase reaction (RT-PCR) method performed in fetal liver, liver cancer, Hep G2, Hep 3B, PLC / PRF / 5 and HeLa cell line in sequence 558 bases were amplified using F1 / B1, a set of initiators containing a common cyclin box with the site (5′UTR) (2-7 lanes in FIG. 2 (b)) and F1 / B2 initiation in cDNA in normal adult liver The base sequence of all tissues and cell line products was determined by amplifying 259 bases (8 lanes in FIG. 2 (b)) using a set of children. As shown in FIG. There was an error.

<Example 4>: Production of human cyclin B2 gene expression vector

1) pMALC-p expression vector construction

First, pHCB2 cloned at EcoR I and Xho I sites of pBluescript SK II (+) was first cleaved with Xho I restriction enzyme and treated with Klenow to make blunt ends. Then, again with EcoRI, electrophoresed in 1% TAE gel, and then a single band corresponding to 1.4 kilobases (kb) was cut to obtain a Gene cleaning kit (Bio 101, Inc., Vista, CA). Was eluted and used as an insert. In the vector production, the multi-linker Hind III site of pMAL-p (NEB, Beverly, MA) was cut and clenowed to convert to blunt ends, and then, eco-RI was cut to gel electrophoresis, and the major segment bands were cut and eluted. Reaction of the recombinant vector is inhibited by reaction at 37 ° C. for 1 hour with alkaline dephosphatase. The molar ratio of the vector and the insert is adjusted to about 1: 3, and the ligation is performed using a ligation enzyme (ligase), a buffer solution, and a diATP (dATP) at 16 ° C. The polylinker site of the pMAL-p vector used is as follows.

After one day, these were introduced into E. coli, and DNA was extracted and digested with various restriction enzymes to confirm human cyclin B2 expression vector pMALC-p (Accession No. KCTC 0458 BP: pMALC-p) as shown in FIG. 3.

2) pRSETC-A expression vector production

First, pHCB2 is double-cut with Bam HI and Xho I restriction enzymes, followed by gel electrophoresis. A single band is cut to elute DNA, and then used as an insert. Vector production of pRSET-A (Invitrogen, San Diago, CA) was cut with Bam HI and Xho I, reacted with alkali dephosphorase and treated at 35 ° C. for 1 hour, followed by gel electrophoresis, extraction, and use as a vector. Vectors and inserts are placed overnight at 16 ° C. in a 1: 3 molar ratio. The polylinker region of pRSET-A used is as follows.

One day later, E. coli was extracted, extracted with DNA, and digested with various restriction enzymes to confirm human cyclin B2 expression vector pRSETC-p (Accession No. KCTC 0459 BP: pRSETC-p) as shown in FIG. 4.

<Example 5>: Human cyclin protein purification

(1) Purification of human cyclin protein by pMALC-p expression vector: pMALC-p expression vector inserted with human cyclin B2 gene was introduced into Escherichia coli (DH5α) and 1 liter of medium (10 g tryptone, 5 g yeast extract 5 g sodium chloride) , 2g glucose, ampicillin 100 ㎍ / ml overnight and cultured 2 × 10 ⁸ bacteria / ml (absorbance A ₆₀₀ ~ 0.5) to grow the IPIP (IPTG; isopropyl β-D-thiogalactopyranoside) inducer to 0.3 mM After incubation for 2 hours, the cells were harvested by centrifugation and 50 ml of column buffer (10 mM phosphate, 30 mM sodium chloride, 0.25% Tween 20, 10 mM betamercaptoethanol, 10 mM idieti, 10 mM easyti) The solution was dissolved in and crushed by ultrasonication in ice water. The supernatant was diluted 1: 5 in column buffer by centrifugation. A 2.5 × 10 cm column filled with amylose 3 mg / ml was prepared, washed with 8x column buffer and diluted supernatant was added. It was washed with column buffer of 8-fold column volume, eluted by adding 10 mM maltose to the column buffer, and collecting 10 to 20 fractions of 3 ml each. Concentrated and stored at a concentration of 1 mg / ml using Amicon concentrated tubes. As a preliminary experiment, 20 μl of the complex protein was added with 1 μl of factor Xa at 4 ° C. for 24 hours, followed by electrophoresis on SD Page Gel to confirm the separation of the complex protein, and then a large amount of the complex protein was digested with Factor Xa. 1 g of hydroxyapatite is called a column buffer and then poured into a 1 x 10 cm column and the complex proteolytic mixture is added to the column. Wash with 80 ml of column buffer, elute with 0.5 M phosphate (pH 7.2) and collect 2 ml fractions to measure protein at absorbance A ₂₈₀ and pour the first 8 ml into 15 ml amylose column. At this time, the maltose binding protein was removed from the eluate, and the human cyclin B2 protein was quantitatively determined by Lory's method (Lowry et al, J Biol Chem 1951; 193: 265-283).

(2) Purification of human cyclin protein by pRSETC-A expression vector: 1 liter of medium (10 g tryptone, 5 g yeast extract 5 g sodium chloride) was introduced into E. coli (DL21) by introducing pRSETC-A expression vector into which human cyclin B2 gene was inserted. , 2g glucose, ampicillin 100 ㎍ / ml overnight and cultured 2 × 10 ⁸ bacteria / ml (absorbance A ₆₀₀ ~ 0.7) to grow the IPIP (IPTG; isopropyl β-D-thiogalactopyranoside) inducer to 0.3 mM After incubation for 2 hours. After centrifugation, the sample was suspended in 2-5 times the volume of ultrasonic buffer (50 mM sodium phosphate buffer pH 8.0, 300 mM sodium chloride) for ultrasonic grinding, and then stored at -20 ° C and ultrasonically crushed on ice water (for 1 minute Grinding / cooling for 1 minute / 200-300 watts). Centrifuge again to collect the supernatant and prepare the column. Equilibrate 8 ml of 50% nickel-anti-resin into the ultrasonic buffer, fill it into a 1.6 cm diameter column, and wash 40-80 ml at approximately 0.5 ml / min with ultrasonic buffer. Again about 80 ml wash buffer (50 mM sodium phosphate, 300 mM sodium chloride, 10% glycerol, pH 6.0) Finally elute with 30 ml wash buffer containing 0.5 M imidazole. Also confirmed by electrophoresis on SDS page gel, purified complex protein was dialyzed at 10 mM Tris pH 8.0 and 10 mM calcium chloride, and 0.5 μg of Boent Enterokinase (sample 1) was used. g was added overnight at 37 ° C. and passed through a nickel anti-resin column to remove the decomposed complex protein. The binding conditions are 40 mM phosphate buffer, pH 8.0 and 500 mM sodium chloride buffer.

Figure 5 (a) is an electrophoretic picture of the protein expressed in E. coli by the expression vector metal binding protein is 3.8 kilodaltons (kDa) (lane 2) and human cyclin B2 and metal binding protein complex is 51.8 kilodaltons (kDa) (4 lanes). The maltose binding protein is 42 kilodaltons (kDa) (lane 6) and the human cyclin B2 and the maltose binding protein complex protein are 90 kDa (lanes 8,9).

Figure 5 (b) is an electrophoresis of the cyclin B2 complex protein eluted from the amylose column and the nickel anti-N (Ni ²⁺ -NTA) column, the left side is 90 kilodaltons (kDa) human cyclin B2 / maltose binding protein The right side shows the complex protein of human cyclin B2 / metal bound protein of 51.8 kilodalton (kDa) size.

<Example 6>: Antibody preparation

To prepare the polyclonal antibody, the eluted protein was loaded with factor Xa or enterokinase for 24 hours at 4 ° C., and 48 kilodalton (kDa) cyclin B2 protein was electrophoresed on SDS gel. Cut, purify, and inoculate the rabbits. In short, 0.5 ml of HBS: Hank's balanced salt solution was added to 8% S.D.Zegel and electrophoresis of the digested maltose-binding protein and cyclin B2 complex protein digested with factor Xa. Pulverize ultrasonically, add 0.5 ml HBS solution, grind with homogenizer to mix with 1 ml of complete freund's adjunvant, and inoculate 100-200 μl of rabbit back into the back of the rabbit. After 1 week of immunization 4 times in the same way as de-adjuvant, blood was collected and stored at -70 ℃ and the polyclonal antibody serum was nitrocellulose. 0.2 M glycine (pH 2.5) is eluted by a strip or cyclin B2 column.

Preparation of monoclonal antibody was performed by immunization of 6 week old BALB / C mice with intraperitoneal injection of 20 μg of maltose-binding protein / cyclin B2 complex protein 1: 1 with complete freund's adjunvant. Two weeks later, 20 µg of maltose binding protein / cycline B2 complex protein was injected intravenously and immunized again. Three days later, the mouse spleens were extracted to form a splenocyte suspension and fused with NS (NS-1) mouse bone marrow cells. Hybridoma was selected as a hat medium, and the isotype of the monoclonal antibody produced by the hybrid was coated with a microtiter plate with a metal binding protein / cycline B2 complex protein (10 ㎍ / ml). It was measured by ELISA method. Positive clones were selected and cloned three times by limiting dilution, stored in a nitrogen tank, thawed and thawed as needed to inject hybridoma cells into the abdominal cavity of BALB / c mice injected with pristane. The ascites was collected and stored at -70 ° C.

<Example 7>: Immunoblot Analysis

In experiments to test whether the produced human cyclin antibody specifically binds to human cyclin antigen, E. coli introduced human cyclin B2 expression vector, and induced expression of maltose-binding protein / human cyclin B2 in E. coli. Gastric mucosa and cancer tissue were treated with 1% Triton X- containing aprotinin (1 trypsin unit / ml), leupeptin (10 μg / ml) and pepstatin A (10 μg / ml). The protein was extracted by 100, electrophoresed on SDS gel, transferred to nitrocellulose membrane, and immunoblot of immune cyclin B2 was performed as shown in FIG. 6 using alkaline phosphatase-binding anti-goat goat immunoglobin (Henikoff). S, Gene (Amst) 1984; 28: 351-59). In Figure 6a E. coli lysate does not show a band by pre-immune serum (lane A1), but anti-cycline B2 serum appears to be a single band by recognizing the 90 kilodalton (kDa) maltose binding protein / cyclin B2 complex protein (A2) lane). In FIG. 6B, no bands were detected by pre-immune sera in normal gastric mucosa and gastric cancer tissues (lanes B1 and 2), but anti-cyclin B2 serum showed a single band of 48 kilodaltons (kDa) (lanes B3 and 4). Cyclin B2 expression levels in gastric cancer tissue are higher than normal stomach.

<Example 8> Immunohistochemical Staining

Immunohistochemical staining was performed using streptavidin-biotin-peroxidase (Biomedia, Foster City, CA). Normal, gastric adenomas and gastric cancer tissues were fixed in 10% formalin, which is a common pathological tissue treatment method, and paraffin-embedded tissues were used to remove paraffins with xylene, washed with 1M Tris buffer, and methanol at 40 ° C. with methanol periodic acid. Inactivate endogenous peroxidase for 2 minutes. Tissue sections were placed in a biotinylated immunoglobin-binding immunoglobin for 4 minutes at 40 ° C. for 9 minutes at 40 ° C. under primary cyclin B2 polyclonal antibody (1: 200 dilution) and aminoethylcarbazole (3-amino-9-ethylcabazole). ) And 0.05% hydrogen peroxide and pH 5.2 buffer. Negative control staining was done using physiological saline or mouse IgG ₁ immunoprotein instead of primary antibody.

7 shows immunohistochemical staining in normal gastric mucosa (A), gastric adenoma (B, C) and malignant gastric cancer (D, E), respectively. It is usually spotted in the cytoplasm or nucleus of normal and cancerous cells and sporadically observed at the base of gastric mucosal crypts in normal gastric mucosa, which is consistent with proliferative epithelial cells. In gastric adenocarcinoma, proliferative epithelial cells are widely scattered sporadically toward the base of the crypts as well as into the gastric lumen and gradually decrease in number toward the gastric lumen. In gastric cancer tissues, cancer cells that are more strongly spotted are observed with diffuse staining of gastric cancer cell lesions. In particular, it showed strong staining in the cytoplasm of malignant Cygnus ring cells. In FIG. 7, immunohistochemical staining in normal colon mucosa (F, G), colorectal adenoma (H), and colorectal cancer (I, J) is also strongly stained on the basal layer of crypts, that is, proliferative mucosal epithelial cells, in normal mucosa. Scattering of epithelial cells was observed in all of the colon cells. In colon cancer cells, strong spot-staining cells were found in the colony of diffuse cancer cells. Diffuse staining reflects the high levels of cyclin B2 in the overall cancer cells, of which dot-stained cells were identified as cells in the dividing cycle. In general, benign cancers are characterized by increased numbers of human cyclin B2 positive cells. In malignant cancers, cancer cells are present in a highly stained division cycle with a diffuse staining of cancer cell lesions and a new and innovative staining.

Human cyclin B2 gene, protein and antibody of the present invention can measure the cell proliferation in normal cells, benign tumor cells, malignant cancer cells at the level of gene and protein expression, the dynamics of other cancer cells and the diagnosis, prognosis, It can be used for test methods such as metastasis. It is also used to treat cancer genes and use anti-cancer treatments for antibodies, which will greatly contribute to medical development.

Claims (10)

Human cell cycle control gene isolated from human fetal liver cDNA library, characterized by the following sequencing, amino acid sequence, 5 prime compared to 5 'UTR and 3 prime compared to 3' UTR Human cyclin B2 gene; The recombinant human cyclin B2 protein expression vector pMALC-p (KCTC 0458 BP), which clones the human cyclin B2 gene of claim 1 and inserts the gene into pMAL-p to express human cyclin B2 protein. Human cyclin B2 protein, which was recombinantly expressed by introducing cyclin B2 protein expression vector pMALC-p and pRESET-A (KCTC 0459 BP) into Escherichia coli and expressing it (Accession No. KCTC 0458BP, KCTC 0459 BP). An antihuman cyclin B2 antibody obtained by immunizing an animal with a recombinant human cyclin B2 protein expressed by a human cyclin B2 expression vector according to claim 3. The method of claim 4, wherein Anti-human cyclin B2 antibody obtained by immunizing an antibody with rabbit or mouse. The method of claim 4, wherein The anti-human cyclin B2 antibody is a polyclonal anti-human cyclin B2 antibody obtained by immunizing rabbits. The method of claim 4, wherein Anti-human cyclin B2 antibody is a monoclonal anti-human cyclin B2 antibody by spleen lymphocyte hybridoma obtained by immunization with mouse. Cloning and determining the nucleotide sequence of the human cyclin B2 gene according to claim 1, preparing a protein expression vector of human cyclin B2, and recombinant expression vector pMALC-p (KCTC 0458BP) or pRESET-A (KCTC 0459BP) Preparing a human cyclin B2 protein by introducing into E. coli, and preparing an anti-human cyclin B2 antibody. A reagent for diagnosing proliferation, division, or cancer differentiation of human tissues or cells using the anti-human cyclin B2 antibody according to claim 4 as an active ingredient. The recombinant human cyclin B2 protein expression vector pRSETC-A (KCTC 0459 BP), which clones the human cyclin B2 gene of claim 1 and inserts the gene into pRSET-A to express human cyclin B2 protein.